Patients double-seropositive for ANCA and anti-GBM antibodies have varied renal survival, frequency of relapse, and outcomes compared to single-seropositive patients
Authors: Stephen P. McAdoo, Anisha Tanna, Zdenka Hrušková, Lisa Holm, Maria Weiner, Nishkantha Arulkumaran, Amy Kang, Veronika Satrapová, Jeremy Levy, Sophie Ohlsson, Vladimir Tesar, Mårten Segelmark, Charles D. Pusey
Journal: Kidney International (2017)

Co-presentation with both ANCA and anti-GBM antibodies is thought to be relatively rare. Current studies of such ‘double-positive’ cases report small numbers and variable outcomes. To study this further we retrospectively analyzed clinical features and long-term outcomes of a large cohort of 568 contemporary patients with ANCA-associated vasculitis, 41 patients with anti-GBM disease, and 37 double-positive patients with ANCA and anti-GBM disease from four European centers. Double-positive patients shared characteristics of ANCA-associated vasculitis (AAV), such as older age distribution and longer symptom duration before diagnosis, and features of anti-GBM disease, such as severe renal disease and high frequency of lung hemorrhage at presentation. Despite having more evidence of chronic injury on renal biopsy compared to patients with anti-GBM disease, double-positive patients had a greater tendency to recover from being dialysis-dependent after treatment and had intermediate long-term renal survival compared to the single-positive patients. However, overall patient survival was similar in all three groups. Predictors of poor patient survival included advanced age, severe renal failure, and lung hemorrhage at presentation. No single-positive anti-GBM patients experienced disease relapse, whereas approximately half of surviving patients with AAV and double-positive patients had recurrent disease during a median follow-up of 4.8 years. Thus, double-positive patients have a truly hybrid disease phenotype, requiring aggressive early treatment for anti-GBM disease, and careful long-term follow-up and consideration for maintenance immunosuppression for AAV. Since double-positivity appears common, further work is required to define the underlying mechanisms of this association and define optimum treatment strategies. Co-presentation with both ANCA and anti-GBM antibodies is thought to be relatively rare. Current studies of such ‘double-positive’ cases report small numbers and variable outcomes. To study this further we retrospectively analyzed clinical features and long-term outcomes of a large cohort of 568 contemporary patients with ANCA-associated vasculitis, 41 patients with anti-GBM disease, and 37 double-positive patients with ANCA and anti-GBM disease from four European centers. Double-positive patients shared characteristics of ANCA-associated vasculitis (AAV), such as older age distribution and longer symptom duration before diagnosis, and features of anti-GBM disease, such as severe renal disease and high frequency of lung hemorrhage at presentation. Despite having more evidence of chronic injury on renal biopsy compared to patients with anti-GBM disease, double-positive patients had a greater tendency to recover from being dialysis-dependent after treatment and had intermediate long-term renal survival compared to the single-positive patients. However, overall patient survival was similar in all three groups. Predictors of poor patient survival included advanced age, severe renal failure, and lung hemorrhage at presentation. No single-positive anti-GBM patients experienced disease relapse, whereas approximately half of surviving patients with AAV and double-positive patients had recurrent disease during a median follow-up of 4.8 years. Thus, double-positive patients have a truly hybrid disease phenotype, requiring aggressive early treatment for anti-GBM disease, and careful long-term follow-up and consideration for maintenance immunosuppression for AAV. Since double-positivity appears common, further work is required to define the underlying mechanisms of this association and define optimum treatment strategies. Anti-glomerular basement membrane (GBM) disease and the anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are rare conditions, with estimated incidences in Europe of 1 and 20 per million population per year, respectively.1Pusey C.D. Anti-glomerular basement membrane disease.Kidney Int. 2003; 64: 1535-1550Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 2Watts R.A. Mahr A. Mohammad A.J. et al.Classification, epidemiology and clinical subgrouping of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis.Nephrol Dial Transplant. 2015; 30: i14-i22Crossref PubMed Scopus (165) Google Scholar The concurrence of both ANCA and anti-GBM antibodies in individual patients, however, is well-recognized, and occurs at a much higher frequency than would be expected by chance alone. This phenomenon was first reported within a few years of the first description of ANCA in the 1980s,3O’Donoghue D.J. Short C.D. Brenchley P.E. et al.Sequential development of systemic vasculitis with anti-neutrophil cytoplasmic antibodies complicating anti-glomerular basement membrane disease.Clin Nephrol. 1989; 32: 251-255PubMed Google Scholar, 4Jayne D.R. Marshall P.D. Jones S.J. et al.Autoantibodies to GBM and neutrophil cytoplasm in rapidly progressive glomerulonephritis.Kidney Int. 1990; 37: 965-970Abstract Full Text PDF PubMed Scopus (234) Google Scholar and has been observed in several series from around the world over the subsequent 30 years.5Levy J.B. Hammad T. Coulthart A. et al.Clinical features and outcome of patients with both ANCA and anti-GBM antibodies.Kidney Int. 2004; 66: 1535-1540Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 6Rutgers A. 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Segal P.E. et al.Characteristics and outcome of crescentic glomerulonephritis in patients with both antineutrophil cytoplasmic antibody and anti-glomerular basement membrane antibody.Clin Rheumatol. 2013; 32: 1317-1322Crossref PubMed Scopus (33) Google Scholar, 15Alchi B. Griffiths M. Sivalingam M. et al.Predictors of renal and patient outcomes in anti-GBM disease: clinicopathologic analysis of a two-centre cohort.Nephrol Dial Transplant. 2015; 30: 814-821Crossref PubMed Scopus (69) Google Scholar, 16Cui Z. Zhao J. Jia X.Y. et al.Anti-glomerular basement membrane disease: outcomes of different therapeutic regimens in a large single-center Chinese cohort study.Medicine (Baltimore). 2011; 90: 303-311Crossref PubMed Scopus (88) Google Scholar These studies, however, have generally been limited by small size (many describing fewer than 20 cases) and variations in the severity of disease at presentation, with between 0% and 100% of patients being dependent on dialysis at diagnosis.8J DEZ Taylor D. Thein H. et al.Incidence and features of dual anti-GBM-positive and ANCA-positive patients.Nephrology. 2011; 16: 725-729Crossref PubMed Scopus (28) Google Scholar, 15Alchi B. Griffiths M. Sivalingam M. et al.Predictors of renal and patient outcomes in anti-GBM disease: clinicopathologic analysis of a two-centre cohort.Nephrol Dial Transplant. 2015; 30: 814-821Crossref PubMed Scopus (69) Google Scholar Furthermore, in the largest series to date, from Chinese centers, fewer than 25% of patients were treated with plasma exchange, and so the applicability of the findings to European patients treated with substantially different therapeutic regimens is limited.7Zhao J. Yang R. Cui Z. et al.Characteristics and outcome of Chinese patients with both antineutrophil cytoplasmic antibody and antiglomerular basement membrane antibodies.Nephron Clin Pract. 2007; 107: c56-c62Crossref PubMed Scopus (27) Google Scholar, 16Cui Z. Zhao J. Jia X.Y. et al.Anti-glomerular basement membrane disease: outcomes of different therapeutic regimens in a large single-center Chinese cohort study.Medicine (Baltimore). 2011; 90: 303-311Crossref PubMed Scopus (88) Google Scholar The aim of the present study is to describe the clinical features and long-term outcomes of a contemporary cohort of patients with double-positive ANCA and anti-GBM disease. Given the rarity of these patients, we have identified cases from 4 large Northern European nephrology centers, which employ comparable treatment protocols for these cases, including plasma exchange, cyclophosphamide, and steroids, unless contraindicated. We have compared clinical features and outcomes to those for single-positive AAV and single-positive anti-GBM disease. Because patients with double-positive disease more closely resemble those with single-positive anti-GBM disease at presentation, we have also compared histopathology and treatment in these 2 groups. Between 2000 and 2013, a total of 646 cases were identified at 4 centers in 3 countries, including 568 patients with single-positive AAV, 41 with single-positive anti-GBM disease, and 37 patients who were double positive for anti-GBM antibodies and ANCA (hereafter AAV, anti-GBM, and double-positive groups, respectively) (Table 1). The ratio of double-positive to single-positive anti-GBM cases was similar in all 3 countries (47% overall); however, patients who were double positive represented a variable proportion of the AAV cases (3% to 10.5%; 6.1% overall). The demographic features of the cohort are summarized in Table 1. The single-positive anti-GBM group demonstrated the typical bimodal age distribution of this disease, whereas patients who were double positive had an age distribution similar to patients with isolated AAV (Figure 1). There was no significant difference in gender ratio between the 3 groups. Notably, 1 patient in the double-positive group had a previous diagnosis of isolated anti-myeloperoxidase (MPO) and AAV 2 years prior to presenting with double-positive disease.Table 1Case identification, demographics, clinical features, and serologyAAVAnti-GBMDouble positiveP valueAAVversusDPversusGBMAAVversusDPGBMversusDPAAVversusGBMCases, n5684137––––•United Kingdom1711920•Sweden100138•Czech Republic29799Cases, %87.9%6.3%5.7%DemographicsAge, yr (range)62.3 (11–95)58.3 (13–91)63.6 (17–88)0.170.990.310.21Gender•Male54%46%38%0.110.060.490.34•Female46%54%62%Clinical FeaturesDuration of symptoms,aCalculated for a sample of 48 ANCA cases. wk (range)12 (0–56)2 (0–20)10 (1–26)<0.010.99<0.01<0.01Lunghemorrhage131/56823%16/4140%14/3738%0.010.040.850.02Required RRT atpresentation132/56823%26/4163%21/3757%<0.01<0.010.55<0.01eGFR,bCensored for patients on RRT. ml/min (range)29 (5–90)20 (5–90)19 (6–76)0.060.110.990.67Serum creatinine,bCensored for patients on RRT. μmol/l (range)186 (39–693)275 (62–667)309 (71–606)0.060.180.990.37SerologyAnti-GBM level, xULN (range)–5.4 (1–29.1)14.2 (1–50.4)–0.06–Proportion seronegative for anti-GBM, %–4/4111%4/3711%–1.00–ANCA serology, %<0.01––•Anti-MPO48%70%•Anti-PR351%27%•Anti-MPO & PR3<1% (n = 2)3%AAV, anti-neutrophil cytoplasm antibody–associated vasculitis; DP, double-positive; eGFR, estimated glomerular filtration rate; GBM, glomerular basement membrane; MPO, myeloperoxidase; PR3, proteinase 3; RRT, renal replacement therapy; xULN, multiples of upper limit of normal.Results expressed as median ± range. Comparison between groups by Kruskall–Wallis test with Dunn’s post-test to ascertain differences between individual groups (for continuous data), or by chi-square test (for categorical data).a Calculated for a sample of 48 ANCA cases.b Censored for patients on RRT. Open table in a new tab AAV, anti-neutrophil cytoplasm antibody–associated vasculitis; DP, double-positive; eGFR, estimated glomerular filtration rate; GBM, glomerular basement membrane; MPO, myeloperoxidase; PR3, proteinase 3; RRT, renal replacement therapy; xULN, multiples of upper limit of normal. Results expressed as median ± range. Comparison between groups by Kruskall–Wallis test with Dunn’s post-test to ascertain differences between individual groups (for continuous data), or by chi-square test (for categorical data). Table 1 summarizes key clinical features and serological findings at presentation. The duration of symptoms prior to receiving a diagnosis was similar in the AAV and double-positive groups (median: 10–12 weeks), and this was significantly longer than in single-positive anti-GBM patients (2 weeks; P < 0.01). Despite shorter duration of symptoms, the severity of disease—whether defined as need for hemodialysis or, in patients who were not dialysis-dependent, by GFR estimations or serum creatinine measurements—in anti-GBM patients was similar to that of patients who were double positive. The frequency of alveolar hemorrhage was similar in anti-GBM and double-positive groups, occurring in about one-third of patients. Severe disease manifestations (dialysis requirement and lung hemorrhage) were less common in patients with AAV, each occurring in approximately one-quarter of cases. Patients who were double positive had additional extrarenal manifestations, including nonhemorrhage lower respiratory tract disease (in 26%), otorhinolaryngological involvement (18%), musculoskeletal symptoms (18%), cutaneous features (13%), neurological (8%), gastrointestinal (5%), and ocular symptoms (3%). Because a variety of anti-GBM assays were used during the study period and at different hospital sites, results were standardized by expressing them as multiples of the upper limit of normal (xULN) for each assay. Patients who were double positive tended to have lower levels of circulating anti-GBM antibodies than did anti-GBM patients who were single positive, although the difference between groups was not statistically significant. A similar proportion of patients in both these groups (approximately 10%) were seronegative for circulating anti-GBM antibodies. In these cases, there was convincing evidence of linear IgG deposition on renal biopsy, in the absence of another attributable cause, in keeping with our definition of anti-GBM disease. In the AAV group, the proportion of patients who were positive for anti-proteinase 3 (PR3) versus anti-MPO antibody was approximately equal. However, in the double-positive groups there was a comparative over-representation of patients with anti-MPO antibodies (70% vs. 48%, P < 0.01). One patient in the double-positive group was triple positive for anti-MPO, anti-PR3, and anti-GBM antibodies. Notably, this patient had a history of recreational drug use and was positive for hepatitis C virus. Because a variety of methodologies, including indirect immunofluorescence and antigen-specific assays, were used to confirm ANCA positivity over the period of the study, it was not possible to standardize comparisons of ANCA titer. Because the anti-GBM and double-positive groups had similar disease severity at presentation, we performed more detailed analysis to compare histopathological and therapeutic differences in these 2 cohorts. Approximately two-thirds of patients in both groups underwent renal biopsy, as described in Table 2. The severity of renal disease in those who underwent biopsy was similar in both groups and equivalent to the severity of renal disease in the parent cohort, suggesting that biopsy findings may be representative and comparable between groups. It was not possible, however, to retrospectively identify the reasons why renal biopsy was not undertaken in all remaining patients. In some cases, this was due to the need for immediate treatment with plasma exchange, and others were considered too clinically unstable for biopsy. In keeping with the age difference between the parent groups, the mean age in the subset of patients who underwent biopsy was lower in the anti-GBM group compared with the double-positive group.Table 2HistopathologyAnti-GBMDouble positiveP valueUnderwent biopsy, n (%)29 (71%)25 (68%)0.81Mean age at biopsy, yr (range)46 (13–91)62 (46–76)<0.01Renal status at biopsy•Required RRT52%54%1.00•eGFR,aCensored for patients on RRT. ml/min (range)21 (5–90)16 (8–73)0.78•Serum creatinineaCensored for patients on RRT. μmol/l (range)275 (62–677)315 (71–606)0.75Glomerular findings•Crescentic glomeruli, %64% (0–100)64% (25–100)0.98•Sclerotic glomeruli, %0% (0–80)15% (0–100)0.19•Normal glomeruli, %5% (0–100)0% (0–67)0.56Tubular atrophy, % (range)5% (0%–30%)27% (0%–80%)<0.01Immunofluorescence pattern0.69•Linear IgG79%80%•Pauci-immune3%8%•Technically inadequate17%12%eGFR, estimated glomerular filtration rate; GBM, glomerular basement membrane; RRT, renal replacement therapy.Results expressed as median ± range. Comparison between groups by Mann-Whitney test (for continuous data), or by chi-square test (for categorical data).a Censored for patients on RRT. Open table in a new tab eGFR, estimated glomerular filtration rate; GBM, glomerular basement membrane; RRT, renal replacement therapy. Results expressed as median ± range. Comparison between groups by Mann-Whitney test (for continuous data), or by chi-square test (for categorical data). The median number of glomeruli in each biopsy was 15 (interquartile range: 10–20). There was no difference in the proportion of crescentic glomeruli between the 2 groups. There was, however, a tendency for more sclerotic glomeruli to be observed in patients who were double positive (median: 15% vs. 0%; P = 0.188). Likewise, the finding of “synchronous” crescent formation tended to be more commonly observed in patients with anti-GBM disease (73% vs. 33% in patients who were double positive; P = 0.092). There was a highly significant difference in the degree of interstitial fibrosis and tubular atrophy between these 2 groups, with more evidence of chronic damage in double-positive cases (median: 27% vs. 5%, P < 0.001). In those cases in which adequate tissue was available for analysis, all but 3 cases (2 double-positive, 1 single-positive for anti-GBM, and all with circulating anti-GBM antibodies) had linear deposition of IgG by immunofluorescence analysis of the kidney biopsy. There was no detectable difference between single-positive anti-GBM and double-positive groups with regard to initial treatment administered, the majority receiving standard of care with steroids (97% vs. 100% in double-positive and anti-GBM cases, respectively; P = 0.47), cyclophosphamide (100% vs. 92%; P = 0.24), and plasma exchange (80% vs. 89%; P = 0.33). In total, 10 patients did not undergo plasma exchange for various reasons. In the double-positive group, 7 patients did not receive plasma exchange. Of these, 2 were dialysis dependent at presentation with 100% crescent formation on kidney biopsy, and in the absence of lung hemorrhage, plasma exchange was deemed futile. These patients received cytotoxic therapy and steroids for nonrenal manifestations. Of the other 5 patients, none had alveolar hemorrhage or required dialysis, and their initial treatment was typical of those presenting with isolated AAV. In the single-positive anti-GBM group, 1 patient was dialysis dependent with no normal glomeruli on kidney biopsy, and so treatment was deemed futile; 1 patient had well-preserved renal function (serum creatinine: 78 μmol/l) and so plasma exchange was initially reserved for nonresponse to immunosuppressive treatment alone, and was ultimately not required; and 1 patient was clinically unstable and therefore unable to undergo plasma exchange. At 6 months, 74% of patients who were double positive were receiving ongoing immunosuppressive treatment with or without corticosteroids (71% with azathioprine, 21% with mycophenolate mofetil, and 8% with methotrexate), whereas only 14% of patients with single-positive anti-GBM disease received ongoing therapy (P < 0.001), of whom 80% received azathioprine and 20% received MMF. Patient and renal survival for all 3 cohorts at 3 and 12 months is summarized in Table 3. Overall patient survival was similar in all groups at both time points. Renal survival was favorable in the AAV group at both time points, although there was no significant difference in the proportion of patients who required dialysis in the anti-GBM and double-positive group at either time point. The proportion of patients who presented with dialysis-dependent renal failure and who recovered renal function and were alive at 1 year was significantly different between groups, varying from 17% in patients with single-positive anti-GBM disease to 29% in patients who were double positive and 49% in AAV cases. As Figure 2 demonstrates, this was due in part to cross-over from dialysis dependence to independence, and vice versa, particularly within the double-positive group, in which a substantial proportion recovered renal function in the first 3 months of follow-up (35% recovery vs. 10% recovery in patients who were single positive; P = 0.11). There was no significant difference in age (mean: 65 vs. 64 years; P = 0.88) or receipt of plasma exchange (100% vs. 82%; P = 0.52) between those patients and who were double-positive and who recovered and those who did not, respectively, although those who recovered renal function tended to have lower levels of anti-GBM antibodies (3.8 vs. 10 xULN, respectively; P = 0.07). Only one-half of the patients who were double-positive who recovered renal function underwent renal biopsy, and so it was not possible to reliably identify histopathological predictors of treatment response.Table 3Patient and renal survival at 3 and 12 months after diagnosisDiagnosis0 Months3 months12 monthsRenal recovery at 1 yearbProportion of patients requiring RRT at presentation who were alive with independent renal function at 1 year.Independent of RRTPatient survivalRenal survivalaCensored for death.Patient survivalRenal survivalaCensored for death.AAV437/56877%540/56895%490/54091%512/56890%452/51288%64/13149%Anti-GBM15/4137%37/4190%15/3642%36/4187%15/3444%4/2417%Double positive16/3743%33/3789%16/3250%31/3783%16/3053%6/2129%P value<0.010.13<0.010.38<0.01<0.01AAV, anti-neutrophil cytoplasm antibody–associated vasculitis; GBM, glomerular basement membrane; RRT, renal replacement therapy.Comparison between groups by chi-square test.a Censored for death.b Proportion of patients requiring RRT at presentation who were alive with independent renal function at 1 year. Open table in a new tab AAV, anti-neutrophil cytoplasm antibody–associated vasculitis; GBM, glomerular basement membrane; RRT, renal replacement therapy. Comparison between groups by chi-square test. The median duration of follow-up was 4.8 years (range: 0–15 years). Long-term patient and renal survival is summarized in Figure 3a and b, respectively. No difference in unadjusted overall patient survival was observed during the study (P = 0.49). Renal survival was favorable in the AAV group compared with both the anti-GBM group (P < 0.01) and the double double-positive groups (P < 0.01). Patients who were double positive tended to have better renal survival than those who were single positive with anti-GBM disease, although this difference was not statistically significant in unadjusted analysis (P = 0.17). Relapse data were available in 316 AAV patients and all patients with anti-GBM disease and double positivity until last follow-up. Within the follow-up period, 116 of 316 patients with AAV (37%) and 8 of 37 patients who were double positive (22%) had a relapse. Two patients with single-positive anti-GBM disease had early recrudescence of anti-GBM antibodies requiring augmented treatment in the first 6 months following initial diagnosis, which we believe reflected inadequate initial disease control rather than disease relapse. No patients with single-positive anti-GBM disease exhibited evidence of relapse or recurrent antibodies beyond 6 months. During long-term follow-up, a significant proportion (n = 8; approximately 50%) of the surviving patients who were double positive developed recurrent disease (Figure 3c). These relapses tended to occur late (median time to first relapse: 4.4 years; range: 1.1–7.9 years), and the majority (5 of 8) were in patients with ANCA directed against PR3, in keeping with the natural history of isolated AAV. Two patients had relapses associated with MPO-ANCA, and 1 notable patient had a relapse of both MPO-ANCA and anti-GBM antibodies. All relapses occurred in patient who were ANCA positive, and the majority (6 of 8) were associated with a rise in ANCA titer of more than 25%, or seroconversion from ANCA-negative to ANCA-positive status, in the 6 months prior to diagnosis of relapsing disease. Table 4 summarizes the clinical features of each relapse and its relation to immunosuppressive treatment. Notably, the majority of patients were not receiving maintenance treatment other than corticosteroids at the time of relapse. As shown in Figure 3c, the frequency of relapse in the double-positive cohort was comparable to that in AAV cases (P = 0.29), whereas there were no relapses in the anti-GBM group (P < 0.01). Two patients who were double positive have undergone renal transplantation. To date, no disease recurrence related to either ANCA or anti-GBM disease has been described in the renal allografts.Table 4Details of double-positive patients with relapseCaseTime to relapse (months)ANCAGBM AbOrgan involvementTreatment at time of relapseTreatment for relapseSubsequent relapse113PR3NegRenal, skinCS only;CYC stopped 9 mo priorCYC, CSYes216PR3NegLRTCS only;AZA stopped 4 mo priorAZA, CSNo322MPONegLRTCS only;AZA stopped 18 mo priorCSYes436PR3NegRenal, SkinMMF, CSRTX, CYC, CSYes571PR3NegRenalAZAAZA, CSNo681PR3NegConstitutionalCS only;AZA stopped 10 mo priorAZA, CSYes787MPONegRenalNone;off treatment 5 yrCSNo895MPOPositiveLRT, RenalNone;off treatment 12 moRTX, CYC, CSNoANCA, anti-neutrophil cytoplasm antibody; AZA, azathioprine; CS, corticosteroids; CYC, cyclophosphamide; GBM, glomerular basement membrane; LRT, lower respiratory tract; MPO, myeloperoxidase; MMF, mycophenolate mofetil; mo, months; PR3, proteinase 3; RTX, rituximab; y, years. Open table in a new tab ANCA, anti-neutrophil cytoplasm antibody; AZA, azathioprine; CS, corticosteroids; CYC, cyclophosphamide; GBM, glomerular basement membrane; LRT, lower respiratory tract; MPO, myeloperoxidase; MMF, mycophenolate mofetil; mo, months; PR3, proteinase 3; RTX, rituximab; y, years. There were significant differences in age, proportion of patients requiring renal replacement therapy (RRT), and proportion of patients with lung hemorrhage at presentation between patients diagnosed with AAV, anti-GBM disease, and double positivity. We therefore performed regression analysis to identify predictors of death and end-stage renal disease (ESRD) in all patients, correcting for these differences in baseline variables (Table 5; Figure 4).Table 5Predictors of death and end-stage renal diseaseUnadjusted analysisDeathESRDDeath or ESRDHR (CI)P valueHR (CI)P valueHR (CI)P valueDiagnosis–0.25–<0.01–<0.01 DPaReference group for estimates of hazard ratios. vers