clinical trial www.kidney-international.org The STARMEN trial indicates that alternating treatment with corticosteroids and see commentary on page 811 cyclophosphamide is superior OPEN to sequential treatment with tacrolimus and rituximab in primary membranous nephropathy Gema Fernández-Juárez1, Jorge Rojas-Rivera2, Anne-Els van de Logt3, Joana Justino4, Angel Sevillano5, Fernando Caravaca-Fontán5, Ana Ávila6, Cristina Rabasco7, Virginia Cabello8, Alfonso Varela9, Montserrat Dı́ez10, Guillermo Martı́n-Reyes11, Marian Goicoechea Diezhandino12, Luis F. Quintana13, Irene Agraz14, Juan Ramón Gómez-Martino15, Mercedes Cao16, Antolina Rodrı́guez-Moreno17, Begoña Rivas18, Cristina Galeano19, Jose Bonet20, Ana Romera21, Amir Shabaka1, Emmanuelle Plaisier22,23, Mario Espinosa7, Jesus Egido2, Alfonso Segarra14, Gérard Lambeau4, Pierre Ronco22,23, Jack Wetzels3 and Manuel Praga5,24; for the STARMEN Investigators25 1 Nephrology Division, Hospital Universitario Fundación Alcorcón, Madrid, Spain; 2Nephrology Division, Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain; 3Nephrology Division, Radboud University Medical Center, Nijmegen, The Netherlands; 4Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Université Côte d’Azur, Centre National de la Recherche Scientifique (CNRS), Valbonne Sophia Antipolis, France; 5Nephrology Division, Instituto de Investigación Hospital Universitario 12 Octubre, Madrid, Spain; 6Nephrology Division, Hospital Dr Peset, Valencia, Spain; 7Nephrology Division, Hospital Reina Sofía, Córdoba, Spain; 8Nephrology Division, Hospital Virgen del Rocío, Sevilla, Spain; 9Nephrology Division, Hospital Virgen de la Victoria de Málaga, Málaga, Spain; 10Fundació Puigvert, Nephrology Division, Institut Investigaci Biosanitaria Sant Pau, Autonomous University of Barcelona (UAB), Barcelona, Spain; 11 Nephrology Division, Hospital Regional Universitario de Málaga, Málaga, Spain; 12Nephrology Division, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 13Centro de Referencia en Enfermedad Glomerular Compleja del Sistema Nacional de Salud (CSUR), Hospital Clínic de Barcelona, Universitat de Barcelona, Barcelona, Spain; 14Nephrology Division, Hospital Universitari Vall d’Hebron, Barcelona, Spain; 15Nephrology Division, Hospital San Pedro de Alcántara, Cáceres, Spain; 16Nephrology Division, Hospital Universitario de A Coruña, A Coruña, Spain; 17Nephrology Division, Hospital Clínico San Carlos, Madrid, Spain; 18Nephrology Division, Hospital Universitario La Paz, Madrid, Spain; 19Nephrology Division, Hospital Universitario Ramón y Cajal, Madrid, Spain; 20Nephrology Division, Hospital Germans Trias i Pujol, Barcelona, Spain; 21Nephrology Division, Hospital de Ciudad Real, Ciudad Real, Spain; 22Sorbonne Université, Université Pierre et Marie Curie Paris 06, and Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S1155, Paris, France; 23Centre de Référence Maladies Rares Syndrome Néphrotique Idiopathique - Hôpital de Jour Néphrologie – Hôpital TENON-Assistance Publique–Hôpitaux de Paris (APHP)- Sorbonne Université, Paris, France; and 24Department of Medicine, Complutense University, Madrid, Spain A cyclical corticosteroid-cyclophosphamide regimen is tapering for another three months) and rituximab (one recommended for patients with primary membranous gram at month six). The primary outcome was complete or nephropathy at high risk of progression. We hypothesized partial remission of nephrotic syndrome at 24 months. This that sequential therapy with tacrolimus and rituximab is composite outcome occurred in 36 patients (83.7%) in the superior to cyclical alternating treatment with corticosteroid-cyclophosphamide group and in 25 patients corticosteroids and cyclophosphamide in inducing (58.1%) in the tacrolimus-rituximab group (relative risk persistent remission in these patients. This was tested in a 1.44; 95% confidence interval 1.08 to 1.92). Complete randomized, open-label controlled trial of 86 patients with remission at 24 months occurred in 26 patients (60%) in the primary membranous nephropathy and persistent corticosteroid-cyclophosphamide group and in 11 patients nephrotic syndrome after six-months observation and (26%) in the tacrolimus-rituximab group (2.36; 1.34 to 4.16). assigned 43 each to receive six-month cyclical treatment Anti-PLA2R titers showed a significant decrease in both with corticosteroid and cyclophosphamide or sequential groups but the proportion of anti-PLA2R-positive patients treatment with tacrolimus (full-dose for six months and who achieved immunological response (depletion of anti- PLA2R antibodies) was significantly higher at three and six Correspondence: Manuel Praga, Instituto de Investigación Hospital Uni- months in the corticosteroid-cyclophosphamide group versitario 12 Octubre, Avda. Córdoba s/n, 28041 Madrid, Spain. E-mail: (77% and 92%, respectively), as compared to the mpragat@senefro.org tacrolimus-rituximab group (45% and 70%, respectively). 25 Members of the STARMEN Investigators are listed in the Appendix. Relapses occurred in one patient in the corticosteroid- Received 13 August 2020; revised 24 September 2020; accepted 11 cyclophosphamide group, and three patients in the October 2020; published online 7 November 2020 tacrolimus-rituximab group. Serious adverse events were 986 Kidney International (2021) 99, 986–998 G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab clinical trial similar in both groups. Thus, treatment with corticosteroid- cyclophosphamide with the newer therapeutic alternatives cyclophosphamide induced remission in a significantly (calcineurin inhibitors, rituximab) was stressed in a recent greater number of patients with primary membranous KDIGO conference.22 We designed the STARMEN (Sequen- nephropathy than tacrolimus-rituximab. tial Treatment with Tacrolimus and Rituximab Versus Alter- Kidney International (2021) 99, 986–998; https://doi.org/10.1016/ nating Corticosteroids and Cyclophosphamide in PMN) j.kint.2020.10.014 study to compare cyclic alternating treatment of corticoste- KEYWORDS: corticosteroids; cyclophosphamide; primary membranous roids and cyclophosphamide with a sequential treatment of nephropathy; rituximab; tacrolimus tacrolimus and rituximab in the induction and maintenance Copyright ª 2020, International Society of Nephrology. Published by of nephrotic syndrome remission for up 24 months. In Elsevier Inc. This is an open access article under the CC BY-NC-ND license addition, we studied the occurrence of relapses after remis- (http://creativecommons.org/licenses/by-nc-nd/4.0/). sion and the role of anti-PLA2R autoantibodies over the course of the treatment. P rimary membranous nephropathy (PMN) is one of the RESULTS most common causes of nephrotic syndrome in adults.1 Patients In 70%–80% of cases, the disease is mediated by auto- From June 2014 through June 2017, 130 patients were antibodies targeting the phospholipase A2 receptor (PLA2R) assessed for eligibility, of whom 44 (33%) were excluded from expressed in podocytes and in 3%–5% by autoantibodies to the study. Main reasons for screening failure were not meeting thrombospondin type 1 domain–containing 7A (THSD7A).2–4 the eligibility criteria (21 patients), unwillingness to partici- Spontaneous remission occurs in one-third of patients,5 and pate in the study (14 patients), and diagnosis of a secondary therefore an observational period of at least 6 months is rec- cause of membranous nephropathy (9 patients). The ommended.6–8 Conversely, about 50% of cases with persistent remaining 86 patients who met the eligibility criteria were nephrotic syndrome eventually progress to end-stage kidney randomly assigned to the corticosteroid-cyclophosphamide disease, and immunosuppressive therapy is recommended for group (43 patients) or to the tacrolimus-rituximab group these patients. (43 patients) (Figure 1). As presented in Table 1, no Controversy persists about the most effective type of significant differences between groups were observed at immunosuppressive regimen. The 2012 Kidney Disease: baseline. Baseline stored serum samples were available in 69 Improving Global Outcomes (KDIGO) guidelines for patients, and anti-PLA2R was positive in 53 (77%). Sensitivity glomerulonephritis recommended a 6-month cyclic regimen analyses showed no differences at baseline between anti- of alternating alkylating agents (usually cyclophosphamide) PLA2R–positive and anti-PLA2R–negative patients nor be- plus corticosteroids for patients at high risk of progression, tween patients with or without anti-PLA2R determinations at since it was the only regimen that was shown to be effective in baseline (Supplementary Tables S1 and S2). One patient was preventing end-stage kidney disease.8–12 However, given the anti-THSD7A positive. Kidney biopsies were performed 8 important number of serious adverse events associated with (range, 6–18) months before randomization. Seventy-three cumulative doses of alkylating agents, treatment alternatives patients (85%) had de novo PMN and 13 (15%) relapsing were introduced. Calcineurin inhibitors (both cyclosporine PMN (Table 1). and tacrolimus) have shown efficacy in inducing remission of All patients received at least 1 month of the assigned ther- nephrotic syndrome in about 70% of patients.13,14 However, apeutic intervention. Two patients in the corticosteroid- the main limitation of these drugs is the high rate of relapse cyclophosphamide group (4.6%) and 6 (13.9%) in the after discontinuation. An observational study found a tacrolimus-rituximab group discontinued the intervention reduction in relapse rates when rituximab was administered at (Figure 1). The remaining 41 in the corticosteroid- the time of tapering off cyclosporine or tacrolimus,15 and a cyclophosphamide group and 37 in the tacrolimus-rituximab pilot study reported encouraging results of a combined group) received the complete intervention. Patients assigned therapy with cyclosporine plus rituximab in high-risk PMN to the corticosteroid-cyclophosphamide group received a patients.16 median dose of oral methylprednisolone of 0.49  0.05 mg/kg/ More recently, the efficacy of rituximab monotherapy has day in months 1, 3, and 5, with a total cumulative dose of 3.4  received great attention.17 A single dose of rituximab was 0.9 g. The total cumulative dose of intravenous methylpred- suggested to be effective for induction of remission in an nisolone was 8.2  1.4 g. The total cumulative dose of cyclo- observational cohort,18 although in recent clinical trials, phosphamide was 10  3.5 g. Doses and blood levels of higher doses were needed for optimal efficacy.19–21 Indeed, tacrolimus in the tacrolimus-rituximab group are presented in the superior efficacy of rituximab versus cyclosporine in the Supplementary Table S3. Three patients in this group received a recent MENTOR (Membranous Nephropathy Trial of Rit- second dose of rituximab (0.5 g in 2 patients, 1 g in 1 patient) at uximab) study was achieved using a total dose of 4 g months 12, 12, and 18, respectively, and 2 others received rituximab.20 additional doses of tacrolimus beyond month 9. The need for head-to-head trials comparing the 6-month During the follow-up period after the end of the cyclic alternating treatment with corticosteroids and assigned intervention, 5 patients in each group were Kidney International (2021) 99, 986–998 987 clinical trial G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab Figure 1 | Enrollment, randomization, and follow-up. Patients were randomly assigned in a 1:1 ratio to receive corticosteroid- cyclophosphamide or tacrolimus-rituximab. Premature discontinuation occurred in 2 patients in the corticosteroid-cyclophosphamide group and 6 patients in the tacrolimus-rituximab group. The remaining patients (41 in the corticosteroid-cyclophosphamide group and 37 in the tacrolimus-rituximab group) received the complete intervention. During the follow-up after the end of the assigned intervention, 5 patients in each group were switched to a nonstudy intervention owing to lack of efficacy of the assigned treatment and were considered to be nonresponders. Relapses occurred in 1 patient in the corticosteroid-cyclophosphamide group and 3 patients in the tacrolimus-rituximab group. switched to a nonstudy intervention owing to a lack of rituximab at month 14, 2 patients received tacrolimus from efficacy of the assigned treatment. In the corticosteroid- month 18, and 1 patient cyclosporine from month 16. In cyclophosphamide group, 2 patients were treated with the tacrolimus-rituximab group, the 5 patients received a 988 Kidney International (2021) 99, 986–998 G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab clinical trial Table 1 | Baseline characteristics according to treatment group Characteristic All patients (n [ 86) Corticosteroid-cyclophosphamide (n [ 43) Tacrolimus-rituximab (n [ 43) P value Age, yr 55.7  11.4 56.2  12.0 55.2  10.8 1 Male sex 55 (64) 24 (55) 31 (72) 0.12 De novo/relapsing PMN 73 (85) / 13 (15) 36 (83)/7 (17) 37 (86)/6 (14) 0.70 Weight, kg 78.5  16.0 76.9  17.0 80.0  15.0 0.37 Blood pressure, mm Hg Systolic 128.3  15.9 129.4  17.7 127.1  14.0 0.25 Diastolic 76.0  9.8 75.1  10.7 77.0  8.8 0.37 Serum creatinine, mg/dl 1.0  0.3 1.0  0.3 1.0  0.28 0.48 eGFR, ml/min per 1.73 m2a 79.8  23.5 79.1  25.5 80.5  21.6 0.78 Serum albumin, g/dl 2.6 (2.2–2.9) 2.6 (2.3–2.9) 2.6 (2.0–2.9) 0.47 Serum cholesterol, mg/dl 263.9  64.0 264.1  57.8 263.8  70.2 0.34 Anti-PLA2R–positive patientsb 53 of 69 (77) 29 of 37 (78)c 24 of 32 (75)d 0.38 Anti-PLA2R, RU/ml 80 (44–149) 59 (37–150) 113 (61–151) 0.1 Urinary protein, g/24 h 7.4 (5.2–11.5) 7.4 (4.8–11.3) 7.4 (6.7–11.6) 0.22 Concomitant treatment ACEis and/or ARBs 82 of 83 (99) 39 of 43 (91) 43 of 43 (100) 0.48 Diuretics 70 of 83 (84) 35 of 43 (81) 35 of 43 (81) 0.45 ACEis, angiotensin-converting enzyme inhibitors; Anti-PLA2R, anti–phospholipase A2 receptor autoantibodies; ARBs, angiotensin II receptor blockers; eGFR, estimated glomerular filtration rate; RU, relative units. a eGFR was calculated according to the Chronic Kidney Disease Epidemiology Collaboration equation. b Anti-PLA2R positivity defined as >14 RU/ml. c In 6 of 43 patients (16%) anti-PLA2R was not determined at baseline. d In 11 of 43 patients (25.5%) anti-PLA2R was not determined at baseline. Values are presented as mean  SD, n (%), or median (interquartile range). 6-month cyclic treatment with corticosteroid and cyclo- found in female patients (Supplementary Figure S1). phosphamide at month 12 (2 patients), month 14 (2 pa- When baseline characteristics of patients were compared tients), or month 21 (1 patient). All the patients who were between those who achieved complete/partial remission switched to a nonstudy intervention were considered to be and those with no response, a significantly higher pro- nonresponders (Figure 1). Follow-up was complete in all of portion of men (80% vs. 57%) and a significantly higher the 86 enrolled patients. Primary outcome Thirty-six patients (83.7%) in the corticosteroid- Table 2 | Composite outcome of complete or partial remission cyclophosphamide group and 25 patients (58.1%) in the at 3 to 24 months tacrolimus-rituximab group had a primary outcome of Intention-to-treat analysis complete/partial remission at 24 months (relative risk [RR] 1.44, 95% confidence interval [CI] 1.08 to 1.92) Corticosteroid- Tacrolimus- Time from cyclophosphamide rituximab Relative risk (Table 2; Figure 2a). The per-protocol analysis confirmed randomization (n [ 43) (n [ 43) (95% CI) significant differences in the primary outcome between groups: 35 of 41 (85%) in the corticosteroid- 3 mo 22 of 43 (51) 12 of 43 (28) 1.83 (1.04–3.22) 6 mo 32 of 43 (74) 19 of 43 (44) 1.68 (1.15–2.46) cyclophosphamide group and 22 of 37 (59%) in the 12 mo 34 of 43 (79) 22 of 43 (51) 1.55 (1.11–2.15) tacrolimus-rituximab group (RR 1.44, 95% CI 1.07 to 18 mo 36 of 43 (84) 23 of 43 (53) 1.57 (1.15–2.13) 1.93) (Table 2). The difference in the number of patients 24 mo 36 of 43 (84) 25 of 43 (58) 1.44 (1.08–1.92) with complete or partial remission in both groups was Per-protocol analysis already significant at month 3 and was maintained Corticosteroid- Tacrolimus- throughout the study (Table 2; Figure 2a). Time from cyclophosphamide rituximab Relative risk As shown in Table 3 and Figure 2b, 26 patients (60%) in randomization (n [ 41) (n [ 37) (95% CI) the corticosteroid-cyclophosphamide group achieved com- 3 mo 22 of 41 (51) 12 of 37 (32) 1.65 (0.96–2.85) plete remission at 24 months. In the tacrolimus-rituximab 6 mo 31 of 41 (75) 18 of 37 (48) 1.55 (1.07–2.26) group, 11 patients (26%) achieved complete remission at 24 12 mo 33 of 41 (80) 20 of 37 (54) 1.49 (1.07–2.08) 18 mo 35 of 41 (85) 20 of 37 (54) 1.58 (1.14–2.18) months (RR 2.36, 95% CI 1.34 to 4.16). 24 mo 35 of 41 (85) 22 of 37 (59) 1.44 (1.07–1.93) A tendency for a greater efficacy of the corticosteroid- CI, confidence interval. cyclophosphamide treatment was found across different The intention-to-treat analyses included all the patients who underwent randomi- non-prespecified subgroups defined by baseline values of zation, whereas the per-protocol analyses included all the patients who received the full course of trial medications. In both analyses, the primary outcome was the proteinuria, serum albumin, serum creatinine, anti- composite of complete or partial remission at 24 months. PLA2R levels. and age, although no differences were Data are n (%) or 95% CI. Kidney International (2021) 99, 986–998 989 clinical trial G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab Figure 2 | Kaplan-Meier analysis of complete or partial remission. Kaplan-Meier estimates of (a) remission (complete or partial) and (b) complete remission in the corticosteroid-cyclophosphamide and tacrolimus-rituximab groups. proteinuria were observed among nonresponders group difference P ¼ 0.005) (Figure 3a; Supplementary (Supplementary Table S4). Table S5). Serum albumin increased from a mean 2.6  0.1 g/dl at baseline to 4  0.1 g/dl at 24 months in the Secondary outcomes corticosteroid-cyclophosphamide group and from 2.6  0.1 Proteinuria decreased from a median 7.4 g/24 h (interquartile g/dl at baseline to 3.9  0.1 g/dl at 24 months in the range 4.8–11.3) at baseline to 0.35 g/24 h (0.2–9) at 24 tacrolimus-rituximab group (between-group difference P ¼ months in the corticosteroid-cyclophosphamide group and 0.2) (Figure 3b; Supplementary Table S5). There was a from 7.4 g/24 h (6.7–11.6) at baseline to 1 g/24 h (0.3–3.3) at nonsignificant trend for higher values of estimated glomer- 24 months in the tacrolimus-rituximab group (between- ular filtration rate (eGFR) in the corticosteroid- 990 Kidney International (2021) 99, 986–998 G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab clinical trial Table 3 | Complete remission at 3 to 24 months compared with patients who achieved a complete or partial Intention-to-treat analysis remission of nephrotic syndrome (Supplementary Table S7). Corticosteroid- Tacrolimus- One out of the 36 patients (2.7%) in the corticosteroid- Time from cyclophosphamide rituximab Relative risk cyclophosphamide group, and 3 out of the 25 patients randomization (n [ 43) (n [ 43) (95% CI) (12%) in the tacrolimus-rituximab group who had achieved 3 mo 1 of 43 (2) 0 of 43 (0) partial remission presented a relapse (Supplementary 6 mo 6 of 43 (14) 0 of 43 (0) Table S8). The 3 relapses in the tacrolimus-rituximab group 12 mo 14 of 43 (33) 4 of 43 (9) 3.50 (1.25–9.78) occurred at month 12, 3 months after tacrolimus discontin- 18 mo 19 of 43 (44) 7 of 43 (16) 2.71 (1.27–5.78) uation. Two of them occurred in anti-PLA2R–positive pa- 24 mo 26 of 43 (60) 11 of 43 (26) 2.36 (1.34–4.16) tients who had reached an immunologic response at month 6 Per-protocol analysis and were not accompanied by a reappearance of anti-PLA2R Corticosteroid- Tacrolimus- antibodies. The remaining relapse occurred in a patient in Time from cyclophosphamide rituximab Relative risk whom anti-PLA2R antibodies had not been measured at randomization (n [ 41) (n [ 37) (95% CI) baseline. Tacrolimus was restarted in 2 patients and the other 3 mo 1 of 41 (2) 0 of 37 (0) one was treated with rituximab. The relapse in the 6 mo 5 of 41 (13) 0 of 37 (0) corticosteroid-cyclophosphamide group occurred at month 9 12 mo 13 of 41 (32) 4 of 37 (11) 2.93 (1.05–8.21) 18 mo 18 of 41 (44) 7 of 37 (19) 2.31 (1.09–4.92) in an anti-PLA2R–positive patient who had reached immu- 24 mo 25 of 41 (61) 10 of 37 (27) 2.26 (1.26–4.04) nologic response at month 3. In this case, the relapse was CI, confidence interval. accompanied by a reappearance of anti-PLA2R antibodies and The intention-to-treat analyses included all the patients who underwent randomi- the patient was treated with tacrolimus. zation, whereas the per-protocol analyses included all the patients who received the full course of trial medications. In both analyses, the primary outcome was the composite of complete or partial remission at 24 months. Adverse events Data are n (%) or 95% CI. All patients except 6 (1 from the corticosteroid- cyclophosphamide group and 5 from the tacrolimus- rituximab group) had at least 1 adverse event (Table 5). cyclophosphamide group than in the tacrolimus-rituximab Most adverse events were of low (345, 84%) or medium (56, group throughout the follow-up (Figure 3c; Supplementary 13%) severity, but 17 (4%) were serious. There were more Table S6). At 24 months, the numbers of patients with adverse events and more adverse events per patient in the a $50% increases of baseline serum creatinine were 1 (2%) in corticosteroid-cyclophosphamide group than in the the corticosteroid-cyclophosphamide group and 5 (12%) in tacrolimus-rituximab group (P ¼ 0.04). More patients in the the tacrolimus-rituximab group (P ¼ 0.2). The numbers of corticosteroid-cyclophosphamide group had leukopenia and patients with preserved renal function (eGFR $45 ml/min Cushing syndrome, while acute kidney injury, hyperkalemia, per 1.73 m2) at 24 months were 40 (93%) in the diarrhea, and distal tremor were more common in the corticosteroid-cyclophosphamide group and 37 (86%) in the tacrolimus-rituximab group. There was a statistically signifi- tacrolimus-rituximab group (P ¼ 0.48). The only patient who cant association between the presence of leukopenia and the developed end-stage kidney disease was a 73-year-old man development of infections, both in the total trial cohort and who had been assigned to the corticosteroid- in each treatment group (P < 0.0001 for the total group and cyclophosphamide group. Three months after treatment for the cyclophosphamide group, P ¼ 0.041 for the tacroli- completion he was excluded from the study because of mus/rituximab group). Most adverse events occurred within persistent massive proteinuria and declining renal function the first 9 months of the trial (304 of 409, 74%), but only 5 of and switched to rituximab (2 doses of 1 g, 15 days apart). No the 17 serious adverse events occurred within this period. response was observed, and chronic dialysis was initiated 16 There were no statistically significant differences in the months after randomization. frequency of serious adverse events between groups. The only Anti-PLA2R levels showed a significant decrease in both case of serious acute kidney injury occurred in the groups (Table 4; Figure 3a). The proportions of anti-PLA2R– tacrolimus-rituximab group, while 4 of the 5 serious in- positive patients who achieved immunologic response at 3 fections occurred in the corticosteroid-cyclophosphamide and 6 months were significantly higher in the corticosteroid- group. Three cases of cancer were reported, although none cyclophosphamide group (77% and 92%, respectively) than of them was deemed to be related to the treatment received. in the tacrolimus-rituximab group (45% and 70%, respec- Two cancers occurred in the corticosteroid- tively) (Table 4). Most patients (80%) who achieved immu- cyclophosphamide group (gastric adenocarcinoma and nologic response during the study presented remission of breast carcinoma, detected at 12 and 11 months, respectively) nephrotic syndrome at 24 months. Immunologic response at and 1 in the tacrolimus-rituximab group (rectal carcinoma, 3 months (P ¼ 0.036) and 6 months (P ¼ 0.005) was asso- detected at 1 month). Anti-PLA2R was positive at baseline in ciated with remission at 24 months. Nonresponding patients the 3 patients with cancer. At the time of tumor detection, the showed a slower decline in anti-PLA2R levels and a signifi- 2 patients in the corticosteroid-cyclophosphamide group were cantly lower proportion of immunologic responses, in clinical remission. Kidney International (2021) 99, 986–998 991 clinical trial G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab Figure 3 | Evolution of proteinuria and anti–phospholipase A2 receptor (PLA2R), serum albumin, and estimated glomerular filtration rate (eGFR). Data are presented as median (interquartile range) over time (albumin, proteinuria, anti-PLA2R) or mean  SD by assigned treatment. DISCUSSION treatment with tacrolimus and rituximab in inducing remis- The study failed to support the hypothesis that sequential sion. The occurrence of remissions was also faster in the therapy with tacrolimus and rituximab was superior to cyclic corticosteroid-cyclophosphamide group, with a significant alternating treatment with corticosteroids and cyclophos- difference in the number of remissions already at 3 months. phamide in inducing persistent remission in patients with Moreover, most remissions were complete in the PMN. In fact, we found that treatment with corticosteroid corticosteroid-cyclophosphamide group, whereas most re- and cyclophosphamide was more effective than sequential missions were partial in the tacrolimus-rituximab group. Table 4 | Evolution of anti-PLA2R antibodies and percentage of immunologic response according to treatment group Anti-PLA2R, RU/ml, median (IQR) Immunologic response, % Time from Corticosteroid- Tacrolimus- P Corticosteroid- Tacrolimus- P randomization All patients cyclophosphamide rituximab value cyclophosphamide rituximab value Baseline 80 (44–149) 59 (37–150) 113 (61–151) 0.1 3 mo 2.3 (1.3–65) 1.4 (0.7–14) 33 (2.6–76) 0.003 77 45 0.03 6 mo 1.4 (0.7–7.4) 1.4 (0–1.7) 2.6 (1.4–30) 0.02 92 70 0.04 12 mo 1.6 (0–8.1) 1.5 (0–5) 2.1 (0–15) 0.5 88 79 0.31 18 mo 1.5 (0–2) 1.3 (0–2.3) 1.6 (0–15) 0.3 88 83 0.88 24 mo 1.6 (1.3–5.1) 1.4 (0.9–3.2) 1.9 (1.7–14) 0.06 88 83 0.91 IQR, interquartile range; PLA2R, phospholipase A2 receptor; RU, relative units. Differences of median were compared with the Mann-Whitney test. 992 Kidney International (2021) 99, 986–998 G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab clinical trial Table 5 | Adverse events according to treatment group Corticosteroid- cyclophosphamide (n [ 43) Tacrolimus-rituximab (n [ 43) Event Patients Events Patients Events P valuea Any adverse event 42 (98) 239 (411) 39 (91) 170 (280) 0.04 Serious adverse events 8 (19) 10 (17) 6 (14) 7 (12) 0.93 Fatal 0 (0) 0 (0) 0 (0) 0 (0) 1.00 Nonfatal 8 (19) 10 (17) 6 (14) 7 (12) 0.56 Nonserious adverse events 34 (79) 229 (394) 33 (77) 163 (268) 0.04 Adverse events in at least 5% of patients or any serious adverse event Systemic/general Headache 3 (7) 5 (9) 5 (12) 7 (12) 0.48 Asthenia 7 (16) 9 (16) 4 (10) 4 (7) 0.52 Depression 3 (7) 3 (5) 1 (2) 2 (3) 0.62 Anxiety 8 (19) 14 (24) 4 (10) 4 (7) 0.35 Metabolic Cushing syndrome 7 (16) 8 (14) 0 (0) 0 (0) 0.01 Hyperglycemia 4 (9) 4 (7) 2 (5) 2 (3) 0.68 Hyperlipidemia 5 (12) 7 (12) 2 (5) 2 (3) 0.43 Gastrointestinal Diarrhea 4 (9) 4 (7) 13 (31) 13 (21) 0.02 Abdominal complaint 7 (16) 7 (12) 3 (7) 4 (7) 0.31 Infections Upper respiratory infection 10 (23) 14 (24) 10 (24) 14 (23) 1.00 Pneumonia 4 (9) 4 (7) 2 (5) 2 (3) 0.68 Urinary tract infection 4 (9) 4 (7) 1 (2) 1 (2) 0.36 Tuberculosis 1 (2) 1 (2) 0 (0) 0 (0) 1.00 Musculoskeletal Bone pain 10 (23) 12 (21) 9 (21) 12 (20) 1.00 Myalgia 7 (16) 9 (16) 2 (5) 2 (3) 0.16 Cramps 5 (12) 6 (10) 5 (12) 5 (8) 1.00 Bone fracture 1 (2) 1 (1) 2 (5) 2 (3) 0.62 Neurologic Tremor 2 (5) 2 (3) 7 (17) 7 (12) 0.09 Paresthesia 2 (5) 2 (3) 2 (5) 2 (3) 1.00 Hematologic Anemia 13 (30) 17 (29) 9 (21) 10 (17) 0.46 Leukopenia 13 (30) 22 (38) 2 (5) 2 (3) 0.003 Cardiovascular and blood pressure Acute coronary syndrome 1 (2) 1 (2) 1 (2) 1 (2) 1.00 Venous thrombosis 5 (12) 5 (9) 2 (5) 2 (3) 0.43 Hypertension 5 (12) 7 (12) 6 (14) 6 (10) 0.76 Hypotension 4 (9) 4 (7) 0 (0) 0 (0) 0.12 Renal Acute kidney injury 8 (19) 8 (14) 14 (33) 16 (26) 0.14 Hyperkalemia 1 (2) 1 (2) 6 (14) 6 (10) 0.06 Edema 6 (14) 6 (10) 4 (10) 5 (8) 0.74 Dermatologic Skin eruption 4 (9) 4 (7) 0 (0) 0 (0) 0.12 Miscellaneous Drug infusion reaction 1 (2) 1 (2) 4 (9) 4 (7) 0.36 Cancer 2 (5) 2 (3) 1 (2) 1 (2) 0.62 Gastric adenocarcinoma 1 (2) 1 (2) 0 (0) 0 (0) Breast carcinoma 1 (2) 1 (2) 0 (0) 0 (0) Rectal carcinoma 0 (0) 0 (0) 1 (2) 1 (2) Results for patients are presented as n (%), for events as n (rate per 100 patient-years). a P values are for the differences in number of events between groups. Previous studies with calcineurin inhibitors and ritux- the favorable effect of calcineurin inhibitors in PMN could imab have demonstrated that these drugs are effective at be largely attributed to their effects on podocyte cyto- inducing remission in PMN. It has been postulated that skeleton, resulting in a nonspecific proteinuria Kidney International (2021) 99, 986–998 993 clinical trial G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab reduction.23 However, we found that tacrolimus induced a although immunologic response occurred faster in the rapid decrease in anti-PLA2R levels, in agreement with corticosteroid-cyclophosphamide group. Cyclophosphamide previous studies.24 induces generalized leukocyte and mature plasma cell abla- The main limitation of calcineurin inhibitors is the high tion, resulting in a more drastic reduction in antibody relapse rate after discontinuation, occurring in 40%–60% of production than more specifically targeted drugs such as patients. In the MENTOR study,20 rituximab (1 g at days 1 rituximab or calcineurin inhibitors, and previous studies and 15 after randomization, and 2 other infusions at month 6 have demonstrated the effectiveness of alkylating agents in if the patient had not reached complete remission) was the most aggressive cases of PMN.32–34 Early immunologic compared with cyclosporine given for 12 months. There were response was followed by clinical remission in the vast no significant differences in the number of remissions at 12 majority of patients, which confirms the usefulness of anti- months. However, a higher proportion of patients in the PLA2R monitoring for a personalized treatment of the cyclosporine group relapsed after treatment withdrawal, and disease. the number of patients in remission at 24 months was The rate of adverse effects was significantly higher among significantly higher in the rituximab group (60% vs. 20%). patients treated with corticosteroid-cyclophosphamide, In our study, the number of remissions at 6 months was although there were no differences in the number of serious lower in the tacrolimus-rituximab group than in the adverse events. Some studies have shown that corticosteroid-cyclophosphamide group. Rituximab infusion cyclophosphamide-based therapies in PMN are accompanied at month 6 did not attenuate the difference in remission rates, by serious untoward complications.35,36 However, cumulative although the number of complete remissions increased after doses of cyclophosphamide were higher than those used in rituximab infusion. An important finding was the low our study. Observational studies have suggested that noncy- number of relapses after tacrolimus discontinuation. This clic schemes of cyclophosphamide-based treatments, using finding agrees with a previous observational study that re- lower doses of the drug, can achieve satisfactory results in ported a beneficial effect of rituximab, infused at the onset of PMN.37 Anti-PLA2R monitoring could help to reduce treat- calcineurin inhibitor tapering, to prevent relapses in PMN ment length in patients in which a rapid immunologic patients who had responded to cyclosporine or tacrolimus.15 response is observed. Other retrospective studies have re- The pathogenesis of relapses after tacrolimus discontinu- ported encouraging results with the use of intravenous pulses ation in PMN and the mechanisms through which rituximab of cyclophosphamide as a substitute for oral cyclophospha- can prevent this complication are unclear. Notably, rituximab mide in patients treated with the alternating cyclic regimen of is also effective in reducing the number of nephrotic corticosteroids and cyclophosphamide.38,39 On the other syndrome relapses in other kidney diseases, such as minimal- hand, it would be important to explore whether the use of change disease and focal and segmental glomerulo- lower doses of corticosteroids can reduce side-effects without sclerosis.25,26 In the present study, the low number of relapses decreasing the effectiveness of the treatment. These new compensated for the lower than expected number of re- schemes of treatment with corticosteroids and cyclophos- missions at 24 months. Consequently, the final response rate phamide should ideally be compared with treatments based of remissions in the tacrolimus-rituximab group was 58%, on calcineurin inhibitors and rituximab. Whether treatment similar to that obtained with rituximab-only in recent tri- with tacrolimus beyond 6 months or higher and earlier doses als19,20 and better than that observed after cyclosporine or of rituximab could increase the remission rates in patients tacrolimus discontinuation without the coverage of rituximab treated with tacrolimus-rituximab should be explored in infusion.13,14,24 On the other hand, it should be considered future studies. that the tacrolimus-rituximab group had 72% male subjects The present study has important limitations. There was (vs. 55% in the corticosteroid-cyclophosphamide group) and lack of blinding regarding interventions and outcome there were trends toward higher anti-PLA2R levels, a higher assessment. The limited sample size prevented analysis by interquartile range of proteinuria, and a lower interquartile prespecified subgroups or detailed analysis of anti-PLA2R range of serum albumin in this group. Although not signifi- kinetics, and anti-PLA2R antibodies were not measured in cant, these differences at baseline may have biased the result a number of patients. Because CD19þ B cells were not toward an inferior outcome in the tacrolimus-rituximab measured, no information about the adequacy of rituximab group. dose was available. However, this study was a head-to-head Since the discovery of anti-PLA2R autoantibodies as a prospective controlled trial that compared the classic cyclic pathogenic driver of the disease in 70%–80% of patients alternating treatment with corticosteroids and cyclophos- with PMN, a cumulative number of studies have confirmed phamide versus the newer therapeutic alternatives the crucial role of serial measurement of anti-PLA2R levels (sequential treatment with tacrolimus and rituximab), with to predict clinical outcomes, to evaluate the therapeutic a sample size and follow-up period that allowed us to draw response, and to help guiding the length of immunosup- important conclusions about the treatment of PMN in pressive therapy.2,27–31 We found that both corticosteroid- clinical practice. cyclophosphamide and tacrolimus-rituximab regimens In conclusion, treatment with corticosteroid- induced a significant reduction in anti-PLA2R levels, cyclophosphamide induced remission (mostly complete) of 994 Kidney International (2021) 99, 986–998 G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab clinical trial Figure 4 | Design of the study. During a 6-month observational period, treatment with angiotensin-converting enzyme inhibitors/ angiotensin receptor blockers and supportive treatment was optimized. Study visits were scheduled at screening (1 month), at enrollment, monthly from month 1 to month 9, and at months 12, 15, 18, 21, and 24 after randomization. Clinical data and medications were recorded at every study visit. Routine laboratory tests were obtained at every study visit. Blood and urine samples were collected at baseline and at months 3, 6, 12, 18, and 24. Primary outcome evaluation was performed at 24 months per intention-to-treat and per-protocol analyses, and secondary outcomes were evaluated at 6, 12, 18, and 24 months. nephrotic syndrome in a significantly greater number of pa- contraindications, or low blood pressure, before the screening tients than treatment with tacrolimus-rituximab, although period. In the case of fertile women, a negative urine pregnancy test side-effects were more frequent in the former group. was mandatory. Exclusion criteria were secondary causes of mem- branous nephropathy (autoimmune or infectious diseases, neo- METHODS plasms, etc.), HIV infection, liver disease, treatment with another Study design investigational drug, suspected or known hypersensitivity or allergy This multicenter, prospective, open-label, randomized, controlled reaction to drugs from study, previous treatment with corticosteroids trial with a 2-group parallel design (Figure 4) was designed by the (3 months before screening), other immunosuppressive agent (6 principal investigators and conducted at 20 sites (19 in Spain, 1 in months before screening), rituximab, or any other biologic agent (2 the Netherlands). A complete list of study sites and investigators years before screening), nonresponse to previous immunosuppres- is provided in the Supplementary material. Members of the sants, other severe condition or abnormal laboratory test with a steering committee and other study committees for the trial are potential risk for the patient outcome, and current drug or alcohol listed in the Supplementary material. The study protocol was dependence. Full eligibility criteria as well as inclusion and exclusion previously published.40 A data and safety monitoring board criteria are provided in Supplementary Methods. ensured the safety of the participants and the integrity of the trial. Appropriately authorized ethics committees approved the trial at Interventions and follow-up all participating sites. The trial is registered at ClinicalTrials.gov: We used a random number–producing algorithm in central com- NCT01955187. puter systems for simple randomization, with an equal allocation ratio (1:1) to intervention with corticosteroid-cyclophosphamide or Patients tacrolimus-rituximab. The subject numbers were assigned sequen- Adult patients ($18 years old) with biopsy-proven PMN were tially as each subject entered the study. eligible. All eligible patients were followed for an observational In the corticosteroid-cyclophosphamide group, patients received period of at least 6 months. Inclusion criteria were eGFR $45 ml/ methylprednisolone at months 1, 3, and 5 (1 g intravenously at days min per 1.73 m2, nephrotic-range proteinuria (>4 g/24 h, without a 1, 2, and 3, then 0.5 mg/kg/day orally from day 4 to day 30). At decrease of >50% during the observational period), and hypo- months 2, 4, and 6, patients received oral cyclophosphamide albuminemia (#3.5 g/dl during the observational period). All pa- adjusted for age and renal function (1.0–2.0 mg/kg/day for 30 days) tients received the standard of care (therapy with angiotensin- (Supplementary Methods). converting enzyme inhibitors/angiotensin receptor blockers for at In the tacrolimus-rituximab group, patients received oral tacro- least 2 months and controlled blood pressure (#150/90 mm Hg) for limus (0.05 mg/kg/day), to reach target blood levels of 5–7 ng/ml, for at least 3 months, with exceptions in case of intolerance, 6 months. At day 180, patients received intravenous rituximab (1 g) Kidney International (2021) 99, 986–998 995 clinical trial G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab and tacrolimus dosage was reduced by 25% per month, with com- categoric variables were analyzed with likelihood c2 or Fisher exact plete withdrawal at the end of month 9 (Supplementary Methods). test as appropriate. Tacrolimus dose was reduced in case of kidney function Longitudinal data, such as serum albumin, serum creatinine, impairment (Supplementary Methods). To minimize infusion re- eGFR, and other repeated measures, from randomization until actions with rituximab, patients received 100 mg methylpredniso- months 3, 6, 9, 12, 18, and 24 were analyzed with the use of lone, 1 g acetaminophen, and 50 mg diphenhydramine. Both groups multivariate linear mixed models. The models included time, received prophylaxis with 160/800 mg oral trimethoprim/sulfa- treatment, and their interaction as fixed effect and subject as random methoxazole 3 times a week during the treatment period. Patients effect, with unstructured covariance matrix. Proteinuria and anti- were switched to nonstudy interventions in case of lack of response, PLA2R levels were analyzed as median and interquartile range. and patients with a relapse of nephrotic syndrome were classified as Time-to-event analyses (time to remission, time to nephrotic “no response.” syndrome relapse) were performed with the use of Kaplan-Meier curves, log-rank test, and Cox proportional hazards regression models. Those patients who dropped out of the study without Outcomes and definitions reaching the primary outcome were censored. The primary outcome was complete or partial remission (composite To test the proportional hazards assumption, a time-dependent endpoint) at 24 months. Secondary end points included the rate of covariate was defined as an interaction of the time variable and complete and partial remission at 3, 6, 12, 18, and 24 months; relapse the covariate in question. The proportional hazards assumption was of nephrotic syndrome at 6, 12, 18, and 24 months; immunologic accepted as reasonable when the significance of the coefficient of the response at 3 to 24 months; and the percentage of patients free time dependent covariate was statistically significant. Baseline factors of $50% increases of serum creatinine and with preserved kidney associated with major outcomes were determined with the use of a function (eGFR $45 ml/min per 1.73 m2) at 24 months; and adverse Cox proportional hazards regression model. The magnitude of as- events. Safety end points were the proportion of patients with drug- sociation was reported as the hazard ratio with 95% CI. related adverse events during the study and total cumulative dose A detailed description of the statistical methods is provided in received of each study treatment. Supplementary Methods. All statistical analyses were performed by Complete remission was defined as a reduction of proteinuria statisticians blinded for treatment groups and using Stata 15 and from baseline to a value #0.3 g/24 h plus stable kidney function SPSS 25. (eGFR $45 ml/min per 1.73 m2); partial remission as a reduction of proteinuria >50% from baseline; and a value <3.5 g/24 h plus stable renal function (eGFR $45 ml/min per 1.73 m2). No response was APPENDIX defined as a proteinuria reduction of <50% from baseline values. STARMEN Investigators Relapses were defined as a reappearance of proteinuria >3.5 g/24 Spain. Hospital Universitario 12 Octubre, Madrid: Manuel Praga, Angel h and at least $50% increase from the lowest value in 3 or more Sevillano, Fernando Caravaca-Fontan, Hernando Trujillo, Eduardo Gutiérrez; consecutive visits in patients with previous partial or complete Hospital Universitario Fundación Alcorcón, Madrid: Gema Fernandez Juarez, remission. Patients were considered to be positive for anti-PLA2R Amir Shabaka; Hospital Universitario Fundacion Jiménez Díaz, Madrid: Jorge Rojas-Rivera, Jesus Egido, Alberto Ortiz; Hospital General Universitario Gregorio when baseline serum levels were >14 RU/ml as measured with a Marañón, Madrid: Marian Goicoechea, Úrsula Verdalles; Hospital Universitari standardized commercial enzyme-linked immunosorbent assay41 Vall d ’Hebron, Barcelona: Alfons Segarra, Irene Agraz; Hospital Regional Uni- (Euroimmune, Lubeck, Germany). Immunologic response was versitario de Málaga: Guillermo Martín-Reyes, Lara Perea; Hospital Virgen de la defined by a level of anti-PLA2R #14 RU/ml. eGFR was calculated Victoria de Málaga: Ildefonso Valera, Mónica Martín; Hospital Virgen del Rocío, with the Chronic Kidney Disease Epidemiology Collaboration Sevilla: Virginia Cabello, Miguel Angel Pérez Valdivia; Hospital Clinic, Barcelona: Luis F. Quintana, Miquel Blasco; Hospital Universitario de A Coruña, A Coruña: equation. Mercedes Cao, Andrés López Muñiz; Hospital Dr. Peset, Valencia: Ana Avila, Tamara Malek; Hospital Reina Sofía, Córdoba: Mario Espinosa, Cristina Rabasco; Fundación Puigvert, Barcelona: Montserrat Diaz, Iara DaSilva; Hospital Germans Statistical analysis Trias i Pujol, Barcelona: Jordi Bonet, Maruja Navarro; Hospital San Pedro de Based on the results of previous studies, we hypothesized a remission Alcántara, Cáceres: Juan Ramón Gómez-Martino; Hospital Universitario La Paz, rate of 60% at 2 years for the corticosteroid-cyclophosphamide Madrid: Begoña Rivas; Hospital Clínico San Carlos, Madrid: Antolina Rodríguez- group and 85% for the tacrolimus-rituximab group. We planned Moreno; Hospital Universitario Ramón y Cajal, Madrid: Cristina Galeano; Hos- pital de Ciudad Real, Ciudad Real: Ana Romera; Hospital Universitario Puerta de to include 94 patients, assuming a statistical power of 80%. Hierro, Madrid: Ana Huerta; Hospital Universitario de Getafe: Ezequiel Rodrí- Primary outcome (complete/partial remission at 24 months), was guez-Paternina; Hospital Universitario Severo Ochoa, Leganes: Ana Vigil; Hos- analyzed by intention-to-treat and per-protocol analyses estimating pital Universitario Infanta Leonor, Madrid: Roberto Alcázar; Hospital del the RR with 95% CI and compared with Pearson c2 or Fisher exact Henares: Vicente Paraíso; Hospital Universitario Infanta Sofía, Madrid: Vicente test. Hazards for complete or partial remission at 3, 6, 12, and 18 Barrio. months were also estimated for the evaluation of secondary objec- The Netherlands. Radboud University Medical Center, Nijmegen: Jack Wetzels, Anne-Els van de Logt, Julia Hofstra. tives. Subgroup analyses of the primary outcome were undertaken to determine whether the difference between treatments varied ac- cording to subgroups of baseline characteristics: sex, age, albumin, DISCLOSURE proteinuria, creatinine, eGFR, and anti-PLA2R. Risk ratios and 2- GL has received royalty fees from Euroimmune. MP has received consultancy side interaction P values were calculated with the use of multivar- and speaker’s fees from Otsuka, Alexion, Fresenius, and Retrophin and research iate modified Poisson regression models (Poisson regression with grants from Alexion. All the other authors declared no competing interests. robust error variance) For secondary outcomes, differences between the 2 groups in DATA SHARING continuous variables were analyzed with unpaired Student t test or Data from the STARMEN trial, including patient-level data, can be made Wilcoxon rank sum test, as appropriate. Differences between available on request from established research groups with an appropriate 996 Kidney International (2021) 99, 986–998 G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab clinical trial data-sharing agreement. Please contact the corresponding author for data 5. Polanco N, Gutiérrez E, Covarsí A, et al. Spontaneous remission of sharing. nephrotic syndrome in idiopathic membranous nephropathy. J Am Soc Nephrol. 2010;21:697–704. 6. Waldman M, Austin HA. Treatment of idiopathic membranous ACKNOWLEDGMENTS nephropathy. J Am Soc Nephrol. 2012;23:1617–1630. Work in this study was supported by the Instituto de Salud Carlos III/Fondo 7. Hofstra JM, Fervenza FC, Wetzels JFM. Treatment of idiopathic Europeo de Desarrollo Regional (ISCIII/FEDER) grants PI13/02495 and ICI14/ membranous nephropathy. Nat Rev Nephrol. 2013;9:443–458. 00350, Red de Investigación Renal (RedInRen) (RD12/0021/0029), European 8. Kidney Disease: Improving Global Outcomes (KDIGO) Glomerulonephritis Renal Association–European Dialysis and Transplant Association (ERA-EDTA), Work Group. KDIGO clinical practice guidelines for glomerulonephritis. Fundación Renal Iñigo Álvarez de Toledo (FRIAT), and Fundación para la Kidney Int Suppl. 2012;2, 139–27. Investigación Biomédica Hospital 12 de Octubre (iþ12). Funding agencies had 9. Ponticelli C, Zucchelli P, Passerini P, et al. A randomized trial of no participation in any of the phases of the study. GL received grants from methylprednisolone and chlorambucil in idiopathic membranous Centre National de la Recherche Scientifique, Fondation Maladies Rares (LAM- nephropathy. N Engl J Med. 1989;320:8–13. RD_20170304), National Research Agency (ANR, grants MNaims ANR-17-CE17- 10. Ponticelli C, Zucchelli P, Passerini P, et al. A 10-year follow-up of a 0012-01), “Investments for the Future” Laboratory of Excellence SIGNALIFE, a randomized study with methylprednisolone and chlorambucil in network for innovation on signal transduction pathways in life sciences (ANR- membranous nephropathy. Kidney Int. 1995;48:1600–1604. 11-LABX-0028-01) with allocated PhD fellowships for JJ, Initiative of Excellence 11. Ponticelli C, Altieri P, Scolari F, et al. A randomized study comparing (IDEX; UCAJedi ANR-15-IDEX-01), and Fondation pour la Recherche Médicale methylprednisolone plus chlorambucil versus methylprednisolone plus (FRM, ING20140129210, DEQ20180339193, and FDT201805005509 [to GL and cyclophosphamide in idiopathic membranous nephropathy. J Am Soc JJ]). Nephrol. 1998;9:444–450. We thank the trial staff at all participating sites and the patients who 12. Jha V, Ganguli A, Saha TK, et al. A randomized, controlled trial of steroids and participated in the trial. cyclophosphamide in adults with nephrotic syndrome caused by idiopathic membranous nephropathy. J Am Soc Nephrol. 2007;18:1899–1904. 13. Cattran DC, Appel GB, Hebert LA, et al. Cyclosporine in patients with AUTHOR CONTRIBUTIONS steroid-resistant membranous nephropathy: a randomized trial. Kidney GF-J, JR-R, GL, PR, JW, and MP drafted the manuscript. GF-J, JR-R, ASevillano, Int. 2001;59:1484–1490. FC-F, AA, CR, VC, AV, MD, GM-R, MGD, LFQ, IA, JRG-M, MC, AR-M, BR, CG, JB, AR, 14. Praga M, Barrio V, Juárez GF, Luño J. Tacrolimus monotherapy in AShabaka, EP, ME, JE, ASegarra, PR, JW, and MP participated in data collection. membranous nephropathy: a randomized controlled trial. Kidney Int. FC-F performed the statistical analysis and contributed to the writing of the 2007;71:924–930. manuscript. All authors agreed on the content of the manuscript, reviewed 15. Segarra A, Praga M, Ramos N, et al. Successful treatment of membranous drafts, and approved the final version. glomerulonephritis with rituximab in calcineurin inhibitor-dependent patients. Clin J Am Soc Nephrol. 2009;4:1083–1088. 16. Waldman M, Beck LH Jr, Braun M, et al. Membranous nephropathy: pilot SUPPLEMENTARY MATERIAL study of a novel regimen combining cyclosporine and Rituximab. Kidney Int Rep. 2016;1:73–84. Supplementary File (PDF) 17. Ruggenenti P, Cravedi P, Chianca A, et al. Rituximab in idiopathic Investigators and Committee Members. membranous nephropathy. J Am Soc Nephrol. 2012;23:1416–1425. Supplementary Methods. 18. Cravedi P, Ruggenenti P, Sghirlanzoni MC, Remuzzi G. Titrating rituximab Table S1. Sensitivity analyses according to anti-PLA2R positivity. to circulating B cells to optimize lymphocytolytic therapy in idiopathic Table S2. Sensitivity analyses in patients with or without anti-PLA2R membranous nephropathy. Clin J Am Soc Nephrol. 2007;2:932–937. determination at baseline. 19. Dahan K, Debiec H, Plaisier E, et al. Rituximab for severe membranous Table S3. Mean doses and mean blood levels of tacrolimus in the nephropathy: a 6-month trial with extended follow-up. J Am Soc Nephrol. 2017;28:348–358. tacrolimus-rituximab group. 20. Fervenza FC, Appel GB, Barbour SJ, et al. Rituximab or cyclosporine in the Table S4. Baseline characteristics of patients who achieved complete treatment of membranous nephropathy. N Engl J Med. 2019;381:36–46. or partial remission at any time of the study and nonresponder 21. Seitz-Polski B, Dahan K, Debiec H, et al. High-dose rituximab and early patients. remission in pla2r1-related membranous nephropathy. Clin J Am Soc Table S5. Proteinuria and serum albumin by group and time from Nephrol. 2019;14:1173–1182. randomization. 22. Floege J, Barbour SJ, Cattran DC, et al. Management and treatment of glomerular diseases (part 1): conclusions from a Kidney Disease: Table S6. Serum creatinine and eGFR by group and time from Improving Global Outcomes (KDIGO) Controversies Conference. Kidney randomization. Int. 2019;95:268–280. Table S7. Evolution of anti-PLA2R and development of immunologic 23. Faul C, Donnelly M, Merscher-Gomez S, et al. The actin cytoskeleton of response in nonresponder patients. kidney podocytes is a direct target of the antiproteinuric effect of Table S8. Evolution of proteinuria and anti-PLA2R in patients who cyclosporine A. Nat Med. 2008;14:931–938. presented a relapse. 24. Ramachandran R, Kumar A, Kumar V, et al. Two-year follow-up study of membranous nephropathy treated with tacrolimus and corticosteroids Figure S1. Subgroup analyses of the primary composite outcome versus cyclical corticosteroids and cyclophosphamide. Kidney Int Rep. (complete/partial remission) at 24 months by non-prespecified char- 2017;2:610–616. acteristics of patients at baseline. 25. Iijima K, Sako M, Nozu K, et al. Rituximab for childhood-onset, CONSORT Checklist. complicated, frequently relapsing nephrotic syndrome or steroid- dependent nephrotic syndrome: a multicentre, double-blind, randomised, placebo-controlled trial. Lancet. 2014;384:1273–1281. REFERENCES 26. Ruggenenti P, Ruggiero B, Cravedi P, et al. Rituximab in steroid- 1. Couser W. Primary membranous nephropathy. Clin J Am Soc Nephrol. dependent or frequently relapsing idiopathic nephrotic syndrome. J Am 2017;12:983–997. Soc Nephrol. 2014;25:850–863. 2. Beck LH Jr, Bonegio RG, Lambeau G, et al. M-Type phospholipase A2 27. van de Logt AE, Fresquet M, Wetzels JF, Brenchley P. The anti-PLA2R receptor as target antigen in idiopathic membranous nephropathy. antibody in membranous nephropathy: what we know and what N Engl J Med. 2009;361:11–21. remains a decade after its discovery. Kidney Int. 2019;96:1292–1302. 3. Ronco P, Debiec H. Molecular pathogenesis of membranous 28. de Vriese AS, Glassock RJ, Nath KA, Sethi S, Fervenza FC. A proposal for a nephropathy. Annu Rev Pathol. 2020;15:287–313. serology-based approach to membranous nephropathy. J Am Soc 4. Tomas NM, Beck LH Jr, Meyer-Schwesinger C, et al. Thrombospondin Nephrol. 2017;28:421–430. type-1 domain–containing 7A in idiopathic membranous nephropathy. 29. Hoxha E, Thiele I, Zahner G, Panzer U, Harendza S, Stahl RA. N Engl J Med. 2014;371:2277–2287. Phospholipase A2 receptor autoantibodies and clinical outcome in Kidney International (2021) 99, 986–998 997 clinical trial G Fernández-Juárez et al.: Corticosteroids-cyclophosphamide versus tacrolimus-rituximab patients with primary membranous nephropathy. J Am Soc Nephrol. 37. Eriguchi M, Oka H, Mizobuchi T, et al. Long-term outcomes of idiopathic 2014;25:1357–1366. membranous nephropathy in Japanese patients treated with low-dose 30. Ruggenenti P, Debiec H, Ruggiero B, et al. Anti–phospholipase a2 cyclophosphamide and prednisolone. Nephrol Dial Transplant. 2009;24: receptor antibody titer predicts post-rituximab outcome of membranous 3082–3088. nephropathy. J Am Soc Nephrol. 2015;26:2545–2558. 38. Mathrani V, Alejmi A, Griffin S, Roberts G. Intravenous 31. Glassock RJ. Antiphospholipase A2 receptor autoantibody guided cyclophosphamide and oral prednisolone is a safe and effective diagnosis and treatment of membranous nephropathy: a new treatment option for idiopathic membranous nephropathy. Clin Kidney personalized medical approach. Clin J Am Soc Nephrol. 2014;9:1341–1343. J. 2017;10:450–454. 32. van de Logt AE, Dahan K, Rousseau A, et al. Immunological remission in 39. Kanigicherla DAK, Hamilton P, Czapla K, Brenchley PE. Intravenous pulse PLA2R-antibody–associated membranous nephropathy: cyclophosphamide and steroids induce immunological and clinical cyclophosphamide versus rituximab. Kidney Int. 2018;93:1016–1017. remission in new-incident and relapsing primary membranous 33. Howman A1, Chapman TL, Langdon MM, et al. Immunosuppression for nephropathy. Nephrology (Carlton). 2018;23:60–68. progressive membranous nephropathy: a UK randomised controlled 40. Rojas-Rivera J, Fernández-Juárez G, Ortiz A, et al. A European trial. Lancet. 2013;381:744–751. multicentre and open-label controlled randomized trial to evaluate 34. Oleinika K, Mauri C, Salama AD. Effector and regulatory B cells in the efficacy of sequential treatment with tacrolimus-rituximab versus immune- mediated kidney disease. Nat Rev Nephrol. 2019;15:11–26. steroids plus cyclophosphamide in patients with primary 35. van den Brand JA, van Dijk PR, Hofstra JM, Wetzels JF. Cancer risk after membranous nephropathy: the STARMEN study. Clin Kidney J. 2015;8: cyclophosphamide treatment in idiopathic membranous nephropathy. 503–510. Clin J Am Soc Nephrol. 2014;9:1066–1073. 41. Dahnrich C, Komorowski L, Probst C, et al. Development of a 36. van den Brand JA, Ruggenenti P, Chianca A, et al. Safety of rituximab standardized ELISA for the determination of autoantibodies against compared with steroids and cyclophosphamide for idiopathic human M-type phospholipase A2 receptor in primary membranous membranous nephropathy. J Am Soc Nephrol. 2017;28:2729–2737. nephropathy. Clin Chim Acta. 2013;421:213–218. 998 Kidney International (2021) 99, 986–998