Clinical trial NCT01180036
Conditions: Idiopathic Membranous Nephropathy
Keywords: Membranous Nephropathy
Sponsor: Mayo Clinic
Start date: 2011-11-01; Completion date: 2017-10-01
Enrollment: 130 participants; Allocation: RANDOMIZED; Intervention model: SINGLE_GROUP

Brief summary:
The primary outcome of this study is to determine whether or not the B cell targeting with Rituximab is non-inferior or more effective than Cyclosporine in inducing long term remission of proteinuria.

Detailed description:
In IMN, experimental data suggests that B cells are involved in the pathogenesis of the disease. To date, the best proven therapy for patients with MN consists of the combined use of corticosteroids and cyclophosphamide (CYC). Since the mechanism of action of CYC includes suppression of various stages of the B cell cycle including B cell activation, proliferation, and differentiation and inhibition of immunoglobulin (IgG) secretion, it lends credence to the hypothesis that B cells abnormalities are involved in the pathogenesis of MN. Given the key role of IgG antibodies in MN, it is reasonable to postulate that suppression of antibody production by depleting B cells may improve or even resolve the glomerular pathology and be reflected by a reduction in proteinuria. Thus, a case could be made for using an agent capable of selectively depleting B cells, and therefore halting the production of immunoglobulins against antigens potentially present in the glomeruli. This approach could stop the initiating sequence of pathogenic events and result in resolution of the. The P.I. believes that the application of selective B cell targeting with Rituximab (RTX) will prove at least equal, or even superior, both in the production of short term and long term control of the nephrotic syndrome (NS) and be safer than any current therapeutic regimen used to treat MN.

Based on this rationale, the investigators conducted a pilot trial in 15 newly-biopsied patients (\<3 years) with IMN and proteinuria \>5g/24h despite ACEi/ARB use for \>3months and systolic BP \<130 millimeter of mercury (mmHg). Mean baseline creatinine was 1.4 mg/dl. Thirteen males and 2 females, median age 47 (range 33-63), were treated with RTX (1g) on days 1 and 15. At six months, patients who remained with proteinuria \>3g/24 received a second identical course of RTX. Baseline proteinuria of 13.0±5.7g/24h (range 8.4-23.5) decreased to 6.0±7.0 g/24h (range 0.2-20) at 12 months (mean ± SD). In the fourteen patients who completed a 12 months follow-up complete remission (proteinuria \<0.3g/24h) was achieved in 2 patients and partial remission (\<3g/24h) in 7 patients. In 5 of these 7 patients, proteinuria was \<1.5g/24h and follow up at 18 months showed that 3 of these 7 patients on PR achieved CR of proteinuria. Five patients did not respond. The mean drop in proteinuria from baseline to 12 months was 6.2± 5.1g (p=.002, paired t-test). There were a limited number of minor side-effects. Initial cluster of differentiation 20 (CD20)+ B cell depletion was seen in all patients. However, at 3 months, CD20+ B cells were starting to recover with five patients \>35 cells/µl (range 35-152).(50) These data contrasts with previous work by Ruggenenti et al. using RTX given weekly (375 mg/m2) for 4 weeks. Pharmacokinetic (PK) analysis showed that RTX levels in this 2-dose regimen were 50% lower compared to non-proteinuric patients, which could potentially result in undertreatment.

Based on these results, the investigators recently conducted a study postulating that in patients with MN, 4 weekly doses of RTX would result in more effective B cell depletion, a higher remission rate and maintaining of the same safety profile compared to patients treated with RTX dosed at 1g x 2.

Arms: arm 1: Patients randomized to the RTX arm will receive 1000 mg IV on Days 1 and 15. Patients who achieve complete remission at 6 months will not be retreated. A second course of RTX 1000 mg IV will be administered at study month 6 for individuals who have not achieved a complete remission, but have achieved a minimum of \>25% reduction in Time 0 proteinuria. Dosing at study month 6 will be independent of cluster of differentiation (CD) 19+ B cell count. arm 2: Patients randomized to the Cyclosporine arm will be started at a dose of CsA = 3.5 mg/kg/day p.o. divided into 2 equal doses given at 12 hour intervals. Target trough CsA blood levels are 125 to 175 ng/ml. Patients will have their doses adjusted according to their blood levels of CSA as monitored every 2 weeks until the target trough level is reached. If a complete remission is achieved by 6 months, CSA will be tapered and discontinued over a three-month period. If after 6 months there has not been a reduction in proteinuria of at least 25% of baseline values, the drug will also be discontinued. If there has been a \>25% reduction in baseline proteinuria (but not complete remission) the CSA will be continued for an additional 6 months.
Interventions: intervention 1: Rituximab intervention 2: Cyclosporine
Intervention descriptions: intervention 1: 1000 mg, I.V. on Days 1 and 15 and will be retreated at month 6 independent of cluster of differentiation (CD) 19+ B cell count intervention 2: Patients randomized to the Cyclosporine arm will be started at a dose of (CsA = 3.5 mg/kg/day p.o. divided into 2 doses for 12 months). Target trough CsA blood levels, as determined in whole blood by High Performance Liquid Chromatography (HPLC), are 125 to 175 ng/ml. A persistent and otherwise unexplained increase in serum creatinine \>30% would prompt an approximate 25% dose reduction of CSA, aiming for a corresponding 25% reduction in CSA trough level. If with this dose reduction the creatinine does not return to within 30% of baseline levels within 3 weeks, then a second dose reduction of approximately 25% with similar reduction in CSA trough level will be used. If the creatinine does not fall to baseline values with this second dose reduction, the drug will be discontinued. At the end of 12 months, Cyclosporine will be tapered by 1/3 of the maintenance dose monthly and hence discontinued after 3 months.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
MEmbranous Nephropathy 
Trial Of Rituximab (MENTOR) 
 
NCT# 01180036 
 
June 17, 2014 


 
0 
            
 
 
STUDY TITLE: 
 
MEmbranous Nephropathy Trial Of Rituximab (MENTOR) 
 
A Multi-Center Randomized Controlled Trial of Rituximab versus Cyclosporine in the 
Treatment of Idiopathic Membranous Nephropathy (IMN) 
 
Study Drugs 
Rituximab and Cyclosporine 
 
Funding 
Fulk Foundation 
Genentech, Incorporated 
 
Study Drug Support Provided By 
Genentech, Incorporated 
 
Sponsor Investigator 
F.C. Fervenza (PI) – Mayo Clinic Rochester 
D.C. Cattran (Co-PI) – University of Toronto 
 
Executive Committee 
F.C. Fervenza (PI) 
D.C. Cattran (Co-PI) 
D. Gipson (Co-I) 
J. Appel (Co-I) 
M. Kretzler (Co-I) 
B. Rovin (Co-I) 
K. Thorpe (Co-I) 
 
Data Management and Coordinating Center 
Applied Health Research Center, St Michael’s Hospital/University of Toronto 
 
Food and Drug Administration IND Exemption  
Number: 109567 
 
NEPTUNE6804 
ClinicalTrials.gov identifier: NCT01180036 
Protocol Number: 
 
10.0 
 
Protocol Version Date: 
17 June 2014 
 
Name: Fernando C. Fervenza (PI) 
Institution: Mayo Clinic – Division of Nephrology and Hypertension 
Address: 200 First Street, SW 
Phone: 507-266-1045 Fax: 507-538-3388 
Email: fervenza.fernando@mayo.edu 


 
1 
 
 
1.1 Overview 
 
This prospective, randomized, controlled phase III nephrology study will determine 
whether rituximab is non-inferior to cyclosporine in inducing long-term remission of 
proteinuria in patients with idiopathic membranous nephropathy (IMN.)  Subjects are 
randomized to open-label courses of treatment: IV rituximab (RTX), 1000 mg (2 
infusions, 14 days apart), or oral cyclosporine (CSA) 3.5 to 5 mg/kg/day.  Subjects are 
evaluated at 6 months; those that meet treatment response criteria will continue study 
medication and receive either 2 further infusions of RTX or a further 6 months of daily 
CSA treatment.  Throughout the study period, laboratory and safety parameters will be 
collected and documented. 
 
 
 
Study Hypothesis: B cell targeting with Rituximab is non-inferior to Cyclosporine in 
inducing long-term remission of proteinuria. 
 
Comparison(s): IV rituximab (RTX), 1000 mg (2 infusions, 14 days apart; repeated at 6 
months if a substantial reduction in proteinuria (equal to or >25%) is seen at 6 months) or 
oral cyclosporine (CSA) 3.5 to 5 mg/kg/day for 6 months (continued for another 6 
months if a substantial reduction in proteinuria (equal to or >25%) is seen at 6 months). 
 


 
2 
 
SPECIFIC AIMS: 
 
The specific aims of this Phase III trial are to test the hypothesis;  
 
1. That B cell targeting with Rituximab is non-inferior to Cyclosporine in inducing long-
term remission (complete or partial) of proteinuria in patients with IMN. 
2. That B cell targeting with Rituximab reduces the number of relapses (efficacy in 
sustaining remission) and increases the time to relapse when compared with 
treatment with Cyclosporine. 
3. That B cell targeting with Rituximab is as effective as Cyclosporine in inducing 
complete or partial remission of proteinuria in patients with IMN during the active 
treatment phase. 
4. That B cell targeting with Rituximab has a better side effect profile and improved 
quality of life when compared with treatment with Cyclosporine in patients with IMN. 
 
BACKGROUND AND SIGNIFICANCE 
IMN is a common immune-mediated glomerular disease and remains the leading cause of 
Nephrotic Syndrome (NS) in Caucasian adults.1  Although in most patients the disease 
progresses relatively slowly, approximately 40% of patients eventually develop End 
Stage Renal Disease (ESRD).2 Because of its frequency, it remains the 2nd or 3rd cause of 
a primary glomerulopathy leading to ESRD.3  Patients with IMN who remain nephrotic 
are at an increased risk for thromboembolic4 and cardiovascular (CV) events.5, 6  
Available immunosuppressive therapies including corticosteroids, alkylating agents, and 
calcineurin inhibitors (Cyclosporine), appear to be at least partially successful in reducing 
proteinuria in IMN, but their use is controversial and is associated with a number of 
adverse effects and a high relapse rate, thus tempering their use in IMN. (reviewed in 7)  
The significant risks associated with current immunosuppressive therapies are important 
in a disease where up to 30% of IMN patients are said to achieve spontaneous remission 
of proteinuria, enjoy long-term renal survival and, should not be treated with 
immunosuppressive therapy.  This information is, however, misleading, since the 
percentage of patients that undergo spontaneous remission is much lower when patients 
are selected with higher grades of proteinuria at presentation e.g. proteinuria >8g/24h.8 
Similarly, although Schieppati et al. reported a 72% renal survival at eight years for 100 
untreated patients with IMN, in this study, 37% of patients were non-nephrotic; a cohort 
well known to rarely progress to renal failure and hence a built-in bias towards a better 
outcome.  Indeed the overall proteinuria was relatively low (56% of patients had 
proteinuria <5g/24h), and the median follow-up was only 39 months.  The follow-up time 
is an important issue given that the natural history of the disease process tends to be slow 
even in the worst cases.  The final limitation was that all deaths were excluded from the 
analysis.9 However despite the favorable elements of this cohort, 25% reached ESRD by 
the end of 8 years. One approach to this has been to provide immunosuppression 
treatment only to those subjects identified as being at higher risk of progression: males, 
severe proteinuria, impaired renal function, a high prevalence of secondary lesions of 
focal and segmental sclerosis, and prominent tubulointerstitial damage on renal biopsy. 
Our experience, and others, however, suggests this approach is seriously flawed.10  There 
is a substantial fraction of patients, who progress to chronic kidney disease (CKD), that 
are not identified by these predictors and are thus unjustifiably excluded from therapy.  A 
second approach has been to treat only those patients who are exhibiting deteriorating 
kidney function.11, 12 These trials have been deemed successful because proteinuria and 


 
3 
azotemia were diminished.11-13 Careful review of the data however, shows that reversal of 
azotemia is almost always incomplete and often transient suggesting that the decline in 
GFR is merely attenuated and not arrested.  In addition, we believe that a prolonged run-
in (i.e., treatment-free) period of 12 or more months would be required to prospectively 
confirm this type of truly progressive IMN, but that this in turn would result in a 
significant and probable irreversible loss of nephrons and ultrafiltration capacity.14  This 
can be illustrated by the study of Polanco et al.8  These authors suggested that patients 
that were treated conservatively and who did not go into spontaneous remission had final 
creatinine = 2.4 ± 2.2 mg/dL, and eGFR 53 ± 35 ml/min.8 However, we believe this 
information is inaccurate. Taking into consideration that the mean age at presentation was 
51 years and the mean follow up was 69 months (making these patients ~56 years at 
follow-up) the eGFR should be ~30 ml/min if male and ~22 ml/min if female, indicating 
a significant loss of function with just waiting. The paper further underestimates chronic 
kidney disease because their end point was restricted to the absence of chronic dialysis or 
need for renal transplantation.  If we add loss of kidney function (as measured by change 
in GFR) their disease course would indicate a significantly higher incidence of CKD over 
time supporting our contention that prolonging the wait time for spontaneous remission 
results in patients paying a heavy price.   
      
Why do so many patients still progress to ESRD? There is growing evidence implicating 
proteinuria as a major player in the development of progressive tubular injury, interstitial 
fibrosis, and GFR loss. The higher the sustained levels of proteinuria, the faster the 
decline in renal function,15 a relationship that is true not only for patients with IMN but 
for other proteinuric renal diseases including FSGS and diabetic nephropathy.16-19 The 
reverse appears also to be true.  There is strong evidence to support that remission of 
proteinuria (either complete or partial remission) is a valid surrogate end point for both 
improved renal survival and slower rate of progression of renal disease, and that this 
reduction is an important therapeutic target for the clinician. This view is in line with the 
position statement of the NKF and NIDDKD indicating that proteinuria can be used as a 
surrogate marker and principal endpoint in clinical trials in proteinuric renal disease 
where GFR-based declines in function take too long to be of practical use.20 Proteinuria is 
also a marker for CV risk.5, 21-24  Recent post-hoc analyses of the diabetic trials 
RENAAL, IDNT and others, have shown that proteinuria determines renal outcome25, 26 
and CV outcome.27-29 The link between chronic renal failure (CRF) and CV disease is so 
strong that over 40% of patients starting dialysis already have evidence of CV disease.30, 
31 This information is important in the present study since all patients will have high 
grade proteinuria >5g/24h, a scenario almost universally associated with marked 
abnormalities in their lipid profile (high total cholesterol, normal or low levels of HDL 
and increased LDL).5, 6  Apart from hyperlipemia, patients with IMN and nephrotic 
syndrome (NS) are also at risk for thromboembolic events, with prevalence rate as high 
as 50% and a mortality rate within this group as high as 42%.4, 32  These data serve to 
emphasize the importance of other common life-defining sequelae of membranous 
nephropathy in these patients, in addition to their risk of renal failure.   
 
There is no standard specific treatment for IMN. Initial therapy should be supportive and 
involves restricting dietary protein intake, controlling blood pressure, hyperlipidemia, and 
edema.  The ideal target for blood pressure is not firmly established but current 
recommendations suggest that 130/80 mm Hg should be the treatment goal. There are 
only limited data to support a lower target of 125/75 mm Hg if there is proteinuria >1 g/d.    
Reducing protein intake to about 0.6-0.8 g/kg ideal body weight per day also tends to 


 
4 
decrease proteinuria.33  ACEi and/or ARBs are effective anti-hypertensive agents that 
may also reduce proteinuria in both diabetic and non-diabetic chronic nephropathy 
patients and slow progression of renal disease independent of blood pressure control.34 
This is the rationale for making these drugs the preferred agents to treat hypertension in 
proteinuric renal diseases.  However, evidence that such therapy is beneficial in IMN is 
weak, largely inferential, and the following issues need to be considered: 1) The degree 
of renal protection is related to the degree of proteinuria reduction and if proteinuria is 
not lowered, the benefit is substantially attenuated.35, 36 In the RENAAL trial the renal 
protective effect of angiotensin II blockade in patients with diabetic nephropathy was 
nearly fully explained by its anti-proteinuric effect.26  2) In patients with IMN, the anti-
proteinuric effect is modest (<30% decrease) and is more significant in patients with 
lower levels of proteinuria.37-39  3) Thus, in contrast to diabetic renal diseases, ACEi may 
not offer the same degree of renal protection to patients with IMN.40  In fact, studies by 
du Buf-Vereijken et al.41 and in a review by Troyanov et al.,42 the use of ACEi or ARBs 
by multivariate analysis did not show an independent value in determining the prognosis 
of patients with IMN. More recently, Praga et al. showed additional evidence that in 
patients with NS, (the majority with IMN), ACEi were ineffective in reducing 
proteinuria, and that this failure to respond in IMN patients was associated with poor 
renal function outcomes.43, 44  4) In patients in which a significant anti-proteinuric 
response is observed, the effect is usually seen within 2 months of initiation of 
angiotensin II blockade therapy.37  Although a relative reduction of proteinuria is always 
a positive result, the aim of anti-proteinuric therapy is to reduce it as close as possible to 
normal levels (complete remission (CR)). Reaching this goal in patients with proteinuria 
> 5g/24 using conservative treatment with ACEi or ARBs seems unrealistic, even when 
these drugs are used at the highest dose. Taken all together, in the past decade relatively 
little progress has been made in the treatment of patients with IMN, and up to 40% of the 
patients will still progress to end stage renal failure.  Agents that result in a higher 
response and lower relapse rates, as well as fewer adverse effects, are truly needed.  
 
Current conservative therapy 
Initial therapy should be supportive and involves restricting dietary protein intake, 
controlling blood pressure, hyperlipidemia and edema (see above).  In patients that do 
have an anti-proteinuric response to angiotensin II blockade therapy, the effect is usually 
seen within 2 months of initiation of the medication and tends to be modest.37  Although 
a relative reduction of proteinuria is an important effect, the aim of anti-proteinuric 
therapy is to reduce as close as possible to normal levels (complete remission).  Reaching 
this goal in patients with sustained proteinuria > 5g/24 using conservative treatment with 
ACEi and/or ARBs is unlikely, even when these drugs are used at the highest dose.  The 
role of immunosuppressive agents in the management of patients with IMN thus remains 
a critical issue.  Our immunosuppressive treatment armamentarium in this regard has 
evolved but is still limited.  Agents that result in a higher response rate with a lower 
relapse rate and fewer adverse effects are truly needed and thus the focus for our 
proposed randomized controlled trial.  
 
Current Options for Immunosuppression in IMN  
 
Cyclosporine (CSA) 
Cyclosporine is a calcineurin inhibitor (CNI) that exerts its immunosuppressive effect by 
blocking the production of IL-2, IL-3 and IFN-γ, resulting in a reduction of T-
lymphocyte helpers/inducers and cytotoxic cell function.45 This immunosuppressive 


 
5 
effect is probably not the only anti-proteinuric effect of CSA since it is known to have 
both hemodynamic effects and to act on the podocyte structure. The latter effect has been 
demonstrated in vitro to alter glomerular permeability46 by a direct effect on the actin 
cytoskeleton (and therefore the shape) of podocytes.47 This is postulated to protect 
podocytes from immune injury and may be part of the explanation behind the observation 
of its significant benefit in certain immunologic glomerular disorders such as in IMN and 
minimal change despite significantly less exposure compared to solid organ transplant 
recipients. Cyclosporine has been extensively used in the treatment of proteinuric 
glomerular diseases and has been proven to be effective in inducing remission of 
idiopathic nephrotic syndrome in children and adults.48 It has been used successfully in 
patients with IMN and nephrotic syndrome resistant to corticosteroids and/or cytotoxic 
drugs and has been tried in some of the glomerular disorders even before exposure to 
cytotoxic therapy because of its significantly different side effect profile.39, 49-55 
 
In the most relevant randomized controlled trial, 51 patients with steroid-resistant 
nephrotic syndrome and preserved renal function (creatinine clearance [CrCl] > 42 
ml/min/1.73m2) were randomized to CSA or placebo treatment, with both groups also 
receiving low-dose prednisone (0.15 mg/kg/day).51 The duration of treatment was 26 
weeks followed by a tapering off period over 4 weeks.  At 26 weeks a significantly 
higher remission rate was observed in patients treated with CSA -compared to placebo- 
(75 % versus 22%, P. <0.001). Among the CSA-treated patients with remission, 90% 
showed partial remission and 10% complete remission.  Twelve months after 
discontinuing the drug a significant percentage of the CSA treated patients remained in 
remission (39%) compared to the placebo treated patients (39 versus 13%; p = 0.007).51  
 
The effectiveness of a more prolonged course of CSA in inducing remission of 
proteinuria has been recently documented in a study where CSA was given for 12 months 
and an 80% remission in proteinuria (complete plus partial) was observed.55  It was 
indicated in this paper that CSA combined with prednisone is more effective than CSA 
monotherapy in maintaining remission in IMN.55  However careful review of their data 
suggests that the group that remained in remission following tapering of the CSA had 
substantially higher trough levels, both in the monotherapy and in the combined CSA 
plus prednisone group, compared to the relapse group suggesting that this may have been 
the reason for the more sustained remission.  This would support our trial proposal i.e. 
CSA alone (no prednisone is required).  In earlier studies51 low dose prednisone was 
added to CSA because historical data in patients with MCD/FSGS had suggested higher 
remission rates with combination therapy and that perhaps the combination would reduce 
the nephrotoxicity of CSA. A recent review of these early studies and the more current 
ones suggest that this combination is not necessary.48  Further support that CNI 
monotherapy is effective is illustrated in the recent RCT in MN using Tacrolimus. This 
study showed virtually identical remission rates in proteinuria as in the original CSA plus 
prednisone trial (see above) using Tacrolimus monotherapy.  Their entry criteria required 
the presence of nephrotic syndrome resistant to ACE inhibitor therapy, steroids or the 
combination of steroids with cytotoxic drugs. Their relapse rate following discontinuation 
of the drug was similar to that of the RCT with CSA.56 CSA has also been shown to be of 
benefit in inducing remission of nephrotic syndrome and preserving renal function in 
patients with IMN associated with progressive renal failure. In a prospective randomized 
controlled trial, 64 patients were placed on a restricted protein diet and followed closely 
for 12 months. Patients that demonstrated significant progression of their disease during 
this observation period (n = 17) determined by an absolute loss in CrCl > 8 ml/min/year 


 
6 
and persistent nephrotic range proteinuria were randomly assigned to either CSA 3.5 
mg/kg/day or placebo for 12 months.57 A significant reduction of proteinuria was 
observed only in CSA-treated patients and the remission was sustained in 75% of these 
patients followed on average for 21 months.57  
 
Thus, CSA represents an effective therapy for inducing remission of nephrotic syndrome 
in patients with IMN.  It leads to remission of proteinuria in the majority of nephrotic 
patients with IMN but with less potential serious toxicity compared to the use of 
cytotoxic/alkylating agents.  There is also more experience with CSA (versus other CNI) 
in the US and Canada and for these reasons it has been chosen as the comparator arm for 
our study.   
 
Patients with IMN that do progress do so over years and follow-up in RCTs with CNI 
therapy have not been of sufficient duration to illustrate improvements in renal survival. 
However there is well documented recent data that indicates that both complete and 
partial remission in proteinuria is associated with improved renal survival.  Complete 
remission was associated with a 100% 10-year survival and partial remission (defined as 
more than a 50% decrease from baseline and attaining a urine protein level of <3.5 g per 
day) was associated with a 90% 10 year renal survival compared to 50% kidney survival 
in those that fail to remit.  In addition, in this same study PR as measured by slope of 
CCR, resulted in a slowing of progression rate of  >80 % compared to non-remitters.42 
 
Toxicity of cyclosporine 
Cyclosporine is a potent immunosuppressive agent and is associated with significant risk 
of both short and long-term toxicity.  In the majority of cases these adverse effects are 
associated with both total daily dose and total exposure.  Precise incidences of these 
toxicities are hard to ascertain from the literature given the rarity of studies where these 
agents are used as monotherapy and their use for a limited duration as proposed in this 
trial.  There is however undoubtedly an acute effect on filtration function and delayed 
effects on renal vasculature that have been well documented and a recent review 
summarized both short and long-term toxicities associated with this agent.58  
Hypertension, overgrowth of body hair and gingiva hyperplasia, mild tremor, infections, 
elevated bilirubin levels and mild nausea represent the most frequently observed short-
term adverse effects related to CSA.58 These are seen in up to 20 to 30% of the treated 
population but are usually tolerated and/or treatable with additional agents (such as 
additional anti-hypertension drugs) or by dose reduction.48   
 
Cyclophosphamide (CYC) 
In IMN, experimental data suggests that B cells are involved in the pathogenesis of the 
disease.59  To date, the best proven long-term therapy for patients with IMN consists of 
the combined use of corticosteroids and CYC, the Ponticelli’s protocol.  However, the 
potential side effects with the use of 9g of methylprednisolone mandated by this protocol 
as well as the potential risk of other serious side effects associated with the use of 
cytotoxic agents (such as bone marrow toxicity, severe infections, gonadal dysfunction) 
combined with the long-term risk of malignancy associated with CYC has left many 
physicians reluctant to use this regimen. It should also be remembered that IMN is a 
relapsing and remitting disease and even with the use of this therapy, relapse rates remain 
high.  In Ponticelli et al.’s randomized 6 month study in patients with IMN comparing 
methylprednisolone plus chlorambucil versus methylprednisolone plus CYC the overall 
relapse rate was 24% (21/87) and similar in both arms.  These relapses occurred between 


 
7 
6 and 30 months post treatment, with the majority within 18 months.60  An additional 
~10% of patients had to stop treatment and were discontinued from the study because of 
adverse effects.  The risk of malignancy with CYC is substantial with the hazard of 
neoplasia roughly correlated to the cumulative dose and duration of cytotoxic therapy.61-
63 Faurschou et al. recently investigated the incidence of malignancies associated with 
CYC exposure in a cohort of 293 patients with Wegener's granulomatosis.64 The risk of 
malignancy was not increased for patients who never received CYC or for patients 
treated with cumulative CYC doses ≤ 36 g.64 In contrast, high risks of leukemia (SIR 
59.0, 95% CI 12-172) and bladder cancer (SIR 9.5, 95% CI 2.6-24) were observed for 
patients treated with cumulative CYC doses >36 g.  These data would suggest that a 
patient who weighs 80kg and is treated with CYC at a dose of 2.5 mg/kg/day would 
exceed this threshold after 2 standard courses of the Ponticelli’s protocol. Since IMN is a 
remitting and relapsing disease with the potential for significant cumulative CYC dosing, 
there is a substantial risk of late-occurring, serious malignancies. The other major 
concern limiting CYC utility is related to its gonadal toxicity.  Data indicates that ovarian 
failure is seen in female patients of any age receiving a cumulative dose of as little as 28 
g of CYC.  In addition, these authors found that age at the onset of therapy was an 
additional independent factor associated with sterility.65, 66  Although male infertility is 
harder to assess, studies have demonstrated that doses above 7.5 g/m2 of CYC can result 
in permanent oligospermia.67 For these reasons, the majority of the academic centers in 
the US have considered CYC treatments too toxic and relegated the use of Ponticelli’s 
protocol to rescue therapy in patients who have failed less toxic immunosuppressive 
therapies.   
 
 
 
INNOVATION 
 
A new approach to therapy:  the case for targeting B cells:  In IMN, experimental 
data suggest that B cells are involved in the pathogenesis of the disease.59  To date, the 
best proven therapy for patients with IMN consists of the combined use of corticosteroids 
and cyclophosphamide (CYC).  The mechanism of action of CYC includes suppression 
of various stages of the B cell cycle including B cell activation, proliferation, and 
differentiation and inhibition of immunoglobulin secretion, supporting the hypothesis that 
B cell abnormalities are involved in the pathogenesis of IMN.68, 69  Given the key role of 
IgG antibodies in IMN70, it is reasonable to postulate that suppression of antibody 
production that targets glomerular antigens by depleting B cells may improve or even 
resolve the glomerular pathology. This approach of stopping the initiating pathogenic 
event could potentially result in resolution of the disease process. This is the theory 
underlying the application of selective B cell targeting with Rituximab (RTX) in 
IMN.  Our hypothesis is that it will prove at least equal to, or even superior to 
current therapies, both in the production of short term and long term control of the 
NS and be safer than any of the other current regimens used to treat IMN.  
 
PRELIMINARY STUDIES 
Based on this rationale, we conducted a pilot trial in 15 newly-biopsied patients (<3 
years) with IMN and proteinuria >5g/24h despite ACEi/ARB use for >3months and 
systolic BP <130mmHg.71 Mean baseline creatinine was 1.4 mg/dl. Thirteen males and 2 
females, median age 47 (range 33-63), were treated with RTX (1g) on days 1 and 15. At 
six months, patients who remained with proteinuria >3g/24 received a second identical 


 
8 
course of RTX. Baseline proteinuria of 13.0±5.7g/24h (range 8.4-23.5) decreased to 
6.0±7.0 g/24h (range 0.2-20) at 12 months 
(mean ± SD) (Figure 1).  In the fourteen 
patients who completed 12 months follow-up 
complete remission (proteinuria <0.3g/24h) 
was achieved in 2 patients and partial 
remission (<3g/24h) in 7 patients. The mean 
drop in proteinuria from baseline to 12 
months was 6.2± 5.1g (p=.002, paired t-test).  
In 5 of these 7 patients, proteinuria was 
<1.5g/24h. 
 
At 18 months follow up 3 of these 7 PR 
patients achieved CR of their proteinuria.  
 
 
 
(Figure 1)    
Five patients did not respond. There were a limited number of minor side-effects (see 
below). 
 
 
 
 
 
 
 
 
 
    
Initial CD20+ B cell depletion was seen in all patients. However, at 3 months, CD20+ B 
cells were starting to recover with five patients demonstrating >35 cells/µl (range 35-
152).71 These data contrast with previous work by Ruggenenti et al. using  the lymphoma 
regimen where RTX is given weekly (375 mg/m2) for 4 weeks.2 Using that regimen, B 
cell depletion was maintained up to 12 months post treatment and their proteinuria 
reduction was more rapid and complete. Similarly our studies using this standard 
lymphoma protocol in patients with ANCA vasculitis demonstrated that peripheral blood 
B-cell depletion was consistently achieved and maintained for at least six months. 
Pharmacokinetic (PK) analysis had showed that RTX levels in our original 2-dose 
regimen were 50% lower compared to studies in non-proteinuric conditions.  This could 
potentially result in undertreatment. Based on these results, we conducted an additional 
study based on the premise that in patients with MN, 4 weekly doses of RTX would 
result in more effective B cell depletion, a higher remission rate while still maintaining 
the same safety profile as patients treated with RTX dosed at 1g x 2. Twenty patients 
(including 11 patients that had failed alternative immunosuppressive therapy) with IMN 
and proteinuria >5g/24h received RTX (375mg/m2 x 4), with retreatment at 6 months 
regardless of proteinuria response. A detailed pharmacokinetics study was conducted in 
concert with immunological analyses of the adaptive immune compartment (T and B 
cells) to ascertain the impact of RTX on lymphocyte subpopulations.  Baseline 
proteinuria of 11.9±4.9g/24h decreased to 4.2±3.8g/24h and 2.0±1.7g/24h at 12 and 24 
months, respectively (p<0.001) while creatinine clearance increased from 72.4±33 at 
baseline to 88.4 ±31.5 ml/min/1.73m2 at 24 months (p=0.02) (Figure 2A/B).  
 


 
9 
 
Figure 2.  A. Box plots of urine protein by months since start of RTX therapy. The top 
and bottom of the box are the estimated 75th and 25th percentiles, respectively. The 
intermediate horizontal line and "+" sign represent the median and mean urine protein, 
respectively. The vertical lines extend to the largest (smallest) data point that is within 1.5 
times the inter quartile range (75th-25th percentile) above the 75th percentile (or below 
the 25th). The square symbol identifies points outside of this range. The number of 
patients with follow-up at 0, 1, 3, 6, 9 12, 18, and 24 months are: 20, 20, 20, 19, 18, and 
18, respectively. * P<0.05, ** P<0.001; B. Longitudinal effect of RTX on proteinuria 
(log transformed). 
 
Of 18 patients who completed 24-months follow up, 4 are in complete remission, 12 are 
in partial remission (CR + PR = 80%), 1 had a limited response (>50% drop in P but 
>3.5g/24h) and 1 patient relapsed from a partial remission. When interpreting these 
results we should take into account that >50% of these patients had failed previous 
immunosuppressive therapy. This study also emphasizes that proteinuria is reduced 
gradually and that it may take several months to reach its nadir. This observation is in 
agreement with previous reports in patients with IMN treated with prednisone in 
combination with a cytotoxic agent, but was seen without the short-term toxicity 
observed with alkylating agents.60  Kidney function remained stable or improved in all 
patients. These results were observed despite the fact that in patients with nephrotic 
syndrome (NS), serum albumin levels influence tubular creatinine secretion resulting in 
an endogenous creatinine clearance that overestimates GFR in NS.72  In addition, 
previous investigators have indicated that the onset of IMN is accompanied by a severe 
depression in hydraulic permeability of the glomerular capillary wall. This is partially 
offset by enhancement of filtration surface area and by profound depression of 
glomerular oncotic pressure. This can result in the GFR initially remaining in the normal 
range or at worst, depressed by <50% in moderate IMN. In severe IMN these 
investigators found this mechanism was significantly worsened by a marked reduction in 
functional glomerular number and a GFR depression of >50%.  These abnormalities were 
presumably reversed with RTX treatment and resolution of the NS. However, an 
additional explanation could have been that a number of patients had their angiotensin II 
blockade reduced or discontinued with time, altering the renal hemodynamics and 
thereby contributing to the significant increase in creatinine clearance. The important 
summary point in this regard is this type of GFR improvement by reduced RASS 
blockade would not be accompanied by a decrease in proteinuria.  RTX therapy 
represents a substantial advantage versus the risk for nephrotoxicity and other short-term 
toxicities associated with the use of calcineurin inhibitors (CNI). In addition RTX 
treatment assures patient adherence (since it is given by IV infusions). This is in contrast 
to the necessary monitoring required during the treatment with Cyclosporine.  This makes 


 
10 
RTX an easier option from the perspective of physician management and potentially 
offers a better quality-of-life for the patient. Cost estimates are also worth addressing. 
Although RTX is more expensive upfront the annual treatment costs compared to 
Cyclosporine are similar.  Perhaps most importantly, the effect of RTX appears to be 
sustained as only 1 patient in our recent pilot (5%) relapsed within the 2 year follow up 
period. A similar long term preservation of proteinuria reduction was observed in our first 
study71 with only 1 patient relapsing at 36 months. Thus, in the 35 patients included in 
our 2 studies, only 2 relapses occurred, and in one case proteinuria relapsed into the sub 
nephrotic range. These results (2/35 = 6% relapse) are similar if not better than those 
reported by Ponticelli et al. (21/87=24%) (63), and are substantially better than those 
reported with CNI treatment.  In addition they are significantly better than the most 
recently suggested IMN treatment option of prednisone combined with MMF.  In these 
studies a >40% relapse rate has been reported shortly after discontinuing therapy.51, 56, 74 
 
Serum RTX levels using the four dose regimen were similar to those obtained with 2 
doses of RTX. Four doses of RTX did result in more effective B cell depletion but 
proteinuria reduction was basically identical to the results obtained using RTX 1000mg 
on days 1 and 15. Thus, we believe that the two dose regimen with retreatment at 6 
months should be used in a randomized-control trial comparing RTX to Cyclosporine 
(the standard of care for IMN in the US). We believe that RTX will prove equal or 
superior to Cyclosporine in the treatment of MN and could represent the new standard of 
care for patients with this disease.  
 
Toxicity of Rituximab 
No dose-limiting effects were observed in our two Phase I/II studies evaluating safety 
and efficacy of RTX in IMN. The most commonly observed side effects were infusion 
related (flu-like symptoms, chills/rigors, fever, fatigue, headache, hypotension, nausea, 
leukopenia, angioedema and pruritus) and typically responded to an interruption of the 
infusion and resumption at a slower rate.71 Other side effects included: 1) serum sickness-
like syndrome (n=1); hair loss and thinning (n=2); one case of community acquired 
pneumonia three months after the first infusion that resolved with oral antibiotic 
treatment (this patient was retreated without complications); 4) herpes zoster reactivation 
(n=1) treated with oral antiviral drugs with full recovery. Similar to the study by 
Ruggenenti et al., no patient had a major drug-related adverse event.2 
 
Anti PLA2R levels and response to treatment 
RTX offers an advantage in MN over nonselective immunosuppressive agents such as 
CYC since it primarily targets B cells. However, changes in CD20+ B cell number in 
general have not consistently paralleled the response in terms of either reduction in 
proteinuria or improvement in the clinical phenotype of the nephrotic syndrome.71, 75 This 
variation strongly suggests that the monitoring of the CD 20 count will not prove to be 
the ideal instrument for determining treatment dose and/or duration.  It would be 
substantially better if we could monitor the specific effect of immunosuppressive therapy 
on the pathogenic antibodies in IMN. A recent discovery indicates that autoantibodies to 
the M-type phospholipase A2 receptor (PLA2R) may represent such a specific marker of 
IMN.  This autoantibody has been found in greater than 70% of IMN patients.76  When 
the analysis is limited to new-onset, nephrotic patients with IMN, this sensitivity 
increases even further.  These investigators have shown that PLA2R is a transmembrane 
constituent of the human podocyte (the most likely target cell in MN) and anti-PLA2R 
antibodies are predominantly of the IgG4 subclass and co-localize with IgG4 in the sub-


 
11 
epithelial deposits which are pathognomonic of the disease.  In their observations, the 
presence of anti-PLA2R correlates well with disease activity, disappearing with a 
spontaneous or treatment-induced remission, and reappearing with a relapse of IMN.  
 
We also have studied whether the immunologic changes in serum anti-PLA2R levels 
parallel the clinical reduction in proteinuria in response to RTX in patients with MN. 
Serial serum samples from our 2 cohorts of MN patients treated with RTX (cohort 1 
treated with 1 g of RTX at d.1 and d.15) and cohort 2 (treated with 4 weekly doses of 375 
mg RTX), were assayed for anti-PLA2R antibodies.77  Ten out of 15 (67%) patients in 
cohort 1 and 16/20 (80%) in cohort 2 had anti-PLA2R reactivity in their baseline serum 
samples. When the 2 cohorts were combined, the baseline distribution of cases with anti-
PLA2R positivity was not significantly different when compared to the final clinical 
groupings of complete remission (CR), partial (PR) limited or no remission (NR), 
(p=0.61). However, an anti-PLA2R level below 500 arbitrary units at 9-12 months after 
initiation of treatment was significantly different among these groups: in the complete 
remission (CR) group 100% had a reduction to this level, in the PR group 88% had 
reduction to this level but in the limited response (LR) group only 50% had a reduction 
and in those with no response only 17% had a reduction to this level. (p=0.006). Median 
proteinuria at 9, 12, and 15-18 months was consistently lower in those subjects with 
undetectable or very low anti-PLA2R levels versus those with higher levels. In the group 
in which anti-PLA2R disappeared, 86% were in complete or partial remission at the final 
time point: 4 complete remissions (CR), 14 partial remissions (PR), 1 limited remission 
(LR), and one non-responder (NR).  Perhaps most relevant was, in those patients with 
decreases in anti-PLA2R, the decline almost always preceded the reduction in proteinuria 
by months.  In addition, 1 patient who had attained remission and who had become anti-
PLA2R negative following RTX became antibody positive at the time of his relapse. 
Thus, in at least these two cohorts of MN patients with anti-PLA2R antibodies at the start 
of treatment, post-RTX anti-PLA2R levels correlated with and seemed to precede clinical 
response to RTX (Figure 3).  These results suggest that monitoring anti-PLA2R 
autoantibody levels may provide a window onto the immunologic effects of treatment on 
the course of IMN, and allow a more specific and an earlier means of determining 
treatment effectiveness compared to the clinical response of decreasing proteinuria. This 
observation is understandable given the nature of the deposits and proposed 
pathophysiology of IMN. Even if the B cells producing anti-PLA2R were completely 
eliminated and all circulating anti-PLA2R removed, the immune deposits would persist in 
the subepithelial space until cleared.  Supportive evidence of this was seen in repeat 
kidney biopsies performed in RTX-treated IMN patients after they had entered complete 
clinical remission.78  There are other important implications to the antibody story to 
consider if they truly parallel immunologic disease activity.  Secondary chronic changes 
in IMN such as focal sclerosis or interstitial damage from prolonged disease activity may 
lead to indefinite persistence of low-level proteinuria, despite full clearance of immune 
deposits. This is the case seen in kidneys made proteinuric in the Heymann nephritis 
model and subsequently transplanted into naïve hosts.79 This may explain our observation 
that not only does the decline in proteinuria lag behind that of anti-PLA2R, but also that 
the median proteinuria never reaches zero even by 24 months. It may also explain why 
most clinical responses are partial remissions (rather than complete) in the group that 
cleared anti-PLA2R. It will be important to see whether these patients will continue to 
have a further slow decline in proteinuria with further follow-up, similar to patients with 
IMN who undergo spontaneous remission.8 It may also help to explain why even patients 
with only partial remissions are still associated with good long term outcomes, i.e. in 


 
12 
these cases the residual proteinuria may be explained by the residual kidney scars rather 
than the persistence of the immunologic disease.42 
 
 
 
Figure 3.  Representative plots of anti-PLA2R (gray squares) and proteinuria (black 
diamonds) versus time following initial RTX treatment. Values are plotted as percent of 
baseline value.  Panel A and B depicts the typical reduction and disappearance of anti-
PLA2R followed by resolution of proteinuria exhibited by the majority of patients. Panel 
C is representative of patients in whom anti-PLA2R did not substantially decline 
following treatment and the associated with persistence of proteinuria. Panel D depicts 
the single patient whose anti-PLA2R level returned with relapse of his disease after 
having initially disappeared. 
 
This incomplete mirroring of immunological improvement by clinical status is an 
important issue. As mentioned, proteinuria could remain at a sub-nephrotic level in spite 
of the absence of immunological disease activity. In this situation, further 
immunosuppression would have no benefit but would have continued toxicity risk. In 
contrast, at a similar level of proteinuria, immunological activity, as detected by the 
continuing presence of circulating anti-PLA2R could be ongoing. In this setting, an 
increase or change in immunosuppressive regimen may be the best strategy.  Ultimately, 
if confirmed anti-PLA2R levels may become an important surrogate assessment of 
disease activity and of treatment outcome. We propose to expand these observations by 
measuring anti-PLA2R levels in all patients enrolled into the current trial. This in itself 
will be a major innovation for the results of these studies have the potential to create a 
new paradigm for monitoring and treatment in patients with IMN.  If confirmed, the 


 
13 
autoantibody level can be added to predictive algorithms and have the potential to 
influence future treatment protocols and lead to improved care of patients with IMN. 
 
 
OVERALL RESEARCH DESIGN AND METHODS: 
The goal of this proposal is to conduct a prospective randomized controlled phase III 
study comparing Rituximab (RTX) to Cyclosporine (CSA) in the treatment of patients 
with IMN. Once a patient with IMN and proteinuria ≥5g/24h is identified, meets other 
entry criteria and consents to the study, he/she will receive a minimum of 3 months of 
conservative therapy aimed at maximizing Angiotensin II blockade (run-in phase). If at 
the end of this period the patient still meets entry criteria he/she will be randomized into a 
12-month treatment period, and a subsequent follow-up of 12 months. Efficacy of 
treatment will be assessed by remission status (based on changes in proteinuria) at 24 
months from randomization. Patient safety will be assessed via collection of adverse 
event data and evaluation of pre- and post-treatment laboratory data. At the 6-month post-
randomization visit, patients who have been randomized to either CSA or RTX but who 
do not have a reduction in proteinuria ≥25% (confirmed on repeat measurements within 2 
weeks) will be considered treatment failures and exit the study. Data from that point 
onward will be censored. Patients in the RTX group who have a reduction in proteinuria 
of equal to or >25% at 6 months will be given another identical course of RTX (1g x 2).  
This treatment regimen was chosen given our initial data that suggested a percentage of 
our cohort appeared to respond to an additional course of therapy. Data from our 2 pilot 
studies showed that RTX is out of the circulation by month 2 in the majority of patients 
and B cells have recovered by month 3 in patients treated with RTX 1g x 2 (the regimen 
chosen for the study). Thus, at 6 months, patients in the RTX arm are completely, at least 
as best as we can measure, free of immunosuppression, and hence the second course 
should not be associated with additive toxicity. The same definition of response will 
apply to the CSA arm at 6 months. An equal to or >25% reduction in proteinuria will 
dictate continuation of the CSA for an additional 6 months (total of 12 months of full 
dose CSA). This assessment at six months allows a balance between risk-benefit in both 
arms. The protocol provides similar exposure to immunosuppression in both arms and 
allows an early exit for patients related to safety issues by providing a lack of efficacy 
(futility) end point at 6 months. 
 
Primary endpoint 
CR or PR (defined as per table 1) at 24 months after randomization will be the primary 
endpoint. This will be assessed in an intention to treat (ITT) analysis.  
 
 
Secondary endpoints 
1. Relapse state at month 24 after randomization (Urine Protein (UP) > 3.5 g/24h after 
earlier CR or PR) 
2. Anti-PLA2R levels 
3. Quality of life as measured by modified KDQOL 
4. Adverse events 
5. ESRD 
6. CR or PR, and CR alone at 6, 12, 18, and 24 months after randomization 
7. Time to CR or PR 
8. Effect of treatment on renal function, as assessed by slope of creatinine clearance from 
baseline to 24 months. 


 
14 
Table 1.  Definition of remission status  
Remission status 
Proteinuria (UP g/24 hours) after treatment 
Complete remission (CR) 
UP ≤ 0.3 g/24h and serum albumin ≥ 3.5g/dl 
Partial remission (PR) 
Reduction in baseline UP of ≥ 50% plus final UP ≤ 3.5 g/24h 
but > 0.3 g/24h 
Non-response (NR) 
Reduction in baseline UP of < 25% (includes increase in UP) 
Relapse 
Development of nephrotic range proteinuria following CR or 
PR, i.e. > 3.5g/24h 
 
Patient recruitment 
The study population will be comprised of individuals with biopsy-proven idiopathic MN. 
Total study size will be 126 patients, enrolled among up to 25 participating sites.   
Potential candidates for the study will be identified by the investigators using existing 
databases and clinical trial networks.  The P.I. or the study coordinator will contact these 
potential candidates and if the preliminary interview indicates their willingness to 
participate, a consent form will be reviewed with the patient.  If a potential candidate 
remains interested, the consent form will be signed, and the patient enrolled in the study.  
The institutional review board at each participating site will approve the recruitment 
process and consent forms.  The Mayo Nephrology Collaborative Group (MNCG) will 
post information on its web site describing the study and identifying local contacts at the 
collaborating centers.   Each site involved may have their own methods of informing and 
recruiting potentially eligible patients but in all cases will have IRB approval before 
embarking on this process.  Each site will be expected to randomize approximately 6-10 
patients over the recruitment period.  If, based on site estimates of the IMN population 
that would be eligible for this study, we enroll approximately 30% of the annual 
population in a single year our recruitment will be complete.  We have assembled a 
consortium for this trial with a population that well exceeds the enrollment needs based 
upon the multi-year enrollment plan for this trial.     
 
 
Recruitment strategy 
Potential candidates for the study will be identified by the P.I. at each site using existing 
research and clinical databases and clinical trial networks. With local IRB approval, 
electronic health records and pathology clinical data bases will be used for cohort 
discovery at each institution. Study investigators will also contact local research 
volunteer registries and local patient advocacy groups and request they provide 
information about this trial to their participants/members. Each participating site will be 
encouraged to accept referrals of study patients identified from national patient volunteer 
registries such as researchmatch.org and the Office of Rare Disease Research sponsored 
patient contact registry. Regional advertising will also be carried out via our ongoing 
participation in the NEPTUNE trial (sponsored by the NIDDK/ODR.) through its 
collaborative network of 18 academic centers in North America. The P.I. or the study 
coordinator will contact potential study candidates and if the preliminary interview 
indicates their willingness to participate, a consent form will be reviewed with the 
patient. If a potential candidate remains interested, the consent form will be signed, and 
the patient enrolled in the study.  According to our site estimates, only 18% of the 
available population will need to be eligible and willing to participate in the trial to 
achieve the target sample size.  
 
 


 
15 
Potential Challenges and Solutions 
Patient recruitment is often slower than anticipated in clinical studies.  The team of 
clinical centers engaged in this trial, including the primary Mayo Clinic site, has 
significant experience in recruitment and conduct of clinical trials. Past recruitment 
success at Mayo into phase I and II trials of RTX in adults with IMN is documented by 
the enrollment of 15 patients in less than 6 months and 20 patients in 11 months.71, 75  At 
Mayo Clinic alone, an average of 50 cases of MN a year have been diagnosed for the past 
5 years based on kidney biopsy registry. The projected available population is > 200% of 
the recruitment goal.  
 
If enrollment at some of the involved clinical sites is slower than expected, we have the 
option to recruit additional patients from the other participating sites or add, for example, 
additional sites from the Neptune Consortium to our trial.  
 
 
Inclusion Criteria 
• Idiopathic MN diagnosed by renal biopsy (original biopsy needs to include light, 
immunofluorescence and electron microscopy); pathology report must be adjudicated 
by a study PI (Dr. Fervenza or Dr. Cattran, or documented delegate) prior to 
randomization.  
• Age 18-80 years inclusive 
• If female, must be post-menopausal, surgically sterile or practicing a medically 
approved method of contraception (with exception of no birth-control pill given the 
potential for increased risk of thromboembolism in the nephrotic setting). 
• Patient must be off prednisone or mycophenolate mofetil for >1 month and alkylating 
agents for >6 months. The rationale is to minimize the potential confounding effect of 
delayed benefits from previous immunosuppressive agents and to reduce the risk of too 
much immunosuppression from the combined previous drug exposure plus trial drug 
exposure, e.g. infections. 
• Treatment with an ACEi and/or ARB, for ≥3 months prior to randomization and 
adequate blood pressure control (target BP is <130/80 mm Hg in >75% of the readings, 
but subjects with BP <140/80 mmHg in >75% of the readings will be eligible).   
OR 
If patient is intolerant to even a very low dose of either ACEi or ARB therapy, approval 
for participation in the trial has been obtained from the study PI(s) prior to 
randomization. 
Patients with documented evidence of ≥3 months treatment with maximal angiotensin 
II blockade, on an HMG-CoA reductase inhibitor, and BP control (BP <140/80 mm Hg 
in >75% of the readings) who remain with proteinuria ≥5g/24h and meet the other 
eligibility criteria (as confirmed at the Time 0 visit by the central lab results) may enter 
the treatment phase of the study and be randomized to RTX/CSA without the need of 
the run-in/conservative phase of the study.  However, in addition these patients must 
have a documented <50%  reduction in proteinuria compared to previous 24 hr 
proteinuria or Uprot/Ucrea ratio estimates during  this period of  ACEi and/or ARB 
treatment otherwise they must fulfill the run-in requirement.  
o  (Please refer to manual of operation for clarification of tests mandated for 
patients who are randomized without the run-in period)   


 
16 
• Proteinuria ≥5g/24h using the average from two 24-hour urine collections collected 
within 14 days of each other despite Ang II blockade for ≥3 months as described above. 
• Estimated GFR ≥40 ml/min/1.73m2 while taking ACEi/ARB therapy OR quantified 
endogenous creatinine clearance ≥40 ml/min based on a 24 hour urine collection.  The 
GFR will be estimated using the 4 variable MDRD equation as published in the NKF-
CKD guidelines.81 This approach is adopted, rather than the much more expensive and 
more invasive techniques (e.g. inulin or iothalamate clearance) since the likelihood of 
detecting significant changes in GFR in this short term study is remote regardless of 
which method is chosen. At entry into the study and at set time points thereafter 
patients will also have a 24h urine collection for calculation of CrCl and proteinuria.  
 
Exclusion Criteria 
• Patients with presence of active infection or a secondary cause of IMN (e.g. hepatitis B, 
SLE, medications, malignancies). Testing for HIV, Hepatitis B and C should have 
occurred <2 years prior to enrollment into the study. Screening for malignancy should 
be carried out according to standard guideline recommendations.  
• Type 1 or 2 diabetes mellitus: to exclude proteinuria secondary to diabetic nephropathy.  
Patients who have recent history of steroid induced diabetes but no evidence on renal 
biopsy performed within 6 months of entry into the study are eligible for enrollment. 
• Pregnancy or breast feeding (for safety reasons). 
• History of resistance to CSA (or other calcineurin inhibitors, e.g. tacrolimus), RTX or 
alkylating agents (e.g. Cytoxan). Patients who previously responded to CSA/CNI, RTX 
or alkylating agents with either a CR or PR but relapsed off CSA/CNI after 3 months, 
or relapsed off RTX or alkylating agent after 6 months, are eligible.  
 
Randomized Treatment Groups: Once all entry criteria have been satisfied and 
confirmed, patients will be randomized to treatment with Rituximab or Cyclosporine.  
Randomization will be performed by study site staff through the electronic case report 
form (eCRF.) The randomization list will be stratified by site, and generated by the Data 
Management and Coordinating Center (DMCC) using random permuted blocks of 
variable size.  
 
Rituximab: Patients randomized to the RTX arm will receive 1000 mg IV on Days 1 and 
15. Patients who achieve complete remission at 6 months will not be retreated. A second 
course of RTX 1000 mg IV will be administered at study month 6 for individuals who 
have not achieved a complete remission, but have achieved at least a ≥ 25% reduction 
compared to their central laboratory (Mayo Clinic Rochester) baseline (Time 0) 
proteinuria (but not a CR). Dosing at study month 6 will be independent of CD19/20+ B 
cell count. The rationale for retreating patients who have had an equal to or > 25% 
reduction in proteinuria at 6 months is based on our experience in our pilot studies (first 
study using 1000 mg on Days 1 and 15 and second study using 4 weekly doses of 
375mg/m2) where an increase in the proteinuria remission rate was achieved after a 
second course of treatment.  In our 2 studies repeated courses of RTX were not associated 
with additive adverse effects in comparison to their first course. If after six months the 
reduction in proteinuria is less than 25% compared to baseline the RTX treatment will not 
be repeated, the patient will exit from the study and will be considered a failure of 
therapy.  
 
Dosage and Administration of Rituximab: The first infusion of both courses (Day 1 
and Day 181) of RTX will be administered IV at an initial rate of 50 mg/hr. All patients 


 
17 
will be premedicated with acetaminophen (1g) and diphenhydramine HCl (50 mg) by 
mouth from 30 to 60 minutes prior to the start of an infusion.  Premedication with 
steroids (100 mg methylprednisolone IV) will also be given 30 minutes prior to the first 
infusion of each series of RTX (Day 1 and Day 181). If a hypersensitivity or infusion-
related reaction does not occur, the infusion rate will be escalated by 50 mg/hr increments 
every 30 minutes, to a maximum of 400 mg/hr.  If a hypersensitivity (non-IgE-mediated) 
or infusion-related reaction develops, the infusion will be temporarily slowed or 
interrupted. Treatment of infusion-related symptoms with additional diphenhydramine 
and acetaminophen will be recommended. Additional treatment with bronchodilators or 
IV saline may be indicated. The infusion may be continued at one-half the previous 
infusion rate upon improvement of the patient’s symptoms.  If the infusion was well 
tolerated, subsequent infusions (Day 15 and Day 195) may be administered at an initial 
rate of 100 mg/hr and increased by 100 mg/hr increments at 30-minute intervals, to a 
maximum of 400 mg/hr as tolerated.  If the first infusion was not tolerated, the guidelines 
for the first infusion should be followed for all subsequent infusions.  Patients 
administered an antihistamine for treatment or prevention of infusion related reactions 
will be given appropriate warnings about drowsiness and impairment of driving ability 
prior to discharge.  Participants will be observed for 1 hour post infusion.  
Although most study visits have a window of +/- 10 days, the 2nd infusion in each series 
(i.e., Day 15 and Day 195) will have a more restricted window of +/- 3 days. Specifically, 
the 2nd infusion in each series should occur 14 days +/- 3 days from the time of the 1st 
infusion in the series.  The window is to account for weekends, holidays, and scheduling 
conflicts.   
   
Monitoring of Rituximab Effects 
The numbers of CD19/20+ B cells in the peripheral blood will be quantified by flow 
cytometry using peripheral blood leukocytes. Flow cytometry will be performed pre- 
RTX treatment and at regular intervals following administration of RTX (see Table 2b: 
Test Schedule and Monitoring for Rituximab Treatment Arm).  These assays will allow 
us to follow the level of B-cell depletion, kinetics of B-cell reconstitution and the 
composition of cells that re-populate the B-cell pool after treatment with RTX as well as 
helping to ensure dosing adequacy in these patients.  
 
 
Cyclosporine: Neoral brand (Novartis) is the preferred Cyclosporine product for this 
trial. Participating sites must contact the DMCC prior to dispensing any other brand to 
study participants as not all brands of Cyclosporine are bio-equivalent. Patients 
randomized to the Cyclosporine arm will be started at a dose of CSA = 3.5 mg/kg/day 
p.o. divided into 2 equal doses given at 12 hour intervals. Target trough CSA blood 
levels, as determined in whole blood by HPLC, are 125 to 175 ng/ml. Patients will have 
their doses adjusted according to their blood levels of CSA as monitored every 2 weeks 
+/- 3 days until the target trough level is reached.  After reaching target, CSA trough 
levels will continue to be checked as per the visit schedule (Table 2a).  If a complete 
remission is achieved by six months, CSA will be tapered by approximately 1/3 of the 
maintenance dose monthly and hence discontinued after two months. If there has been at 
least an equal to or >25% reduction compared to their central laboratory (Mayo Clinic 
Rochester) baseline (Time 0) proteinuria (but not complete remission) the CSA will be 
continued for an additional six months. A persistent and otherwise unexplained increase 
in serum creatinine >30% will prompt an approximate 25% dose reduction of CSA, 
aiming for a corresponding 25% reduction in CSA trough level (for example, if trough 


 
18 
CSA was 175 ng/ml the goal would be to reduce it to 130 ng/ml by reducing the dose by 
approximately 25% - dose reduction is approximate because CSA is only available in 
specific dose strengths (25mg, 100mg) and therefore some rounding may be required).  If 
with this dose reduction the creatinine does not return to within 30% of baseline levels 
within 3 weeks, then a second dose reduction of approximately 25% with a similar 
reduction in CSA trough level (e.g. if CSA trough level was 100 ng/ml, reduce to 75 
ng/ml) will be used.  If the creatinine does not fall to within 30% of baseline values with 
this second dose reduction, the drug will be discontinued.  If after six months the 
reduction in proteinuria is less than 25% compared to baseline the drug will be 
discontinued, the patient will exit from the study and be considered a failure of therapy.  
At the end of 12 months, Cyclosporine will be tapered by 1/3 of the maintenance dose 
monthly and hence discontinued after 2 months. Serum potassium and serum creatinine 
levels will be checked at the initiation of Cyclosporine in conjunction with blood draw 
for CSA level. If Cyclosporine dose is changed during treatment, potassium, creatinine 
and CSA levels will be rechecked every two weeks +/- 3 days post increase/decrease in 
CSA dosage until levels are stable and at target.  
 
 
Figure 4. Schematic representation of the study design.  
 
 
 
Blinding 
This will be an open-label study with neither patients nor physicians blinded. Blinding, 
for this study, was considered difficult and unnecessary and would significantly add to 
the cost of the study. The need to provide RTX intravenously would require that the CSA 
arm patients would have to receive an IV infusion of saline which seems unjustifiable 
from the scientific and budgetary points of view. Furthermore, with the exception of 


 
19 
quality of life measures, all endpoints are based on objective (laboratory) criteria. Having 
the physicians in the study blinded to CSA level also introduces a potential risk factor 
that’s unnecessary in our estimation. Documentation of cyclosporine levels will ensure 
that patients are taking the drug at the appropriate time and achieving the prescribed 
levels indicated in the protocol.  
 
Pharmaceutical logistics 
RTX will be provided by Genentech.  Although Cyclosporine is recognized by insurance 
service providers as standard treatment for patients with IMN, participants randomized to 
the cyclosporine arm will receive the drug free of charge.  Neoral brand (Novartis) is the 
preferred Cyclosporine product for this trial.  If it is not possible to source Neoral from 
the local hospital pharmacy, please contact the DMCC prior to dispensing any study drug 
as not all brands of Cyclosporine are bio-equivalent.  Appropriate quantities of the 
medication will be supplied to the individual from the individual sites following 
randomization and again at months 3, 6, 9, and 12 and may also be supplied at other time 
points should dose adjustments be required.  
 
Screening visit  
The Screening visit will consist of meeting with any potential candidate for the study, 
reviewing eligibility criteria, including laboratory requirements (as outlined below and in 
Tables 2a and 2b), and reviewing and signing the informed consent form.   
 
Candidates who have not yet been exposed to conservative therapy will be started on 
the initial step of the protocol, i.e. maximize angiotensin II blockade as described in the 
run-in phase below. If any of the exams, tests or procedures involved in this visit were 
completed as routine standard of care within 30 days of screening, participants do not 
need to repeat them. It will be up to the study doctor to review and consider if the 
previous findings are appropriate/adequate to use otherwise they will be repeated.  In 
addition to data from this screening visit, retrospective data from at least one additional 
time point within the previous year will be collected whenever possible.  Retrospective 
data collection needs to include proteinuria, urine creatinine, creatinine clearance, 
Uprot/Ucrea ratio, systolic blood pressure, diastolic blood pressure, serum creatinine, and 
serum albumin values. 
 
Candidates who have already been on conservative therapy for 3 months or more 
without a documented decrease in proteinuria of >50% as compared to a value from 
within the last 3 to 12 months do not need to fulfill the run-in requirement as long as their 
local labs indicate that proteinuria remains ≥5g/24h. These individuals should proceed 
directly to full screening as part of the Time 0 visit and do not require a formal screening 
visit. Review of the eligibility criteria, lab requirements, and the review and signing of 
the informed consent form will take place at the Time 0 visit for these individuals. 
However, retrospective data from at least two previous time points (approximately 3 
months prior to obtaining consent plus one more time point within the previous year) will 
still be collected whenever possible.  Retrospective data collection needs to include 
proteinuria, urine creatinine, creatinine clearance, Uprot/Ucrea ratio, systolic blood 
pressure, diastolic blood pressure, serum creatinine, and serum albumin values. 
 
Patient monitoring and evaluation  
Patients will be followed for 2 years following randomization to monitor for the 
occurrence of adverse events, late remissions, relapses, GFR changes and development of 


 
20 
end stage renal disease.  The first 12 months of the study will be considered as the 
treatment period while the remaining 12 months will be considered as an observational 
period including the period of tapering to discontinuation in the Cyclosporine arm.  In 
addition, as described later in the protocol (see ancillary studies) we will seek permission 
to follow the remission cohort (i.e., those in PR or CR at 12 months or CR at 6 months as 
well as those who are not in remission but have a ≥50% reduction in proteinuria from 
baseline by month 12) for an additional 12 months of observation beyond the previous 
end point of observation of 24 months to monitor for relapses and changes in PLA2R.   
 
For those patients in the Cyclosporine arm, Cyclosporine trough level measurements 
will be done every 2 weeks +/- 3 days after initiation of Cyclosporine until levels are 
stable and at target. Potassium and creatinine will also be checked in conjunction 
with CSA level. If Cyclosporine dose is changed during treatment, potassium, 
creatinine and CSA level will be rechecked every 2 weeks +/- 3 days until levels are 
stable and at target. Cyclosporine levels will also be assessed whenever serum 
creatinine rises by >30% without a recognizable reason such as volume depletion, 
additional drugs, sepsis, etc.  Patients who cannot tolerate the medications and/or who are 
treatment failures at 6 months will exit the study at 6 months (i.e., the 6 month visit and 
accompanying data collection should still occur).  Patients who go in to complete 
remission will discontinue study drug but will continue with full visit follow-ups until 
month 24 as scheduled.  However, the therapeutic/management plan for these patients 
will be solely at the discretion of the managing nephrologist although while in CR no 
further immunosuppressive therapy is recommended.  Likewise, patients who relapse 
from either a partial or complete remission or who are not in partial or complete 
remission by the end of the treatment period (i.e., 12 months) will continue with the visit 
schedule, but the therapeutic/management plan will be solely at the discretion of the 
managing nephrologist. Finally, for patients who temporarily stop taking Cyclosporine, 
there are three possible scenarios: 
1. Patients who discontinue Cyclosporine for < 2 weeks may simply be restarted on 
the study drug and continue to fully participate in the trial.  The treatment is to be 
restarted at its former dose and the patient returned to the regularly scheduled 
follow-ups including the periodic sampling for assessing potassium, creatinine 
and CSA trough levels. 
2. Patients who discontinue Cyclosporine for ≥2 weeks, but < 4 weeks may also be 
restarted on the study drug at its former dose and fully participate in the trial, but 
the temporary discontinuance will be logged as a protocol deviation and the 
missed treatment days will be made up at the end of month 12.  This is to ensure 
that all patients receive approximately 12 months of treatment with study drug. 
3. Patients who discontinue Cyclosporine for ≥ 4 weeks will not be restarted on 
study drug, but will continue with full visit follow-ups until month 24 as 
scheduled.  The therapeutic/management plan will be solely at the discretion of 
the managing nephrologist. 
 
Laboratory testing  
In order to contain costs all clinical tests will be carried out locally at the recruiting 
centers.  However, at critical time points: Time 0, and 6, 12, 18, and 24 months from start 
of treatment, recruiting centers will ship to Mayo Clinic, Rochester, Minnesota, 2 blood 
samples and 2 urine samples (aliquots obtained from two 24h urine collections) for 
determination of serum creatinines, serum albumins and urinary protein/creatinine ratios.  
Blood and urine kits will be provided by Mayo Clinic, Rochester, Minnesota, for these 


 
21 
samples along with shipping costs (see Lab Manual).  The Central Lab results (Mayo 
Clinic, Rochester, MN) will be used for statistical purposes since this will ensure a 
standard methodology is applied to the critical (for the trial analysis) laboratory 
assessments. By measuring the volume of the 24-hour urine collection we will also be 
able to calculate both clearance and the 24-hour urine protein from the aliquot. 
Proteinuria results at Time 0, 6, 12, 18 and 24 months will be taken as the mean of two 
24-hour urine collections done back to back (within 14 days of each other), and the mean 
of these 2 collections will be used to determine CR, PR, or NR. 
 
In order to control for the possibility of under collection or over collection of urine, a 
maximum 20% variation in total 24h urine creatinine (mg/specimen) will be allowed 
between the two urine collections required at Time 0, 6  months, 12 months, 18 months, 
and 24 months.  A difference of greater than 20% will necessitate a third urine collection 
(also to be sent to the central lab), with the values from one of the two previous 
collections being discarded. We will use the subject’s weight to approximate the expected 
total urine creatinine (mg/kg) to determine which of the previous collections is closest to 
the truth and which should be discarded.  It is anticipated that a 3rd urine collection will 
be needed in <10% of cases.  Proteinuria will be calculated as the mean of these two 24-
hour urine collections. 
 
In all patients, CBC with differential, serum creatinine, electrolytes, albumin, urinalysis, 
24-hour Uprot/Ucrea ratio, and creatinine clearance will be evaluated as noted in Tables 
2a and 2b.  In patients receiving RTX, additional tests will include: quantification of 
lymphocyte subsets by flow cytometry, serum immunoglobulin levels, and determination 
of humanized anti-chimeric antibodies (HACA) at study visits Day 1, 6 months, and 9 
months. HACA will be performed free of charge by Genentech using established 
techniques.   
 
Anti-PLA2R Assay 
Recent evidence suggests that the presence of auto-antibodies to the M-type 
phospholipase A2 receptor (PLA2R) is a specific marker of idiopathic MN, found in 
greater than 70% of patients.76 When the analysis is limited to new-onset, nephrotic 
patients with idiopathic MN, this sensitivity increases further. The presence of anti-
PLA2R correlates well with disease activity, disappearing with a spontaneous or 
treatment-induced remission, and reappearing with a relapse of MN.  
 
We intend to explore the correlation of anti-PLA2R and disease activity by carefully 
looking at anti-PLA2R titer in response to RTX/CSA and the achievement of partial or 
complete remission. In those patients that have no remission from their disease in 
response to RTX/CSA, we will determine if circulating anti-PLA2R is still present which 
might suggest the need for a second course of RTX, continued treatment with CSA, or 
alternate immunosuppressive therapy.  Furthermore, in those patients that are in complete 
remission at 6 months, partial or complete remission at 12 months, , or have a ≥50% 
reduction in proteinuria from baseline by 12 months (i.e., the MENTOR remission 
cohort), we intend to explore the following critically important questions: 
1. The relationship between the rate of increase of anti-PLA2R titer and patient 
relapse 
2. The relationship between the absolute change in anti-PLA2R titer (from 
baseline/remission point) and clinical phenotype (i.e., response/relapse time) 


 
22 
3. The predictive capacity of changes in anti-PLA2R titer with regard to relapse 
and/or durability of remission 
4. The timing between changes in anti-PLA2R titer and any subsequent clinical 
relapse 
  
Serum samples from all 126 subjects will be collected at the Screening visit (when 
applicable), Time 0, and months 3, 6, 9, 12, 18, and 24. Serum samples will be collected 
at the individual sites by the study coordinator and stored in a dedicated -20° freezer until 
a point when they can be shipped in bulk to Dr. Fervenza at Mayo Clinic, Rochester, 
Minnesota.  Mayo Clinic will then send the samples in bulk to Dr. Paul Brenchley at the 
Manchester Royal Infirmary in the UK.  The sera will be tested in batches for reactivity 
toward immobilized recombinant PLA2R by ELISA, with appropriate positive and 
negative controls. Upon completion of the study, we will correlate response to treatment 
in each group to the baseline presence and/or titer of anti-PLA2R. A major hypothesis to 
be addressed is whether there is a difference in treatment response between those subjects 
who are initially anti-PLA2R negative versus those who possess circulating anti-PLA2R. 
In those subjects who are initially positive for anti-PLA2R, we will also correlate the 
primary endpoint (remission status) to anti-PLA2R titers throughout and at the end of the 
study. 
 
For those in the remission cohort (see ancillary studies), additional serum samples will be 
collected from those that achieve partial or complete remission or ≥ 50% reduction in 
proteinuria by 12 months at additional time points as follows:  
 
• For patients in the CSA arm that are in partial or complete remission or ≥ 50% 
reduction in proteinuria at 12 months additional samples will be collected at 
months 13, 14, 15, 16, 17, 20, 22, 26, 28, 30, 32, 34, and 36; for those who 
achieve complete remission at 6 months additional samples will be collected at  
months 7, 8, 10, and 11 then at 14, 16, 20, 22, 26, 28, 30, 32, 34, and 36.   
 
• For patients in the RTX arm that achieve partial or complete remission or ≥ 50% 
reduction in proteinuria at 12 months additional samples will be collected  at 
months 14, 16, 20, 22, 26, 28, 30, 32, 34, and 36; for those in the RTX arm that 
have achieved complete remission at 6 months, additional samples will be 
collected at  months  10, 14, 16, 20, 22, 26, 28, 30, 32, 34, and 36.   
 
Whole blood for these particular serum samples may be collected at a peripheral lab and 
shipped to the individual study site within 48 hours for processing and storage in a -20° 
freezer as described in the ancillary studies section later in this protocol.  
 
Investigators in the study will be blind to the results of anti-PLA2R antibodies until the 
study is completed.  The rationale is to avoid bias in the treatment of these patients.  
Although ideally patients would be stratified to the different arms based on the results of 
their initial anti-PLA2R test in order to avoid potential imbalance this will not be possible 
for practical and financial reasons.  Given the relatively small number expected to be 
negative, it is unlikely this will bias the balance between positive and negative patients at 
randomization. 
 
Histopathology 


 
23 
Prior to randomization, a report documenting the results of the histology review 
including, light microscopy, immunofluorescence and electron microscopy results, as 
well as percentage of global and segmental glomerular sclerosis, tubulo-interstitial index, 
and immunofluorescence findings will be submitted to the DMCC and reviewed by the 
study P.I.   
Following randomization, biopsy slides will be centrally reviewed on an on-going basis 
(but as soon as possible following randomization) by the Pathology Department at Mayo 
Clinic Rochester and/or the Pathology Department at the University Hospital Network, 
Toronto. 
 
 
Quality of life measures  
Psychosocial response to illness and its treatment may play an important role in overall 
adjustment and illness management. Research from a variety of chronic illness 
populations, including steroid resistant FSGS and chronic kidney disease (CKD), has 
consistently linked physical and psychosocial variables with health outcomes. These 
findings highlight the significant burden experienced by adults with uncontrolled 
nephrotic syndrome. Little is known about the impact of MN or its treatment on patient 
reported outcomes. Due to the severity of this disease and the potential risk for 
progression in these patients, psychosocial factors may play an important role in 
understanding their response to treatment.  In addition to evaluating the safety and 
efficacy of RTX and CSA, it is essential to assess the individual's perception of his/her 
quality of life relative to disease status and therapeutic response.  Quality of life will be 
assessed by patient self-report using the modified KDQOL questionnaire form at Time 0, 
6, 12 and 24 months.  
 
 


 
24 
Table 2a: Test Schedule and Monitoring for Cyclosporine Treatment Arm 
1. Potassium, Cr checked at 2 weeks +/- 3 days post Cyclosporine initiation. If Cyclosporine is increased or decreased during treatment, potassium, Cr and CSA will be rechecked after 2 weeks +/- 3 days  
†Patient will have blood levels of CSA monitored every 2 weeks +/- 3 days until target is reached and then as per above schedule. 
a Only for participants who continue on CSA at 6 months 
b Telephone or in-clinic visit to confirm successful down-titration of CSA for patients who continue on medication to 12 months (if by phone, medication should be returned at 18 month visit) 
Tests/Assessments  
Screen 
T0 
Day 
15 
Day 
28 
Day 
90/ 
3m 
Day 
180/ 
6m 
Day 
210/ 
7m 
Day 
240/ 
8m 
Day 
270/ 
9m 
Day 
300/ 
10m 
Day 
330/ 
11m 
Day 
365/ 
12m 
Day 
395/ 
13m 
Day 
425/ 
14m 
Day 
455/ 
15m 
Day 
485/ 
16m 
Day 
515/ 
17m 
Day 
545/ 
18m 
Day 
605/ 
20m 
Day 
665/ 
22m 
Day 
730/ 
24m 
Day 
790/ 
26m 
Day 
850/ 
28m 
Day 
910/ 
30m 
Day 
970/ 
32m 
Day 
1030/ 
34m 
Day 
1095/36m 
Randomization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
History/Exam 
X 
X 
 
 
X 
X 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
CBC 
with 
differential 
X 
X 
 
 
X 
X 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
BUN, Cr, Lytes, 
Albumin 
(including 
potassium) 
X 
X 
 
 
X 
X 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
CPK 
 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CSA level † 
 
 
X 
X 
X 
X 
 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lipid panel  
 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
Serum Pregnancy 
X 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anti-PLA2R 
antibodies 
X 
X 
 
 
X 
X 
Xd 
Xd 
X 
Xd 
Xd 
X 
Xc 
Xc,d 
Xc 
Xc,d 
Xc 
X 
Xc,d 
Xc,d 
X 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
DNA sample 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Gene Expression 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
Potassium and Cr 
1 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
UA 
X 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
Uprot/Ucrea 24h 
X 
XX 
 
 
X 
XX 
 
 
X 
 
 
XX 
 
 
 
 
 
XX 
 
 
XX 
 
 
 
 
 
 
Uprot/Ucrea (spot 
urine) 
 
 
 
 
 
 
Xd 
Xd 
 
Xd 
Xd 
 
Xc 
Xc,d 
Xc 
Xc,d 
Xc 
 
Xc,d 
Xc,d 
 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
24h creatinine for 
CrCl 
X 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
24h urine urea 
 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
24h urinary Na+ 
 
X 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
Quality of life-
KDQOL 
 
X 
 
 
  
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
Adverse Events 
 
 
X 
X 
X 
X 
 
 
X 
 
 
X 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
Dispense/Return 
Medication 
 
X 
X 
X 
X 
X 
 
 
Xa 
 
 
Xa 
Xa 
Xa,b 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
25 
c For participants in the MENTOR remission cohort who are in PR or CR at 12 months or have a ≥50% reduction in proteinuria from baseline by 12 months 
d For participants in the MENTOR remission cohort who are in CR at 6 months 


 
26 
Table 2b: Test Schedule and Monitoring for Rituximab Treatment Arm 
Tests/Assessments  
 
Screen 
T0 
Day 
1 
Day 
15 
Day 
28 
Day 
90/ 
3m 
Day 
180/ 
6m 
Day 
181 
Day 
195 
Day 
270/ 
9m 
Day 
300/ 
10m 
Day 
365/ 
12m 
Day 
425/
14m 
Day 
485/
16m 
Day 
545/ 
18m 
Day 
605/
20m 
Day 
665/
22m 
Day 
730/ 
24m 
Day 
790/ 
26m 
Day 
850/
28m 
Day 
910/ 
30m 
Day 
970/ 
32m 
Day 
1030/ 
34m 
Day 
1095/ 
36m 
Randomization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
History/Exam 
X 
X 
 
 
 
X 
X 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
 
CBC with differential 
X 
X 
 
 
 
X 
X 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
 
BUN, Cr, Lytes 
(including potassium) / 
Albumin  
X 
X 
 
 
 
X 
X 
 
 
X 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
 
PBFC1  
 
 
X 
 
X 
 
X 
 
 
 
 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
Serum IgG, IgA and 
IgM 
 
 
X 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
Lipid panel  
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
HACA2 
 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum Pregnancy 
X 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Pregnancy 
 
 
X 
X 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anti-PLA2R 
antibodies 
X 
X 
 
 
 
X 
X 
 
 
X 
Xd 
X 
Xc,d 
Xc,d 
X 
Xc,d 
Xc,d 
X 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
DNA Sample 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Gene Expression 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
UA 
X 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
Uprot/Ucrea 24h 
X 
XX 
 
 
 
X 
XX 
 
 
X 
 
XX 
 
 
XX 
 
 
XX 
 
 
 
 
 
 
Uprot/Ucrea (spot 
urine) 
 
 
 
 
 
 
 
 
 
 
Xd 
 
Xc,d 
Xc,d 
 
Xc,d 
Xc,d 
 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
Xc,d 
24h creatinine for CrCl 
X 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
24h urine urea 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
24h urinary Na+ 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
Pre-infusion 
medication 
(i.e.Tylenol, Benadryl) 
 
 
X 
X 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pre-infusion 
medication (i.e. 
methylprednisolone)  
 
 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Rituximab Infusion 
 
 
X 
X 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Quality of life-
KDQOL 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
Adverse Events 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
 
1. PBFC: Peripheral blood flow cytometry. Quantitate B and T cell subsets;  
2. HACA: human anti-chimeric antibodies; pre-infusion on Days 1 and at 6 months and 9 months. 
3. In the rare case where B cells (CD19/20+) are not replete at Month 12, PBFC will be repeated at Month 18 
C For participants in the MENTOR remission cohort who are in PR or CR at 12 months or have a ≥50% reduction in proteinuria from baseline by 12 months 
d For participants in the MENTOR remission cohort who are in CR at 6 months 


 
27 
Run In Phase – Common Therapy for Both Arms 
The purpose of the run-in phase is to determine if an individual’s IMN can be adequately 
controlled through conservative, non-immunosuppressive treatment.  If proteinuria can be 
brought below 5g/24h after 3 months or more of conservative therapy, the individual will 
not be randomized to one of the study’s treatment arms.  In the rare case where a patient 
is intolerant to even a very low dose of ACEi or ARB therapy, participation in the 
randomized treatment component of the trial may still be possible (provided the patient 
still fulfills the other entry criteria), but will require review and approval by the study 
PI(s) prior to randomization. . 
 
Blood pressure control and angiotensin II blockade: The target blood pressure 
(<130/80mmHg in >75% of the readings; but not <100 mmHg systolic) is chosen based 
on recent recommendations by the JNC VII.82 The first step will be the introduction of an 
ACE Inhibitor (ACEi). Because this part of the study aims to maximize Ang II blockade, 
ACEi dose will be increased every 2 weeks until the maximum tolerated/FDA approved 
dose is achieved or until intolerable side effects occur (e.g. development of postural 
hypotension, light headed, hyperkalemia, etc). Once ACEi dose has been maximized and 
there are no observable side-effects and/or blood pressure is not at target, a long acting 
ARB will be added.  The ARB dose will be increased every 2 weeks to achieve the 
maximum tolerated or maximum approved dosage.  
 
For patients whose blood pressure control is still not at target, it is recommended that 
they receive additional medications in the following order: a loop diuretic, a 
cardioselective β-blocker, a non-dihydropyridine calcium channel blocker (CCB), and 
clonidine.  The selection of these drugs adheres to the recommendations of the JNC VII.82 
The choice of a non-dihydropyridine CCB was made because of concerns that 
dihydropyridine-type CCB may obscure the anti-proteinuric effects of the above therapy. 
In order to further ensure that any potential adverse effect is minimized we have limited 
CCB to be used as a fifth agent, and to be used only when the combination of an 
ARB/ACEi, a diuretic, and a β-blocker have failed to bring BP to target level. Recent 
data have shown that an aldosterone or renin antagonist have significant antiproteinuric 
effects. The reason we do not include these agents as part of the treatment protocol is out 
of concern for the development of severe hyperkalemia when all (ACEi, ARB, a direct 
renin inhibitor and aldosterone antagonist) these agents are used in combination. This 
side effect is likely to be further accentuated in the Cyclosporine group and we want to 
avoid an imbalance of RAS blockade between the arms of the study.  
 
Concomitant Treatment 
At the start of the run-in/conservative phase of the study (or at the start of the treatment 
phase of the study for those patients who are run-in exempt) and as part of the standard of 
care for patients with nephrotic syndrome and significant hyperlipidemia, patients will be 
started on atorvastatin 10 mg a day (or its equivalent with the exception of rosuvastatin 
(Crestor) which is to be avoided given the associated marked increase in the area under 
the curve (AUC) when used in conjunction with CSA). Patients who are currently taking 
rosuvastatin (Crestor) and are randomized to the CSA treatment arm will need to be 
changed to atorvastatin or its equivalent prior to or at the time of starting the 


 
28 
cyclosporine.  If tolerated clinically and, as evidenced by the lack of persistent elevation 
of liver transaminase >3x upper limit of normal or high CK, or clinical rhabdomyolysis, 
the dose will be increased according to the recently published KDOQI-dyslipidemia 
guidelines.83 The dose should not be increased above the maximum of 40 mg/day. The 
rationale for not using a higher statin dose is because of the risk of developing proteinuria 
with the use of statins at high doses, and the added risk of rhabdomyolysis in the CSA 
group. Patients will remain at the highest tolerated dose for the entire duration of the 
study. Serum lipids will be measured at Time 0 and every 3 months thereafter. High 
sodium intake (e.g. >200 mm Na/d or 4.6 g sodium/d) can significantly impair the 
beneficial effects of Ang II blockade.84 Therefore patients will be instructed to follow a 
low salt diet (2-3g/day). Patients will also receive dietary counseling at enrollment as part 
of their standard of care. Patients will be advised of a dietary protein target intake of 0.8-
1.0 g/kg ideal body weight/day of high quality protein and will be encouraged to maintain 
the same diet throughout the duration of the study. Patients with proteinuria >10g/24h 
and serum albumin <2g/dl should be considered for prophylactic anticoagulation.   
 
Patients randomized to receive Rituximab should be started on single strength Bactrim 
one a day (or its equivalent) for pneumocystis pneumonia prophylaxis. This treatment 
will continue until the end of the study treatment phase (12 months) and the B cells 
(CD19/20+) have been repleted (>15 cells/microliter on peripheral blood flow 
cytometry).  B cells (CD19/20+) are expected to be replete by Month 12.  In the rare 
event that B cells (CD19/20+) are not replete by Month 12, flow cytometry should be 
repeated at Month 18 (see table 2b) and Bactrim (or its equivalent)  continued to that 
point of repletion. 
 
Stopping points for medications: 
1. For Cyclosporine: At 6 months, patients in complete remission (as previously defined) 
will have CSA dose tapered and discontinued while those who fail therapy will have CSA 
discontinued immediately (no need to taper the dose).  
After 12 months of therapy, regardless of the degree of proteinuria, Cyclosporine will be 
tapered and discontinued as described above. 
2. Patients in the RTX arm who have either failed therapy or are in complete remission at 
6 months (as previously defined) will not have a repeat treatment course.  
After the second course of RTX therapy at six months they will have no further RTX 
therapy regardless of their proteinuria level 
3. Significant potential life threatening infection. 
4. Persistent elevation of liver enzymes >2 x normal (despite CSA arm dose reduction). 
5. Persistent elevation of serum potassium >6.0 mEq/l despite diet changes, ACEi/ARB 
reduction, diuretic dose increase (and in the CSA arm dose reduction). 
6. A rise in serum creatinine by >30% above baseline that persists in the absence of 
secondary causes such as volume depletion or use of potential nephrotoxic agents (and 
despite adjustments in the CSA dose) 
7. Persistent hypertension in either arm, i.e. supine blood pressure >160 mmHg systolic 
or >90 mmHg diastolic, despite institution of maximum antihypertensive therapy (and 
despite adjustments of CSA dose) 


 
29 
8. Other clinically relevant adverse effects not resolved by reduction in dosage of test 
medications. 
9. Development of any malignancy or lymphoproliferative disorder. 
10. Pregnancy. 
11. The appearance of an independent disease whose standard therapy requires 
continuous administration of significant amounts of corticosteroids, other 
immunosuppressive agents or plasma exchange therapy. 
12. Serious concomitant disease with an expected survival of less than 4 years. 
13. At the wish of the patient. 
14. The study will be placed on hold if 3 of the first 5 patients enrolled and treated with 
RTX or CSA develop severe opportunistic infections, or experience Grade 3-4 
SAEs.  
 
Patients who choose to discontinue study medication or are withdrawn because of 
adverse events will be strongly encouraged to continue follow up examination to the 
study termination. 
 
Ancillary studies: 
 
1. DNA Testing 
Recent data show that in patients with membranous nephropathy, mutations in HLA-
DQA1 and PLA2R1 alleles are associated with an increased risk for developing 
membranous nephropathy.80  One of these alleles, the PLA2R1, is in fact the gene for the 
PLA2 receptor.  In our pilot studies, ~70% of the patients with membranous nephropathy 
had antibodies present in circulation against PLA2 receptor.77  Thus we would like to 
evaluate a linkage between the presence of these antibodies and potential genetic 
mutations in this group of patients.  We will ask for the patients consent to further 
evaluate this linkage by collecting samples of blood to perform additional testing as part 
of their informed consent form (ICF). 
 
2. Quantitative gene expression analysis 
Quantitative gene expression analysis using real-time PCR and Microarray technology in 
peripheral blood mononuclear cells:  There is little information on global gene expression 
changes that occur during active nephrotic syndrome secondary to IMN and subsequent 
to remission of this disease.  Furthermore, there is limited information on how Rituximab 
and Cyclosporine affects gene expression of potentially pathogenic genes.  As part of this 
study, peripheral blood will be collected at Time 0, 12 and 24 months after therapy.  
Following treatment with Rituximab and Cyclosporine administration, B-cell-specific 
genes will be assessed to track the extent of treatment in the B-cell pool gene expression.  
Both quantitative TaqMan real-time polymerase chain reaction and gene microarray 
(Affymetrix U133 chips) will be used to evaluate gene expression profiles.  Real-time 
PCR will be used only to confirm the findings of the gene chip for genes with statistical 
significance.  
 
3. Staining original renal biopsy for CD20 + B cells 


 
30 
We will obtain each patient’s permission to review renal biopsy slides and to obtain 3 
unstained slides, 3 microns thick for staining with antibody against CD20 positive cells 
and anti-APL2R.  The purpose is to stain sections from biopsy tissue with an antibody 
against CD20 positive cells and to look for receptor density to APL2R.  These may be the 
same cells that are in circulation and that are depleted by Rituximab. We want to examine 
the hypothesis that there is a correlation between the number of CD20 positive B cells 
infiltrating the kidney and the patient’s response to therapy.  Preliminary results suggest 
that staining for CD20 and APL2R antibodies are increased in kidney biopsy of patients 
with MN. All patients by this juncture will have completed the therapeutic part of the 
study and will have already received rituximab or CSA treatment as part of study so this 
ancillary project will have no therapeutic or management implications.  
 
Immunohistochemistry methods 
Immunohistochemical staining will be performed at the Department of Pathology, Mayo 
Clinic, Rochester, Minnesota, by Dr. Sanjeev Sethi and/or by Dr. Carmen Avila-Casado 
at University Health Network, Toronto, Ontario.  Formalin-fixed, paraffin embedded 
sections will be cut onto coated glass slides. Slides will be incubated with monoclonal 
anti-CD20 and APL2R primary antibody at 1:1000 dilution (DAKO).  Heat induced 
antigen retrieval will be used. Sections will be incubated at 20C overnight and rinsed.  
Endogenous peroxidase activity will be prevented by pretreating all sections with 3% 
hydrogen peroxide.  Sections will be incubated with a secondary rabbit anti-mouse 
antibody linked with avidin-biotin complex.  Sections will be counterstained with 
hematoxylin.  CD20 positive cells (B-cells) will be counted and the density of positive 
cells per area of renal cortex will be calculated utilizing Image Pro Plus (Media 
Cybernetics) computer image analysis software. 
 
4. The MENTOR Remission Cohort 
Participation in this ancillary study will be restricted to patients in the MENTOR cohort 
who respond to immunosuppression with partial or complete remission at month 12 (as 
defined in Table 1), complete remission at 6 months, or ≥50% reduction in proteinuria 
from baseline by month 12. At the completion of the immunosuppression treatment 
component of the protocol, the expectation is that 70% of all randomized IMN cases 
(N=126 x 70%= 88 patients) will experience both a clinical (complete or partial 
proteinuria remission) and immunological remission.  The immunologic remission is 
expected to be documented by anti-PLA2R levels significantly reduced from baseline 
with most levels in the normal range (less than 40U).  This population is estimated to be 
75% of the initial PLA2R + cohort (75% x 88 = 66 patients).  The great percentage of 
these patients will reach this remission at the 12 month study point.  There will be rare 
cases of CR by 6 months.  Our anticipated/expected rate of proteinuria relapse will be 
between 30% and 50% between month 12 and month 36 (20-30% in those with CR at 6 
months).  Although the clinical proteinuria remission status at 24 months remains the 
main determinant of the RCT, the remission cohort (with an extension up to month 36 for 
periodic sampling of anti-PLA2R titer and urinary protein/creatinine ratios only) will 
provide a unique and probably one-time only opportunity in which to study a number of 
biomarkers of the immunological mechanism as predictors of clinical relapse.  Patients 
will be followed as per protocol for at least 12 months of observation (i.e., to month 24) 


 
31 
after achieving proteinuria remission or ≥50% proteinuria reduction, but for those in the 
remission cohort we will extend this up to month 36 (a maximum observation time of 24 
months post treatment phase) in those that remain in CR or PR.  Those who relapse 
between months 24 and 36 will only be followed until their relapse which is defined as 
development of nephrotic range proteinuria (> 3.5g/day) following CR or PR. 
 
In addition, we are now able to measure other components of the immunological 
elements of primary/idiopathic MN activity and are now better able to more precisely 
define the remission state when combining these additional biomarkers of the immune 
remission state with PLA2R titres. These additional biomarkers include soluble PLA2R.  
A detectable level of soluble PLA2R antigen (sPLA2R) is present in normal serum as a 
result of specific secretion of PLA2R and likely cleavage of the extracellular membrane 
receptor, and is likely to be a characteristic of the normal, remission state of patients with 
idiopathic MN. We hypothesize that as patients experience immunological relapse, their 
biomarker status will change from sPLA2R +ve, anti-PLA2R –ve to sPLA2R –ve , anti-
PLA2R+ve. The timeframe of this change needs to be established and our remission 
cohort provides a unique opportunity to study this hypothesis.  This change could vary 
over weeks dependent on the level of sPLA2R and the rate of production of anti-PLA2R. 
In addition, there may be a distinct phase of soluble circulating immune complexes where 
the patient is apparently seronegative for both free sPLA2R and free anti-PLA2R until 
excess of anti-PLA2R antibodies dominate. This MENTOR Remission cohort offers a 
unique opportunity to investigate: 
a) the transition from immunological remission to relapse  
b) the link between immunological relapse and clinical relapse (proteinuria)  
c) additional biomarkers of relapse.  
The benefit of this study will be the improved capacity to predict those patients at high 
risk of clinical relapse and the potential to institute therapy potentially in advance of 
clinical relapse.  It may also allow us to  determine if  the changing immunologic profile 
can predict  the  severity of relapse and  provide the opportunity to  consider, in the 
future, matching this profile to  preventive strategies  and/or early and less aggressive 
immunosuppressive interventions and/or gauge when risk-benefit favours reducing 
immunosuppressive therapy.  
 
We will obtain each patient’s permission to obtain 5mL of blood at designated time 
points (up to a maximum of 13 extra samples in total) up until month 36 or until relapse 
(see Tables 2a and 2b for visit schedule) to be used for PLA2R and sPLA2R testing.  In 
addition, a urine sample will be collected to determine relapse by measuring urine 
protein/creatinine ratios.  Samples may be collected at a peripheral lab where possible in 
order to minimize the demands on patient travel time, but blood will need to be sent to 
study sites for processing and storage (-20 degrees C) within 48 hours of being drawn.  
Serum samples will be kept in a dedicated freezer until a point when they can be shipped 
in bulk to Dr. Fervenza at Mayo Clinic, Rochester, Minnesota.  Mayo Clinic will then 
send the samples in bulk to Dr. Paul Brenchley at the Manchester Royal Infirmary in the 
UK.   
 
 


 
32 
 
Statistical Methods and Analysis: 
 
Sample size estimation:  The primary study goal is to compare the long-term efficacy of 
RTX (given at baseline and repeated at 6 months) with CSA (12 months treatment + 2 
months taper) using proteinuria response 24 months after randomization. Proteinuria 
positive response will be defined as attaining and maintaining CR or PR at 24 months 
after randomization. We propose to establish that the RTX treatment is non-inferior to the 
CSA. RTX will be considered non-inferior to CSA if the response rate of the RTX arm is 
at most 15% worse than that of CSA arm. In addition we will compare the quality of life 
(QOL) and adverse event (AE) profile over the course of the treatment period. The 
preliminary data has indicated that CSA is effective in inducing a CR/PR of proteinuria in 
between 60 and 75% of MN cases.48, 51 However, nephrotic syndrome (NS) relapses may 
be as high as 50% once CSA is discontinued.85 Thus, we estimate a CR/PR rate in 
patients treated with CSA of 30-50% at 24 months after randomization.85 Similar 
remission and relapse rates with the use of Tacrolimus (another calcineurin inhibitor) 
have been reported by Praga et al.56 In this latter study, almost half of the MN patients 
had a relapse of the nephrotic syndrome after tacrolimus was discontinued.56  On the 
other hand, based on the long term follow up on the 35 patients treated with RTX from 
our 2 studies, we estimate the relapse rate to be <10% at 24 months.71, 75  We observed a 
CR/PR rate at 12 months, very similar to the rate for calcineurin inhibitors i.e. 60%, and 
in our second study completed recently, we observed an 80% CR/PR rate at 24 months, 
implying that RTX had a ~20% failure rate with 95% (CI 6% to 44%). To be 
conservative for the sample size estimation, we considered a maintained CR/PR rate at 
the low end of our previous experience, 55% CR/PR for RTX at 24 months and at the 
high end of our experience with CSA (CNI therapy) CR/PR for CSA of 45%. We propose 
a non-inferiority trial with non-inferiority margin δ =15%, with the null hypothesis of 
πRTX – πcyclosprine < δ, where πRTX and πcyclosprine are the proportions of patients 
with CR/PR at 24 months in treatment arms of RTX and CSA, respectively. Under these 
assumptions, and a one sided alpha of 0.025, enrollment of 63 evaluable patients per 
study arm is required to achieve 80% power to show that the RTX is not inferior. For the 
intent to treat (ITT) analysis we will consider all patients lost to follow up to be non-
responders so no correction to the sample size is needed for dropouts. With 63 patients 
per arm we will have 80% power to detect moderate differences of .6 standard deviations 
for continuous measures or around .25 for proportions for the comparisons of the QOL 
and AEs. 
 
 
Statistical and Analytical Plan: All demographics and entry laboratory data will be 
summarized by treatment group. Frequency distributions will be used to describe 
categorical values and basic summary statistics (mean, standard deviation, median, and 
inter-quartile range) will be used to describe continuous values.  To provide more reliable 
estimates and minimize the “regression to the mean effect” duplicate urine measurements 
obtained at baseline (Time 0), 6, 12, 18 and 24 months will be averaged.   The chi-square 
test and logistic regression will also be used to compare the percent of patients with 
remission at 6, 12, 18, and 24 months adjusting for treatment center and the baseline 


 
33 
proteinuria (UP<8g/24h vs >=8g/24h).  Odds ratios and associated 95% confidence 
intervals will be estimated.  The formal test for the primary endpoint will be based on the 
significance of the treatment group factor in the logistic regression model for UP failure 
at month 24. The Wilcoxon rank-sum test and ordinal logistic regression will be used to 
compare the ordered remission status outcome (1=CR, 2=PR, 3=NR) between treatment 
groups.  Longitudinal methods for categorical outcomes (e.g. generalized estimating 
equation or GEE models) will be used to compare remission status profiles between 
treatment arms. Unless otherwise noted, all tests will be two-sided with alpha level 0.05.   
 
For repeated measures such as urine protein, individual rates of change will be estimated 
using within-patient linear regression analyses (including data from all visits). Because of 
the non-linearity of the changes in proteinuria log transformation estimates will be used. 
Renal function readings will be censored at initiation of dialysis or renal transplant.  
Additionally, mixed effects models, assuming a random center effect, and a random slope 
of creatinine clearance and/or reciprocal of creatinine and intercept for each patient will 
be fit using data from all visits.  These models allow comparison of the average slope 
between groups, while taking into account that each patient’s slope may be based on a 
variable number of readings.  Treatment group comparisons regarding quality-of-life 
scales will be done using repeated measures analyses and mixed effects models. 
 
Adverse events (both patient and event counts) will be tabulated by body system, severity 
and, for each severity, by investigator-assessed relationship to study drug.  Group 
comparisons for adverse events with 4 or more occurrences will be done using chi-square 
or Fisher’s exact test.  The last RTX injection is at 6 months and the tapering of 
Cyclosporine will be completed by the end of month 14.  Hence, the adverse events 
analyses will focus on events through month 18, allowing these additional months for 
potential lingering Cyclosporine or RTX complications. 
 
The analysis of the primary endpoint (urine protein failure at month 24) will be intent-to-
treat (ITT), and will include all randomized subjects in the analysis.  For those without a 
24 month visit, the 18 month visit results will be used if available, otherwise they will be 
assumed to have failed at 24 months.  Per protocol (PP) analyses will also be done 
including only those subjects who receive a full course of study medication and who have 
a 24 month visit.  
 
Interim analyses:  No interim analyses for efficacy will be performed during the trial, 
however safety data will be compiled and reviewed approximately every six months or at 
the request of the Data and Safety Monitoring Board. 
 
 
 
Investigator and Coordinator Training: In order to make efficient use of funding, 
annual investigator and coordinator meetings will be coordinated with national 
nephrology meetings where possible.  It is expected that the majority of the training will 
occur through web-based technology and will be coordinated through the data 
management and coordinating Center.  
 


 
34 
The Data Management and Coordinating Center (DMCC) is located at the Applied 
Health Research Center (AHRC) at St. Michael’s Hospital/University of Toronto. The 
DMCC will be responsible for facilitating all aspects of the study planning, training, 
implementation, data collection and analysis.  
 
The clinical information collected for this study will be stored at the DMCC at the 
Applied Health Research Centre of St. Michael’s Hospital in Toronto, ON, Canada and 
also sent to a federal data repository held by the Office of Rare Diseases Research 
(ORDR) and located at the University of South Florida (USF) in Tampa, FL.  
 
The DMCC database and the USF database use several layers of protection for the 
clinical data stored there. These databases meet all of the local and federal security 
requirements for research datacenters. Participant information is stored only using a study 
ID. 
 
Data management and quality assurance:  Study data, including enrollment and 
monitoring data, will be maintained on a secure password protected database accessible 
to all study centers from the world-wide-web. Each center will enter data for their 
patients.  Quality control will be ensured by oversight by the AHRC coordinating center, 
who will review the electronic files of all patients on a regular basis for completeness.  
 
Quality and completeness of data entry will be monitored on a weekly basis during the 
initial 6 months of study enrollment and biweekly thereafter.  Data quality reports will be 
generated monthly for review by the study Data Quality Assurance Committee, St. 
Michael’s Hospital/University of Toronto. Data queries generated by identification of 
incomplete or inconsistent data will be raised directly within the electronic eCRF and 
should be resolved by the study coordinator or PI in a timely manner. Corrections or 
changes in the data management system are tracked with the retention of the original data 
and the corrected data with the date of data entry and submitting personnel. Sites with 
persistent delays or difficulties in data capture will be provided additional study based 
training. Study data will be stored within the data centre of St. Michael’s 
Hospital/University of Toronto with an off-site daily back-up system. 
 
Analysis of the primary and secondary outcomes will be conducted at the DMCC. 
Additional analyses requested by ancillary investigators will be conducted when feasible 
within the scope of this trial funding. The DMCC will implement the trial policy for data 
sharing and ancillary studies. A fee structure will be established for ancillary studies that 
are beyond the scope of this trial.   
 
 
Data security: 
All clinical data will be processed in a secure electronic environment that includes virus 
protection, and restricted access. Electronically stored data are subject to extensive 
security measures including virus detection, and restricted access.  Security measures in 
place for the database management system proposed for this study include: browser 
security, firewall protection, user name/password protection, user re-authentication, and 


 
35 
inactivity time-out.  The database will not contain study participant name, address or 
medical record number.  Patient data will be identified only by study code. Study data, 
including enrollment and monitoring data, will be maintained on a secure password 
protected database. Quality control will take place at time of data entry (range, 
consistency checks) and will be ensured by oversight by the P.I. and Quality Assurance 
and Clinical Management Workgroup which will review the files of all patients on a 
regular basis for completeness. 
 
Registration of participants on this protocol will employ an interactive data system in 
which the clinical site will attest to the participant’s eligibility as per protocol criteria and 
obtain appropriate informed consent. IRB approval for the protocol must be on file at the 
DMCC and the University of South Florida before accrual can occur from the clinical 
site. 
 
Demographic and adverse event data collected as part of this trial will be transferred to 
the USF on a monthly basis throughout the trial, and uploaded into the data repository 
dbGaP (database of Genotypes and Phenotypes).  
 
A system of coded identifiers will be used to protect participant confidentiality and 
safety.  Each participant enrolled will be assigned a local subject identifier by the DMCC 
which is a combination of the site location code and a serial accession number.  Only the 
registering site will have access to the linkage between this number and the personal 
identifier of the subject.  When the participant data is sent to the USF, the system will 
assign a participant ID number.  Thus each participant will have two codes: the local code 
used to identify the patient in the eCRF and in source documentation and which is linked 
to personal identifiers, and a second code assigned by the USF.  For all data transfers to 
the USF both numbers will be required to uniquely identify the subject.  In this fashion, it 
is possible to protect against data keying errors, digit transposition or other mistakes 
when identifying a participant for data entry since the numbers should match to properly 
identify the participant.  In this fashion, no personal identifiers would be accessible to the 
USF.  
 
 
PROTECTION FOR HUMAN SUBJECTS 
 
This clinical trial will be conducted in accordance with the ethical principles that have 
their origin in the Declaration of Helsinki, and that are consistent with Good Clinical 
Practice and all applicable regulatory requirements. 
 
The study protocol will be reviewed and approved by the National Institutes of Health 
(NIH) before submission to individual center IRBs for approval.  Participant enrollment 
may only begin with IRB approved consent forms. 
 
One hundred and twenty-six patients with IMN will participate in the study. Their 
participation will consist of up to 13 visits over a 2-year period. The only clinical 
specimens obtained will be blood and urine. The age range for participation is 18-80 


 
36 
years. RTX will be provided to the study participants by Genentech and the cyclosporine 
will be provided to that arm free of charge as per protocol.   
 
Protection of human subjects:  
 
1. Informed consent process. Patients who are candidates for the study will review and 
sign an informed consent form, to allow review of their history and physical exam 
including blood pressure estimates, as well as serum/urinary chemistries and medication 
history.  
 
Written informed consent will be obtained from each participant before any study-
specific procedures or assessments are done and after the aims, methods, anticipated 
benefits, and potential hazards are explained.  The participant’s willingness to participate 
in the study will be documented in writing in a consent form, which will be signed by the 
participant with the date of that signature indicated.  The investigator will keep the 
original consent forms and signed copies will be given to the participants.  It will also be 
explained to the participants that they are free to refuse entry into the study and free to 
withdraw from the study at any time without prejudice to future treatment.  Written 
and/or oral information about the study in a language understandable by the participant 
will be given to all participants. 
 
 
2. Measures to reduce risks and discomforts associated with study drug.  
RTX infusions will be administered at centers with the following patient safety 
equipment and supplies: oxygen, oral and endotracheal airways and intubation 
equipment, epinephrine 1:1000 solution for intravenous or endotracheal injection, 
antihistamines, corticosteroids, intravenous infusion solutions, tubing, catheters, tape, and 
defibrillator with electrocardiogram monitor.  Patients will be pre-medicated with 
acetaminophen (1g) and diphenhydramine HCl (50 mg) by mouth from 30 to 60 minutes 
prior to the start of an infusion. Premedication with steroids (100 mg methylprednisolone 
IV) will also be given 30 minutes prior to the first infusion of each series of RTX (Day 1 
and Day 181). Patients administered an antihistamine for the treatment or prevention of 
infusion-related reactions will be given appropriate warnings about drowsiness and 
impairment of driving ability prior to discharge.  Patients will be instructed how and 
when to take their cyclosporine, taught about the most common drug interactions and to 
contact their coordinator/study physician about potential interaction with over-the-
counter medications and / or intercurrent illnesses requiring additional treatment 
prescribed by others. They will also be informed about the need to maintain the proper 
time interval prior to assessment of their cyclosporine drug levels. 
 
3. Adverse event reporting.  Each site’s Principal Investigator and their research team 
(co-Investigators, research nurses, clinical trial coordinators, and data managers) are 
responsible for identifying adverse events. Aggregate report- detailed by severity, 
attribution (expected or unexpected), and relationship to the study drug/study procedures 
– will be available from the DMCC for site review.  Adverse events will be reviewed at 


 
37 
least every 6 months by the research team.  The research team will then evaluate whether 
the protocol or informed consent document requires revision based on the reports. 
 
An adverse event is defined as:  “…an unfavorable and unintended sign, symptom or 
disease associated with a participant’s participation in the study.” 
 
Serious adverse events include those events that:  “result in death; are life-threatening; 
require inpatient hospitalization or prolongation of existing hospitalization; create 
persistent or significant disability/incapacity, or a congenital anomaly/birth defects.”  
 
An unexpected adverse event is defined as any adverse experience…the specificity or 
severity of which is not consistent with the risks of information described in the protocol 
or product monograph.  
 
Expected adverse events are those that are identified in the research protocol or product 
monograph as having been previously associated with or having the potential to arise as a 
consequence of participation in the study. 
 
All adverse events that occur between Time 0 and study termination will be reported.  All 
reported adverse events will be classified using the current version of the Common 
Terminology Criteria for Adverse Events (CTCAE) developed and maintained by CTEP 
at National Cancer Institute. 
 
• Within 24 hours (of learning of the event), investigators must report any Serious 
Adverse Event (SAE) that: 
o 
Is considered life-threatening/disabling or results in death of subject 
-OR- 
o 
Is Unexpected/Unanticipated  
• Investigators must report all other SAEs within 5 working days (of learning of the 
event). 
• All other AEs must be reported to the DMCC within 20 working days of the 
notification of the event or of the site becoming aware of the event. 
 
Local institutional reporting requirements to IRBs, any GCRC oversight committee and 
the FDA, if appropriate, remain the responsibility of the treating physician. 
 
 
As part of the reporting structure, SAEs will be reported to Health Canada, Genentech 
Drug Safety and the Genentech medical science liaison. An adverse event summary form 
will be completed and will be summarized for each IRB and Health Canada annually.   
 
Sites will enter all Adverse Event data into the study eCRF.  Upon entry of a serious 
adverse event, the system will notify the DMCC by email.  The DMCC will in turn notify 
the study PIs, the designated medical monitor and any other designated personnel by 
email. The medical monitor will then review the SAE data to determine causality 
(definitely not related, probably not related, possibly related, probably related, definitely 


 
38 
related) of the serious adverse event. The medical monitor [and, if applicable, sponsor] 
may request further information if necessary and possibly request changes to the protocol 
or consent form as a consequence of the serious adverse event. A back-up notification 
system is in place so that any delays in review by the medical monitor beyond a specified 
period of time are forwarded to a secondary reviewer. The eCRF maintains audit trails 
and stores data (and data updated) related to any adverse event in the study.  
 
 
Study Discontinuation  
The Study Sponsor, DSMB and local IRBs (at their local site) have the authority to stop 
or suspend this trial at any time. This study may be suspended or closed if: 
• Accrual has been met 
• The study objectives have been met 
• The Study Investigators believe it is not safe for the study to continue 
• The DSMB suspends or closes the trial 
  
Subject Discontinuation 
An intent to treat approach will be used. All data acquired prior to termination for the 
reasons outlined below will be included in the primary analysis unless patient withdraws 
consent. Every effort will be made to conduct a final study visit with the participant and 
participants will be followed clinically until, if applicable, all adverse events resolve. 
Reasons for subject discontinuation include: 
 
• Withdrawal of consent 
• Withdrawal by the participant 
• Withdrawal by the investigator 
• Intercurrent illness or event that precludes further visits to the study site  
 
Regulatory issues: 
1.  Inclusion of women: Women make up approximately 30-50% of the population of 
patients with IMN and are expected to be enrolled in numbers in proportion to this 
normal distribution.  
2.  Inclusion of minorities: Individuals of all races and ethnicities are at risk for MN.  
Based on the ethnic composition of the patients followed at, and referred to the Mayo 
Clinic the population served will include African-Americans, Asian, and Hispanic 
subjects proportional to that in the general US and Canadian population. This will 
ensure adequate enrollment within the different racial groups.  
3.  Inclusion of children: It is desirable to include children whenever possible in clinical 
research studies. The rationale to exclude children in this trial is that: a) the disease is 
rare in children; b) there are no published natural history studies in children but the 
general impression is that the disease is benign in this patient population; c) no 
studies on specific immunosuppressive treatment in this age group have been 
published, d) many cases of MN in children are secondary to SLE and positive 
laboratory markers are often delayed beyond the point of clinical presentation. We do 
not want this possibility to contaminate and confound the study population. In the 


 
39 
absence of safety and efficacy data we would not like to expose children to an 
unproven therapy. 
 
Data Safety Monitoring Board (DSMB): The DSMB will include 1 chair person, 
clinical experts in nephrology, epidemiology and trial design, and a biostatistician.  We 
will develop a charter for the DSMB and the board will be responsible for evaluating the 
study design and progress of the study.  The board will be provided data on a regular 
basis to monitor patient safety.  Meetings will be conducted on a semi-annual schedule by 
conference call.  
 
Benefits: 
 
Patients might expect to gain the following benefit from study participation: an 
opportunity to be exposed to experimental medicine that may be effective for their kidney 
disease.  Patients will be made aware of the possibility of adverse events known to be 
associated with RTX (and CSA) and the possibility of previously unrecognized adverse 
events.  All patients will benefit from close follow-up and monitoring of their disease 
process in terms of both efficacy and safety in both arms of the study.   
 
Data sharing plan: 
 
Results from the study will be published in both abstract and in manuscript form as soon 
as feasible. Three years after completion of the study, data and remaining samples will be 
made available to the research community for appropriate and well-designed post hoc 
studies.  Proposals for research projects will be reviewed by the Operations committee 
and presented to all the study investigators for approval. 
 
ESTIMATED DURATION OF THE STUDY 
The estimated duration of the study, given a 24-month enrollment period, a 2-year 
follow-up for each patient, and 1 year for data analysis is approximately 60 months.  For 
those patients in the MENTOR remission cohort, their participation will be extended up 
to 3-years follow-up from thefirst day of study drug.  
 
ANTICIPATED RESULTS 
We believe RTX will be as effective as Cyclosporine in inducing complete and partial 
remissions in this patient population with membranous nephropathy.  
 
PUBLICATION POLICY 
A policy similar to the NIH policy on publication of study results will apply to this study. 
Details 
regarding 
policy 
statements 
may 
be 
found 
on 
the 
website 
at 
http://www1.od.nih.gov/oma/manualchapters. Any abstract or manuscript must be 
submitted to Genentech four weeks prior to submission as per contract. 
 


 
40 
 
REFERENCES 
 
 
1. 
Medawar W, Green A, Campbell E, et al. Clinical and histopathologic findings in 
adults with the nephrotic syndrome. Irish Journal of Medical Science 1990; 159: 
137-140. 
 
2. 
Ruggenenti P, Chiurchiu C, Brusegan V, et al. Rituximab in idiopathic 
membranous nephropathy: a one-year prospective study. Journal of the American 
Society of Nephrology 2003; 14: 1851-1857. 
 
3. 
Cattran 
DC. 
Membranous 
nephropathy: 
quo 
vadis?[comment]. 
Kidney 
international 2002; 61: 349-350. 
 
4. 
Wagoner RD, Stanson AW, Holley KE, et al. Renal vein thrombosis in idiopathic 
membranous 
glomerulopathy 
and 
nephrotic 
syndrome: 
incidence 
and 
significance. Kidney international 1983; 23: 368-374. 
 
5. 
Ordonez JD, Hiatt RA, Killebrew EJ, et al. The increased risk of coronary heart 
disease associated with nephrotic syndrome. Kidney international 1993; 44: 638-
642. 
 
6. 
Wheeler DC, Bernard DB. Lipid abnormalities in the nephrotic syndrome: causes, 
consequences, and treatment. American Journal of Kidney Diseases 1994; 23: 
331-346. 
 
7. 
Fervenza FC, Sethi S, Specks U. Idiopathic membranous nephropathy: diagnosis 
and treatment. Clin J Am Soc Nephrol 2008; 3: 905-919. 
 
8. 
Polanco N, Gutierrez E, Covarsi A, et al. Spontaneous Remission of Nephrotic 
Syndrome in Idiopathic Membranous Nephropathy. J Am Soc Nephrol 2010. 
 
9. 
Schieppati A, Mosconi L, Perna A, et al. Prognosis of untreated patients with 
idiopathic membranous nephropathy.[comment]. New England Journal of 
Medicine 1993; 329: 85-89. 
 
10. 
Safai-Infanger M MB. Management of membranous nephropathy. Nephrology 
Rounds - Stanford University 2000; 3: 1-5. 
 
11. 
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