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Eculizumab deposits in vessel walls in thrombotic microangiopathy
clinical investigation
www.kidney-international.org
Eculizumab deposits in vessel walls in thrombotic microangiopathy Clarissa A. Cassol1, Sergey V. Brodsky1, Anjali A. Satoskar1, Angela R. Blissett1, Spero Cataland2 and Tibor Nadasdy1 1
Department of Pathology, The Ohio State University, Columbus, Ohio, USA; and 2Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA
Terminal complement inhibition therapy with eculizumab (a humanized monoclonal antibody to C5) has revolutionized the treatment of patients with thrombotic microangiopathy (TMA). Successful responders are often placed on long-standing therapy to prevent disease recurrence in the native kidney or allograft. The tissue deposition of eculizumab in patients with C3 glomerulopathy has been described but no studies have yet investigated tissue deposition of eculizumab in cases where it was indicated for thrombotic microangiopathy which, unlike C3 glomerulopathy, does not usually show immune-type electron dense deposits. To evaluate this, we reviewed biopsies from 13 patients who received eculizumab for TMA treatment or prevention of recurrence. We found IgG2, IgG4, and kappa positivity within arterioles corresponding to eculizumab deposits, with similar distribution to C5b-9, in all but one patient. In that patient eculizumab therapy had been discontinued 24 months prior to biopsy. Deposits in arterioles could be seen as early as one day after infusion and after a single dose of eculizumab, and were detected up to 162 days after therapy discontinuation. This may play a role in controlling local complement activation-associated vascular changes in these patients. Thus, IgG subclass staining by immunofluorescence is important to avoid misdiagnoses of immune-complex or monoclonal immunoglobulin deposition disease in patients with TMA who received eculizumab. Kidney International (2019) j.kint.2019.05.008
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https://doi.org/10.1016/
KEYWORDS: eculizumab; IgG subclasses; thrombotic microangiopathy Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
Correspondence: Clarissa A. Cassol, The Ohio State University Wexner Medical Center, Department of Pathology, Renal Pathology Division, 320 W 10th Avenue, M018 Starling Loving Hall, Columbus, OH 43210, USA. E-mail: [email protected]; or Tibor Nadasdy, The Ohio State University Wexner Medical Center, Department of Pathology, Renal Pathology Division, 320 W 10th Avenue, M018 Starling Loving Hall, Columbus, OH 43210 USA. E-mail: [email protected] Received 7 December 2018; revised 30 April 2019; accepted 2 May 2019; published online 24 May 2019 Kidney International (2019) -, -–-
E
culizumab is a humanized murine recombinant monoclonal antibody engineered to minimize immunogenicity, proinflammatory effects, and complement activation while successfully inhibiting the terminal complement component C5 to prevent the generation of the membrane attack complex.1 To achieve these objectives, the antibody consists of a hybrid human IgG2 heavy chain hinge region, which does not bind Fc receptors, and the IgG4 heavy chain CH2 and CH3 regions, which lack the ability to activate the complement cascade, associated with a k light chain. This structure retains the high affinity gained from the variable regions derived from the murine antibody while at the same time ensuring low immunogenicity and few proinflammatory effects. Eculizumab was approved initially for the treatment of paroxysmal nocturnal hemoglobinuria in 2007.1,2 Since then, its use has been expanded to other complement-mediated disorders, including TMA, for which it was approved for clinical use in 2011.3 TMA is a devastating disease characterized by prominent microvascular injury. Historically, TMA has been divided into hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. HUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Meanwhile, patients with a diagnosis of thrombocytopenic purpura have deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) enzyme, and the nonimmune hemolytic anemia and thrombocytopenia primarily are associated with central nervous system symptoms rather than renal disease. In typical forms of HUS, renal disease follows hemorrhagic colitis and diarrhea, and most cases are a result of Shiga toxin–producing Escherichia coli HUS. In contrast, in atypical HUS (aHUS), the TMA usually is not associated with diarrhea, and microangiopathic hemolytic anemia may be absent.4–6 Complement alternative pathway dysregulation resulting from either mutations or acquired autoantibodies against C3-convertase regulatory factors is present in at least 50% of cases of aHUS,7 leading to endothelial injury through generation of the terminal complement components C5b-9 on endothelial cell surfaces. Eculizumab was shown to successfully prevent renal deterioration and reverse microangiopathic hemolytic anemia, when present, in most patients with complementmediated aHUS.3,8 In addition, eculizumab has shown 1
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promising results of “noncomplement”-mediated aHUS, including drug-induced TMA9; TMA after stem cell, renal, or other solid organ transplantation10; TMA associated with other glomerular diseases11–13; and even Shiga toxin– associated typical HUS.14 In the renal allograft setting, treatment with eculizumab was successful in preventing TMA recurrence.15 The tissue deposition of eculizumab (IgG2, IgG4, and k deposits) has been described in patients receiving this drug for C3 glomerulopathy, where it was seen within glomerular capillaries and mesangium, vessel walls, and tubular basement membranes.16 With the expanded use of eculizumab for treatment of different conditions that could benefit from terminal complement blockade,17 pathologists are expected to encounter more biopsy specimens from patients receiving this therapy. Given its low immunogenicity and slow tissue clearance,1 prolonged tissue deposition of the antibody is expected,16 which may generate confusing results (e.g., positive IgG k deposits in the absence of obvious immune complex or monoclonal Ig deposition), especially if IgG subclass staining is not performed or if the pathologist is not informed that the patient has received eculizumab. Increasingly in our practice we have encountered renal native or allograft biopsy specimens from patients who had received or were currently undergoing eculizumab therapy. This article describes the clinical characteristics and pathologic findings of a series of these cases encountered in our practice, highlighting the time course and length of tissue deposition after initiation and discontinuation of therapy. RESULTS
Since 2011, renal biopsy specimens from a total of 17 patients who underwent a renal biopsy during or after eculizumab therapy for the treatment of TMA or prevention of recurrence of TMA were reviewed at our institution (Table 1). Four specimens were excluded because of lack of frozen tissue for IgG subclass staining (these were consult cases for which frozen slides were not received for review). Of the remaining 13 cases, 10 patients were receiving eculizumab at the time of renal biopsy and 3 patients had received the therapy previously (Table 2). Seven biopsy specimens were from native kidneys and 6 were from renal allografts. In 3 renal allograft recipients, the cause of end-stage renal disease (ESRD) was listed as hypertension. TMA developed in these patients shortly after transplant, and therefore it is possible that the original cause of ESRD was in fact TMA; however, none of these patients had a native kidney biopsy to confirm this hypothesis. Pathologic diagnoses are summarized in Table 2. All patients showed at least mild arteriolar IgG2, IgG4, and k deposits by immunofluorescence (IF; Figure 1, Table 2) except patient 5, who showed no k, and patient 3, for whom eculizumab had been discontinued 695 days prior and who had no IgG2 and IgG4 arteriolar staining. Six of the 13 patients treated with eculizumab had morphologic findings of 2
CA Cassol et al.: Eculizumab deposits in TMA
ongoing active TMA (patients 1, 6, 8, and 12) or chronic TMA (patients 5 and 13) in the renal biopsy, and all of these biopsy specimens showed bright arteriolar deposits by IF (2þ or 3þ; Table 2). Even though TMA was noted to involve predominantly the glomeruli in patients 6 and 12, glomerular deposits were absent (patient 6) or mild and segmental (patient 12) in these cases (Table 2). Interestingly, those 2 cases showed the brightest (3þ) staining for IgG2 and IgG4 in arterioles. Biopsy specimens that did not display morphologic changes of ongoing TMA had similar vascular staining for IgG2, IgG4, and k. Glomerular positivity for IgG2, IgG4, and k also was noted in specimens for 8 of the 12 patients that showed arteriolar staining and was mostly mesangial, mild, and segmental, with the exception of one patient with possible concomitant C3 glomerulonephritis (C3GN), whose specimen showed moderate mesangial staining (patient 1). Four patients had pre-eculizumab treatment biopsy specimens, all of which showed negative staining for IgG2, IgG4, and k in arterioles and glomeruli as depicted in Supplementary Figure S1. Positive arteriolar deposits by IF were seen as early as 1 day after eculizumab infusion (patients 2 and 8) and after a single dose of the drug in many cases (patients 1, 4, 8, 9, and 10). Positive IgG2, IgG4, and k could still be detected in the arterioles of patients 5 and 6, 155 and 162 days after the last dose of eculizumab. Three cases (1, 9, and 10) showed additional focal positivity for IgG2, IgG4, and k along Bowman’s capsule and tubular basement membranes. To confirm that IgG2, IgG4, and k deposits corresponding to eculizumab were occurring at sites of complement activation, C5b-9 IF was performed and showed arteriolar positivity in all cases (Table 2, Figure 1). C5b-9 positivity also was observed in the mesangium, ranging from mild (1þ) to prominent (3þ), as well as in the tubular basement membranes and Bowman’s capsule (Figure 1). Tissue distribution and intensity of staining for C5b-9 showed no correlation with the presence or absence of active TMA. For cases in which pre- and post-eculizumab treatment biopsies were available, no significant difference in C5b-9 staining was observed. Electron microscopy was performed in 10 cases (3 cases were early post-transplant allograft biopsy specimens that did not undergo ultrastructural examination) and showed no electron-dense immune-type or finely granular punctate deposits in arteries or arterioles. Three cases showed a few glomerular deposits that were intramembranous with possible concomitant C3GN from patient 1 and mesangial in the biopsy specimen with concomitant class II lupus nephritis from patient 5. The biopsy specimen for patient 3, which showed advanced chronic changes and no arteriolar staining corresponding to eculizumab, also showed a few mesangial deposits. DISCUSSION
For the first time we have demonstrated tissue deposition of the humanized monoclonal antibody eculizumab in patients Kidney International (2019) -, -–-
Sex
Race
1
26
F
C
Preeclampsia
2
42
F
C
Second renal transplant 2 d prior for ESRD due to PKD; first allograft lost due to TMA
294
Negative
C3 187, C4 35, C2 12 (Y, consistent with heterozygous C2 deficiency) BB 987 ([), C4d 989, C5a 284 , SC5b-9 293 ([)
3
27
M
C
aHUS following food poisoning diagnosed at an outside institution 2 yr prior
120
Thrombomodulin mutation
C3 78 (Y), C4 29, CH50 43, C4d 3142 ([), BB Complement 622, SC5b-9 480 ([), C5a 134 ([), ADAMTS13 activity 89.8%
4
38
F
AA
7
Not performed
Not performed
ATG induction; maintenance IS with prednisone, sirolimus; later switched to cyclosporine and sirolimus due to ABMR
2.7
5
22
F
AA
Renal transplant 19 d prior for ESRD secondary to HTN; patient was presensitized and developed DSAs, ABMR rejection and severe TMA with cortical necrosis shortly after transplant Familial HUS, SLE
365
CFHR3 and CFHR1 mutations
C3 76 (Y), C4 6 (Y)
Hydralazine, clonidine, atorvastatin, amlodipine
Currently on dialysis
6
41
M
C
Allogeneic stem cell transplant for aplastic anemia
119
Not performed
C3 109, C4 34, CH50 <10 (Y), BB Complement 1209 ([) , C4d 2176 ([), C5a 83 ([), SC5b-9 265 ([), ADAMTS13 activity 85.1%
2.55
7
72
M
C
Renal transplant 37 d prior for ESRD due to PKD
29
Not performed
Not performed
ATG and CSA pre-BMT, BMT induction with fludarabine, cyclophosphamide, methotrexate and methylprednisolone; maintenance IS prednisone Basiliximab induction, maintenance IS with FK and MMF but had been on FK for only 2 d prior to TMA presentation
Patient
Eculizumab therapy length, d
Genetic testinga
10
Complement workupb
Medications prior to TMA development
Serum creatinine at last follow-up (mg/dl)
Negative
C3 65 (Y), C4 26
Methadone
1.4
ATG induction; maintenance IS with prednisone, MPS, and sirolimus
None
Currently on dialysis
1.5
1.2
Follow-up Responded well to eculizumab and dialysis was discontinued after 6 wk Had delayed graft function and ultimately transplant failure due to antibody-mediated rejection; still on dialysis 5.5 yr after Progressed to ESRD, received a renal transplant 5 mo later, was maintained on eculizumab and doing well but lost to followup 1 yr after transplant Stable allograft function, no longer on eculizumab
Was off dialysis for 3 yr, missed a few doses, had a relapse and is now on dialysis again No TMA relapse, still on eculizumab biweekly
Stable allograft function, no longer on eculizumab, no TMA relapse (Continued on next page)
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Age
TMA-associated conditions/ precipitating factors
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Table 1 | Clinical characteristics
Patient
Sex
Race
8
32
F
C
9
61
F
C
10
54
F
A
11
42
M
H
12
65
M
C
13
40
F
C
Second renal transplant 2 d prior for ESRD due to chronic pyelonephritis; first renal transplant lost because of pregnancyassociated TMA Renal transplant 12 d prior for ESRD due to pauciimmune crescentic and necrotizing GN Renal transplant 32 d prior for ESRD due to hypertensive nephrosclerosis Renal transplant due to hypertensive nephrosclerosis 22 mo prior; previous early posttransplant biopsy showing TMA Allogeneic stem cell transplant for T-cell lymphoma 1 yr prior
SLE, APLS
Eculizumab therapy length, d
Medications prior to TMA development
Serum creatinine at last follow-up (mg/dl)
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Genetic testinga
Complement workupb
1
Negative
Normal C3, C4, CH50
ATG induction; maintenance IS with prednisone, MMF
1.1
Stable allograft function, on eculizumab every 2 mo, no TMA recurrence
5
Not performed
Not performed
1.2
Stable allograft function, on monthly eculizumab, no TMA relapse
14
Not performed
Not performed
ATG induction; maintenance IS with FK and MMF but had been on FK for only 1 d prior to TMA presentation ATG induction; maintenance IS with MMF/prednisone
1.7
Stable allograft function, on biweekly eculizumab, no TMA relapse
601
Not performed
Not performed
ATG induction; maintenance IS with MMF/prednisone
1.3
Stable allograft function, on eculizumab every 3 mo, no TMA relapse
359
Not performed
Fludarabine and melphalan induction; maintenance IS with FK and methotrexate
2.1
Stable allograft function, still on eculizumab, no TMA relapse
229
Single deletion of CFHR3-CFHR1 gene
BB Complement 1814 ([), C4d 2325 ([), C5a 130 ([), SC5b-9 359 ([), CH50 <10 (Y), ADAMTS13 activity 86.2 C3 76 (Y), C4 27, CH50 57, BB Complement 1461 ([), C4d 4011 ([), C5a 44, SC5b-9 294 ([), ADAMTS13 activity 52.5 (Y)
Prednisone, hydroxychloroquine, MMF, leflunomide
2.24
Proteinuria improved from 5.2 to 0.6 g/g; still on eculizumab biweekly
Follow-up
A, Asian; AA, African American; ABMR, antibody-mediated rejection; ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; aHUS, atypical hemolytic-uremic syndrome; APLS, antiphospholipid antibody syndrome; ATG, antithymocyte globulin; BMT, bone marrow transplantation; C, Caucasian; CFHR, complement factor H related; CSA, colony-stimulating activity; DSA, donor-specific antibodies; ESRD, endstage renal disease; F, female; FK, tacrolimus; GN, glomerulonephritis; H, Hispanic; HTN, hypertension; IS, immunosuppresion; M, male; MMF, mycophenolate mofetil; MPS, mycophenolate sodium; PKD, polycystic kidney disease; SLE, systemic lupus erythematosus; TMA, thrombotic microangiopathy. a Genetic testing for genes involved in alternative complement pathway regulation, performed at the University of Iowa. b C3 and C4 values are expressed in mg/dl; CH50 and C2 are expressed is in U/ml; all others are in ng/ml. Reference ranges: ADAMTS13 activity > 67.9%; BB Complement, 244–961 ng/ml; C3, 87–200 mg/dl; C4, 18–52 mg/dl; C2, 25–47 U/ml; CH50, 30–75 U/ml; SC5b-9, 34–248 ng/ml; C5a, 19–48 ng/dl; C4d, 279–1846 ng/ml.
CA Cassol et al.: Eculizumab deposits in TMA
Age
TMA-associated conditions/ precipitating factors
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Table 1 | (Continued) Clinical characteristics
Interval between last eculizumab Biopsy dose and Biopsy type biopsy, d N
10
2
A
1
3
N
695
4a
A
Pre-eculizumab
4b
A
7
5
N
162
6
N
155
7
A
5
8 9a 9b
A A A
1 Pre-eculizumab 5
10a
A
Pre-eculizumab
10
A
13
11 12a 12b
A N N
14 Pre-eculizumab 14
13
N
13
TMA þ possible C3GN No rejection, no TMA Advanced chronic kidney injury, no TMA Mixed antibody and cell-mediated rejection, no TMA Ongoing mixed antibody and cellmediated rejection, no TMA Chronic TMA þ class II LN Ongoing TMA primarily affecting glomeruli ATN, no rejection, no TMA TMA TMA, no rejection ATN, no rejection, no TMA TMA ATN, no rejection, no TMA No rejection, no TMA TMA Ongoing TMA involving primarily the glomeruli FSGS and chronic TMA
C5b-9 arteriolar C5b-9 glomerular IgG2 glomerular
IgG2 arteriolar wall
IgG4 glomerular
IgG4 arteriolar wall
k glomerular
k arteriolar
3þ
3þ mesangial
1þ mesangial
1þ
2þ mesangial
2þ
1þ mesangial
2þ
3þ
1þ mesangial
1þ
1þ mesangial
1 to 2þ
Negative
2þ
3þ
2þ mesangial and GBM 1þ mesangial
Negative
Negative
Negative
Negative
Negative
3þ
3þ
2þ mesangial
Negative
Negative
Negative
Negative
Negative
Negative
3þ
1þ mesangial and GBM
1þ mesangiala
2þ
Trace mesangiala
2þ
Negative
1þ
2þ
1þ mesangial and GBM 2þ mesangial and GBM
1þ mesangial
1þ
Negative
1þ
Negative
Negative
Negative
3þ
Negative
3þ
Trace mesangial
3þ
3þ
3þ mesangial
Trace mesangial
1þ
1þ mesangial
2þ
1þ mesangial
2þ
2þ 3þ 2þ
Trace mesangial Trace mesangial 1þ mesangial
Negative Negative Trace mesangiala
1þ Negative 1 to 2þ
Negative Negative Trace mesangiala
1þ Negative 1 to 2þ
Negative Negative Trace mesangial
2þ Negative 2þ
2þ
No glomeruli present Trace mesangiala
Negative
No glomeruli present Trace mesangiala
Negative
No glomeruli present Trace mesangial
Negative
3þ
No glomeruli present 1þ mesangial
3þ 3þ 3þ
1þ mesangial 1þ mesangial Trace mesangial
1þ Negative 3þ
3þ mesangial
Negativea Negative 2þ segmental mesangial (vascular pole)a Negative
1þ Negative 2þ
3þ
Negativea Negative 1þ segmental mesangial (vascular pole)a Negative
3þ
1þ
2þ
2þ
2þ
Negative Negative 1þ segmental mesangial (vascular pole)a Negative
A, allograft; C3GN, C3 glomerulonephritis; FSGS, focal segmental glomerulosclerosis; GBM, glomerular basement membrane; LN, lupus nephritis; N, native kidney; TMA, TMA, thrombotic microangiopathy. a Focal Bowman’s capsule and tubular basement membrane deposits were also noted in these cases.
2þ 1þ Negative 3þ
2þ
5
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Diagnosis
CA Cassol et al.: Eculizumab deposits in TMA
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Table 2 | Pathologic findings
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CA Cassol et al.: Eculizumab deposits in TMA
Figure 1 | Illustrative case (patient 12) showing positive arteriolar staining for IgG, C5b-9, and k, with no l staining, mimicking monoclonal Ig deposition (top row). IgG subclasses reveals IgG2 and IgG4 restricted positivity within vessel walls, consistent with eculizumab deposition. Immunofluorescence: top row 400, bottom row 200. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
treated for TMA, a disease state that, unlike C3 glomerulopathy, is not associated with immune-type electron-dense deposits. Deposition occurred primarily in arterioles, with IgG2, IgG4, and k positivity by IF detected as early as 1 day after infusion and after a single dose of the drug and lasting as long as 5 months after the last eculizumab infusion, with complete disappearance 24 months after therapy. Less prominent mesangial deposition also was observed in a few cases, as well as focal positivity within Bowman’s capsule and tubular basement membranes. During the development of eculizumab, tissue cross-reactivity studies showed no nonspecific or unexpected binding of eculizumab to human tissues,1 which supports the supposition that the positivity detected in our cases indeed reflects specific binding of the drug to C5, present in the vascular wall, as opposed to nonspecific staining. The absence of IgG2 and IgG4 deposits in one biopsy taken before initiation of therapy and their presence in a biopsy specimen from the same patient taken 10 days later also supports this interpretation. In a previous article describing renal biopsy findings of patients with C3GN who were treated with eculizumab, deposits corresponding to this hybrid monoclonal antibody (IgG2, IgG4, and k) were noted in all patients within glomeruli, tubular basement membranes, and vessel walls after a year of treatment.16 Deposits also were noted by electron microscopy, occasionally with a granular powdery appearance reminiscent of monoclonal Ig deposition disease, and were postulated to represent binding of the drug to preexisting deposits.16 The current series adds to this knowledge by detailing the pathologic findings after eculizumab therapy in native and transplant biopsy specimens from patients with TMA, taken at different time points during eculizumab treatment. Unlike in C3 glomerulopathy, in biopsy specimens with TMA, glomerular electron-dense deposits are not seen, except in cases with other concomitant immune-complex or complement6
mediated (C3GN) diseases, where the expected glomerular electron-dense deposits are evident. In our series, a more restricted tissue distribution of IF staining was observed, with positivity most prominently noted in vessel walls and only mild mesangial deposition in a minority of cases (including one with possible concomitant C3GN and one with class II lupus nephropathy). As opposed to previously described in biopsies from patients with C3GN after eculizumab therapy,16 we were unable to detect any granular punctate powdery deposits similar to monoclonal Ig deposition disease in our cases. One possible explanation for this discrepancy is that TMA does not course with preexisting discrete electron-dense immune-type deposits. In their article, Herlitz et al.16 postulate that the powdery appearance of deposits observed could in fact represent a transformation of the appearance of pre-existing deposits as a result of binding of eculizumab. Alternatively, given the frequent association of C3GN with monoclonal gammopathy, it is possible that the deposits observed in the C3GN cases could represent early monoclonal Ig deposition disease. Although our case numbers may be too small to draw definitive conclusions, it appears that the intensity of IgG2/ IgG4 and k IF staining (and thus eculizumab deposition) is brighter in cases with active ongoing TMA, as opposed to cases with no morphologic evidence of TMA (cases in remission after treatment). In contrast, C5b-9 staining intensity and distribution showed no correlation with disease activity, which is similar to what was observed by Herlitz et al.16 in C3GN biopsies 1 year after eculizumab therapy, as well as in biopsy specimens of persons with lupus nephritis,18 in which similar C5b-9 staining was observed in both active and inactive disease cases. These findings could either represent slow tissue clearance as a result of a prolonged half-life of C5b-9 when bound to extracellular matrix or could indicate incomplete complement inhibition by eculizumab.19 Kidney International (2019) -, -–-
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CA Cassol et al.: Eculizumab deposits in TMA
Clinically, the role of measuring complement biomarkers such as soluble C5b-9 and C5a in patients with TMA is also controversial. When compared with patients who had idiopathic thrombocytopenic purpura, patients with aHUS had significantly higher levels of C5a and SC5b-9, helping to differentiate aHUS from thrombocytopenic purpura.20 However, Noris et al.21 found that a substantial fraction of aHUS did not have elevated levels of C3, SC5b-9, and C5a during the acute phase of disease. These data emphasize the fact that aHUS is a disorder characterized by localized, endothelial cell complement activation rather than fluid phase complement activation and are in keeping with observation by us and others16 of prolonged tissue deposition of C5b-9 and hence eculizumab at sites of complement activation. Therefore, although elevated biomarkers of the alternative and terminal complement pathways may be used to confirm a clinical diagnosis of aHUS, normal levels cannot be used to exclude the diagnosis and withhold complement inhibition therapy. The increasing recognition of the role of complement system in a large variety of systemic and local disorders, together with the development and clinically verified therapeutic success of the terminal pathway complement inhibitor, eculizumab, has reignited clinical and research interest in the complement system and its components as therapeutic targets. Within the kidney, complement activation is known to play an important role in the pathogenesis not only of atypical HUS and C3 glomerulopathy but also of immune complex-mediated diseases such as lupus nephritis22 and IgA nephropathy23; and even non–immune-complex–mediated diseases such as antineutrophilic cytoplasmic autoantibody–associated vasculitis24 and diabetic nephropathy.25 Therefore, it is reasonable to expect that in the future, more and more diseases will be found to benefit from complement-targeted therapies, including eculizumab. Eculizumab not only efficiently binds to C5 and prevents its proteolytic cleavage but also has been engineered to not elicit proinflammatory responses. The tissue deposition of eculizumab in the vascular wall may be beneficial in patients with TMA by controlling local complement activation in arteries/arterioles that otherwise would lead to endothelialvascular wall damage, causing TMA. This highly desirable property, however, may create diagnostic challenges for pathologists, because the drug remains in tissue for prolonged periods and can yield positive IF results resembling monoclonal gammopathy-related renal disease (IgG and k restricted IF positivity), unless IgG subclass staining is performed to reveal the typical IgG2 and IgG4 positivity characteristic of tissue eculizumab deposits. Conclusions
In this study, we have demonstrated that IgG2, IgG4, and k positive deposits corresponding to eculizumab in renal arterioles can be detected as early as 1 day after treatment initiation and can remain detectable in the tissue for as long as 5 months after therapy discontinuation in patients who received this drug for TMA treatment or prevention of TMA recurrence in the renal allograft. Knowledge of this finding is Kidney International (2019) -, -–-
important because the detection of Ig-positive deposits in patients with clinically suspected TMA (which does not usually course with immune-type deposits) could lead to misdiagnoses, especially if therapy was discontinued a while prior to the biopsy and the pathologist was not informed of antecedent eculizumab use and if IgG subclass staining is not performed. METHODS Study design and participants Institution approval was obtained from The Ohio State University Office of Responsible Research Practices. This retrospective cohort study included patients who received eculizumab for the treatment or prevention of recurrence in the allograft of TMA between January 1, 2011, and October 18, 2018, at the Wexner Medical Center at The Ohio State University. Cases were identified through a natural language search in Copath using the term “eculizumab” in the clinical history field of pathologic reports. Reports were reviewed to ensure that eculizumab therapy indication was treatment or prevention of recurrence of TMA. Patients who received eculizumab for the treatment of C3GN were not included.16 Only cases with material available for IgG subclasses staining by immunofluorescence were included. Tissue processing and immunostaining Native and allograft renal biopsies were processed according to standard techniques for light microscopy, IF, and electron microscopy. Direct IF was performed on frozen sections with fluorescein isothiocyanate-conjugated polyclonal rabbit antibodies to IgG1, IgG2, IgG3, and IgG4 (Binding Site, Inc., San Diego, CA) as described previously.26 To confirm that eculizumab deposition was occurring at sites of complement activation, staining for C5b-9 was performed using a mouse anti-human C5b-9 antibody (clone ae11, M0777, Agilent Technologies, Santa Clara, CA) at 1:25 dilution for 1 hour, followed by fluorescein isothiocyanate–conjugated polyclonal rabbit anti-mouse (F0261, Agilent) for 1 hour. Additional Data Sources Clinical data were obtained from electronic medical records or faxed clinical notes sent from outside nephrologists. Definitions Intensity of IF staining was graded semiquantitatively on a scale of 0 to 3þ: 0, absent; 1þ, weak; 2þ, moderate; and 3þ, strong. DISCLOSURE SC has been a consultant for Alexion Pharmaceuticals since 2018 and has received consulting and lecture fees. All the other authors declared no competing interests.
ACKNOWLEDGMENTS
We thank Dr. Natalia Maroz for providing clinical details on 5 patients, Cherri Bott and Kyle Ware for technical assistance in performing IgG subclass immunofluorescence, and Shawn Scully, Senior Systems Consultant at the Department of Pathology from the Ohio State University, for assistance with figure preparation. SUPPLEMENTARY MATERIAL Figure S1. Patient 4 underwent a biopsy on the same day as but prior to infusion of eculizumab. The specimen shows only tubular casts and background staining for IgG1 to IgG4 (top row; biopsy 4a in Table 2). A subsequent biopsy specimen 6 days later showed moderate IgG2 7
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and IgG4 deposition within arteriolar walls (bottom row; biopsy 4b in Table 2). Immunofluorescence 200. Supplementary material is linked to the online version of the paper at www.kidney-international.org. REFERENCES 1. Rother RP, Rollins SA, Mojcik CF, et al. Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. Nat Biotech. 2007;25:1256. 2. Hillmen P, Young NS, Schubert J, et al. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2006;355:1233–1243. 3. Legendre CM, Licht C, Muus P, et al. Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome. N Engl J Med. 2013;368:2169–2181. 4. George JN, Nester CM. Syndromes of thrombotic microangiopathy. N Engl J Med. 2014;371:654–666. 5. Brocklebank V, Wood KM, Kavanagh D. Thrombotic microangiopathy and the kidney. Clin J Am Soc Nephrol. 2018;13:300–317. 6. Tsai HM. Atypical hemolytic uremic syndrome: beyond hemolysis and uremia. Am J Med. 2019;132:161–167. 7. Goodship TH, Cook HT, Fakhouri F, et al. Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference. Kidney Int. 2017;91:539–551. 8. Zuber J, Fakhouri F, Roumenina LT, et al. Use of eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies. Nat Rev Nephrol. 2012;8:643–657. 9. Cavero T, Rabasco C, Lopez A, et al. Eculizumab in secondary atypical haemolytic uraemic syndrome. Nephrol Dial Transplant. 2017;32:466–474. 10. Dhakal P, Giri S, Pathak R, Bhatt VR. Eculizumab in transplant-associated thrombotic microangiopathy 2017;. 2017;23:175–180. 11. Matsumura D, Tanaka A, Nakamura T, et al. Coexistence of atypical hemolytic uremic syndrome and crescentic IgA nephropathy treated with eculizumab: a case report. Clin Nephrol Case Stud. 2016;4:24–28. 12. El-Husseini A, Hannan S, Awad A, et al. Thrombotic microangiopathy in systemic lupus erythematosus: efficacy of eculizumab. Am J Kidney Dis. 2015;65:127–130.
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13. Sciascia S, Radin M, Yazdany J, et al. Expanding the therapeutic options for renal involvement in lupus: eculizumab, available evidence. Rheumatol Int. 2017;37:1249–1255. 14. Pape L, Hartmann H, Bange FC, et al. Eculizumab in typical hemolytic uremic syndrome (HUS) with neurological involvement. Medicine. 2015;94:e1000. 15. Levi C, Fremeaux-Bacchi V, Zuber J, et al. Midterm outcomes of 12 renal transplant recipients treated with eculizumab to prevent atypical hemolytic syndrome recurrence. Transplantation. 2017;101:2924–2930. 16. Herlitz LC, Bomback AS, Markowitz GS, et al. Pathology after eculizumab in dense deposit disease and C3 GN. J Am Soc Nephrol. 2012;23:1229– 1237. 17. Ricklin D, Mastellos DC, Reis ES, Lambris JD. The renaissance of complement therapeutics. Nat Rev Nephrol. 2018;14:26–47. 18. Wilson HR, Medjeral-Thomas NR, Gilmore AC, et al. Glomerular membrane attack complex is not a reliable marker of ongoing C5 activation in lupus nephritis. Kidney Int. 2019;95:655–665. 19. Harder MJ, Kuhn N, Schrezenmeier H, et al. Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation. Blood. 2017;129:970–980. 20. Cataland SR, Holers VM, Geyer S, et al. Biomarkers of terminal complement activation confirm the diagnosis of aHUS and differentiate aHUS from TTP. Blood. 2014;123:3733–3738. 21. Noris M, Galbusera M, Gastoldi S, et al. Dynamics of complement activation in aHUS and how to monitor eculizumab therapy. Blood. 2014;124:1715–1726. 22. Yu F, Haas M, Glassock R, Zhao MH. Redefining lupus nephritis: clinical implications of pathophysiologic subtypes. Nat Rev Nephrol. 2017;13: 483–495. 23. Lai KN, Tang SC, Schena FP, et al. IgA nephropathy. Nat Rev Dis Primers. 2016;2:16001. 24. Chen M, Jayne DRW, Zhao MH. Complement in ANCA-associated vasculitis: mechanisms and implications for management. Nat Rev Nephrol. 2017;13:359–367. 25. Flyvbjerg A. The role of the complement system in diabetic nephropathy. Nat Rev Nephrol. 2017;13:311–318. 26. Satoskar AA, Brodsky SV, Nadasdy G, et al. Discrepancies in glomerular and tubulointerstitial/vascular immune complex IgG subclasses in lupus nephritis. Lupus. 2011;20:1396–1403.
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Eculizumab deposits in vessel walls in thrombotic microangiopathy Clarissa A. Cassol1, Sergey V. Brodsky1, Anjali A. Satoskar1, Angela R. Blissett1, Spero Cataland2 and Tibor Nadasdy1 1
Department of Pathology, The Ohio State University, Columbus, Ohio, USA; and 2Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA
Terminal complement inhibition therapy with eculizumab (a humanized monoclonal antibody to C5) has revolutionized the treatment of patients with thrombotic microangiopathy (TMA). Successful responders are often placed on long-standing therapy to prevent disease recurrence in the native kidney or allograft. The tissue deposition of eculizumab in patients with C3 glomerulopathy has been described but no studies have yet investigated tissue deposition of eculizumab in cases where it was indicated for thrombotic microangiopathy which, unlike C3 glomerulopathy, does not usually show immune-type electron dense deposits. To evaluate this, we reviewed biopsies from 13 patients who received eculizumab for TMA treatment or prevention of recurrence. We found IgG2, IgG4, and kappa positivity within arterioles corresponding to eculizumab deposits, with similar distribution to C5b-9, in all but one patient. In that patient eculizumab therapy had been discontinued 24 months prior to biopsy. Deposits in arterioles could be seen as early as one day after infusion and after a single dose of eculizumab, and were detected up to 162 days after therapy discontinuation. This may play a role in controlling local complement activation-associated vascular changes in these patients. Thus, IgG subclass staining by immunofluorescence is important to avoid misdiagnoses of immune-complex or monoclonal immunoglobulin deposition disease in patients with TMA who received eculizumab. Kidney International (2019) j.kint.2019.05.008
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https://doi.org/10.1016/
KEYWORDS: eculizumab; IgG subclasses; thrombotic microangiopathy Copyright ª 2019, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
Correspondence: Clarissa A. Cassol, The Ohio State University Wexner Medical Center, Department of Pathology, Renal Pathology Division, 320 W 10th Avenue, M018 Starling Loving Hall, Columbus, OH 43210, USA. E-mail: [email protected]; or Tibor Nadasdy, The Ohio State University Wexner Medical Center, Department of Pathology, Renal Pathology Division, 320 W 10th Avenue, M018 Starling Loving Hall, Columbus, OH 43210 USA. E-mail: [email protected] Received 7 December 2018; revised 30 April 2019; accepted 2 May 2019; published online 24 May 2019 Kidney International (2019) -, -–-
E
culizumab is a humanized murine recombinant monoclonal antibody engineered to minimize immunogenicity, proinflammatory effects, and complement activation while successfully inhibiting the terminal complement component C5 to prevent the generation of the membrane attack complex.1 To achieve these objectives, the antibody consists of a hybrid human IgG2 heavy chain hinge region, which does not bind Fc receptors, and the IgG4 heavy chain CH2 and CH3 regions, which lack the ability to activate the complement cascade, associated with a k light chain. This structure retains the high affinity gained from the variable regions derived from the murine antibody while at the same time ensuring low immunogenicity and few proinflammatory effects. Eculizumab was approved initially for the treatment of paroxysmal nocturnal hemoglobinuria in 2007.1,2 Since then, its use has been expanded to other complement-mediated disorders, including TMA, for which it was approved for clinical use in 2011.3 TMA is a devastating disease characterized by prominent microvascular injury. Historically, TMA has been divided into hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura. HUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Meanwhile, patients with a diagnosis of thrombocytopenic purpura have deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) enzyme, and the nonimmune hemolytic anemia and thrombocytopenia primarily are associated with central nervous system symptoms rather than renal disease. In typical forms of HUS, renal disease follows hemorrhagic colitis and diarrhea, and most cases are a result of Shiga toxin–producing Escherichia coli HUS. In contrast, in atypical HUS (aHUS), the TMA usually is not associated with diarrhea, and microangiopathic hemolytic anemia may be absent.4–6 Complement alternative pathway dysregulation resulting from either mutations or acquired autoantibodies against C3-convertase regulatory factors is present in at least 50% of cases of aHUS,7 leading to endothelial injury through generation of the terminal complement components C5b-9 on endothelial cell surfaces. Eculizumab was shown to successfully prevent renal deterioration and reverse microangiopathic hemolytic anemia, when present, in most patients with complementmediated aHUS.3,8 In addition, eculizumab has shown 1
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promising results of “noncomplement”-mediated aHUS, including drug-induced TMA9; TMA after stem cell, renal, or other solid organ transplantation10; TMA associated with other glomerular diseases11–13; and even Shiga toxin– associated typical HUS.14 In the renal allograft setting, treatment with eculizumab was successful in preventing TMA recurrence.15 The tissue deposition of eculizumab (IgG2, IgG4, and k deposits) has been described in patients receiving this drug for C3 glomerulopathy, where it was seen within glomerular capillaries and mesangium, vessel walls, and tubular basement membranes.16 With the expanded use of eculizumab for treatment of different conditions that could benefit from terminal complement blockade,17 pathologists are expected to encounter more biopsy specimens from patients receiving this therapy. Given its low immunogenicity and slow tissue clearance,1 prolonged tissue deposition of the antibody is expected,16 which may generate confusing results (e.g., positive IgG k deposits in the absence of obvious immune complex or monoclonal Ig deposition), especially if IgG subclass staining is not performed or if the pathologist is not informed that the patient has received eculizumab. Increasingly in our practice we have encountered renal native or allograft biopsy specimens from patients who had received or were currently undergoing eculizumab therapy. This article describes the clinical characteristics and pathologic findings of a series of these cases encountered in our practice, highlighting the time course and length of tissue deposition after initiation and discontinuation of therapy. RESULTS
Since 2011, renal biopsy specimens from a total of 17 patients who underwent a renal biopsy during or after eculizumab therapy for the treatment of TMA or prevention of recurrence of TMA were reviewed at our institution (Table 1). Four specimens were excluded because of lack of frozen tissue for IgG subclass staining (these were consult cases for which frozen slides were not received for review). Of the remaining 13 cases, 10 patients were receiving eculizumab at the time of renal biopsy and 3 patients had received the therapy previously (Table 2). Seven biopsy specimens were from native kidneys and 6 were from renal allografts. In 3 renal allograft recipients, the cause of end-stage renal disease (ESRD) was listed as hypertension. TMA developed in these patients shortly after transplant, and therefore it is possible that the original cause of ESRD was in fact TMA; however, none of these patients had a native kidney biopsy to confirm this hypothesis. Pathologic diagnoses are summarized in Table 2. All patients showed at least mild arteriolar IgG2, IgG4, and k deposits by immunofluorescence (IF; Figure 1, Table 2) except patient 5, who showed no k, and patient 3, for whom eculizumab had been discontinued 695 days prior and who had no IgG2 and IgG4 arteriolar staining. Six of the 13 patients treated with eculizumab had morphologic findings of 2
CA Cassol et al.: Eculizumab deposits in TMA
ongoing active TMA (patients 1, 6, 8, and 12) or chronic TMA (patients 5 and 13) in the renal biopsy, and all of these biopsy specimens showed bright arteriolar deposits by IF (2þ or 3þ; Table 2). Even though TMA was noted to involve predominantly the glomeruli in patients 6 and 12, glomerular deposits were absent (patient 6) or mild and segmental (patient 12) in these cases (Table 2). Interestingly, those 2 cases showed the brightest (3þ) staining for IgG2 and IgG4 in arterioles. Biopsy specimens that did not display morphologic changes of ongoing TMA had similar vascular staining for IgG2, IgG4, and k. Glomerular positivity for IgG2, IgG4, and k also was noted in specimens for 8 of the 12 patients that showed arteriolar staining and was mostly mesangial, mild, and segmental, with the exception of one patient with possible concomitant C3 glomerulonephritis (C3GN), whose specimen showed moderate mesangial staining (patient 1). Four patients had pre-eculizumab treatment biopsy specimens, all of which showed negative staining for IgG2, IgG4, and k in arterioles and glomeruli as depicted in Supplementary Figure S1. Positive arteriolar deposits by IF were seen as early as 1 day after eculizumab infusion (patients 2 and 8) and after a single dose of the drug in many cases (patients 1, 4, 8, 9, and 10). Positive IgG2, IgG4, and k could still be detected in the arterioles of patients 5 and 6, 155 and 162 days after the last dose of eculizumab. Three cases (1, 9, and 10) showed additional focal positivity for IgG2, IgG4, and k along Bowman’s capsule and tubular basement membranes. To confirm that IgG2, IgG4, and k deposits corresponding to eculizumab were occurring at sites of complement activation, C5b-9 IF was performed and showed arteriolar positivity in all cases (Table 2, Figure 1). C5b-9 positivity also was observed in the mesangium, ranging from mild (1þ) to prominent (3þ), as well as in the tubular basement membranes and Bowman’s capsule (Figure 1). Tissue distribution and intensity of staining for C5b-9 showed no correlation with the presence or absence of active TMA. For cases in which pre- and post-eculizumab treatment biopsies were available, no significant difference in C5b-9 staining was observed. Electron microscopy was performed in 10 cases (3 cases were early post-transplant allograft biopsy specimens that did not undergo ultrastructural examination) and showed no electron-dense immune-type or finely granular punctate deposits in arteries or arterioles. Three cases showed a few glomerular deposits that were intramembranous with possible concomitant C3GN from patient 1 and mesangial in the biopsy specimen with concomitant class II lupus nephritis from patient 5. The biopsy specimen for patient 3, which showed advanced chronic changes and no arteriolar staining corresponding to eculizumab, also showed a few mesangial deposits. DISCUSSION
For the first time we have demonstrated tissue deposition of the humanized monoclonal antibody eculizumab in patients Kidney International (2019) -, -–-
Sex
Race
1
26
F
C
Preeclampsia
2
42
F
C
Second renal transplant 2 d prior for ESRD due to PKD; first allograft lost due to TMA
294
Negative
C3 187, C4 35, C2 12 (Y, consistent with heterozygous C2 deficiency) BB 987 ([), C4d 989, C5a 284 , SC5b-9 293 ([)
3
27
M
C
aHUS following food poisoning diagnosed at an outside institution 2 yr prior
120
Thrombomodulin mutation
C3 78 (Y), C4 29, CH50 43, C4d 3142 ([), BB Complement 622, SC5b-9 480 ([), C5a 134 ([), ADAMTS13 activity 89.8%
4
38
F
AA
7
Not performed
Not performed
ATG induction; maintenance IS with prednisone, sirolimus; later switched to cyclosporine and sirolimus due to ABMR
2.7
5
22
F
AA
Renal transplant 19 d prior for ESRD secondary to HTN; patient was presensitized and developed DSAs, ABMR rejection and severe TMA with cortical necrosis shortly after transplant Familial HUS, SLE
365
CFHR3 and CFHR1 mutations
C3 76 (Y), C4 6 (Y)
Hydralazine, clonidine, atorvastatin, amlodipine
Currently on dialysis
6
41
M
C
Allogeneic stem cell transplant for aplastic anemia
119
Not performed
C3 109, C4 34, CH50 <10 (Y), BB Complement 1209 ([) , C4d 2176 ([), C5a 83 ([), SC5b-9 265 ([), ADAMTS13 activity 85.1%
2.55
7
72
M
C
Renal transplant 37 d prior for ESRD due to PKD
29
Not performed
Not performed
ATG and CSA pre-BMT, BMT induction with fludarabine, cyclophosphamide, methotrexate and methylprednisolone; maintenance IS prednisone Basiliximab induction, maintenance IS with FK and MMF but had been on FK for only 2 d prior to TMA presentation
Patient
Eculizumab therapy length, d
Genetic testinga
10
Complement workupb
Medications prior to TMA development
Serum creatinine at last follow-up (mg/dl)
Negative
C3 65 (Y), C4 26
Methadone
1.4
ATG induction; maintenance IS with prednisone, MPS, and sirolimus
None
Currently on dialysis
1.5
1.2
Follow-up Responded well to eculizumab and dialysis was discontinued after 6 wk Had delayed graft function and ultimately transplant failure due to antibody-mediated rejection; still on dialysis 5.5 yr after Progressed to ESRD, received a renal transplant 5 mo later, was maintained on eculizumab and doing well but lost to followup 1 yr after transplant Stable allograft function, no longer on eculizumab
Was off dialysis for 3 yr, missed a few doses, had a relapse and is now on dialysis again No TMA relapse, still on eculizumab biweekly
Stable allograft function, no longer on eculizumab, no TMA relapse (Continued on next page)
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3
Age
TMA-associated conditions/ precipitating factors
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Table 1 | Clinical characteristics
Patient
Sex
Race
8
32
F
C
9
61
F
C
10
54
F
A
11
42
M
H
12
65
M
C
13
40
F
C
Second renal transplant 2 d prior for ESRD due to chronic pyelonephritis; first renal transplant lost because of pregnancyassociated TMA Renal transplant 12 d prior for ESRD due to pauciimmune crescentic and necrotizing GN Renal transplant 32 d prior for ESRD due to hypertensive nephrosclerosis Renal transplant due to hypertensive nephrosclerosis 22 mo prior; previous early posttransplant biopsy showing TMA Allogeneic stem cell transplant for T-cell lymphoma 1 yr prior
SLE, APLS
Eculizumab therapy length, d
Medications prior to TMA development
Serum creatinine at last follow-up (mg/dl)
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Genetic testinga
Complement workupb
1
Negative
Normal C3, C4, CH50
ATG induction; maintenance IS with prednisone, MMF
1.1
Stable allograft function, on eculizumab every 2 mo, no TMA recurrence
5
Not performed
Not performed
1.2
Stable allograft function, on monthly eculizumab, no TMA relapse
14
Not performed
Not performed
ATG induction; maintenance IS with FK and MMF but had been on FK for only 1 d prior to TMA presentation ATG induction; maintenance IS with MMF/prednisone
1.7
Stable allograft function, on biweekly eculizumab, no TMA relapse
601
Not performed
Not performed
ATG induction; maintenance IS with MMF/prednisone
1.3
Stable allograft function, on eculizumab every 3 mo, no TMA relapse
359
Not performed
Fludarabine and melphalan induction; maintenance IS with FK and methotrexate
2.1
Stable allograft function, still on eculizumab, no TMA relapse
229
Single deletion of CFHR3-CFHR1 gene
BB Complement 1814 ([), C4d 2325 ([), C5a 130 ([), SC5b-9 359 ([), CH50 <10 (Y), ADAMTS13 activity 86.2 C3 76 (Y), C4 27, CH50 57, BB Complement 1461 ([), C4d 4011 ([), C5a 44, SC5b-9 294 ([), ADAMTS13 activity 52.5 (Y)
Prednisone, hydroxychloroquine, MMF, leflunomide
2.24
Proteinuria improved from 5.2 to 0.6 g/g; still on eculizumab biweekly
Follow-up
A, Asian; AA, African American; ABMR, antibody-mediated rejection; ADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; aHUS, atypical hemolytic-uremic syndrome; APLS, antiphospholipid antibody syndrome; ATG, antithymocyte globulin; BMT, bone marrow transplantation; C, Caucasian; CFHR, complement factor H related; CSA, colony-stimulating activity; DSA, donor-specific antibodies; ESRD, endstage renal disease; F, female; FK, tacrolimus; GN, glomerulonephritis; H, Hispanic; HTN, hypertension; IS, immunosuppresion; M, male; MMF, mycophenolate mofetil; MPS, mycophenolate sodium; PKD, polycystic kidney disease; SLE, systemic lupus erythematosus; TMA, thrombotic microangiopathy. a Genetic testing for genes involved in alternative complement pathway regulation, performed at the University of Iowa. b C3 and C4 values are expressed in mg/dl; CH50 and C2 are expressed is in U/ml; all others are in ng/ml. Reference ranges: ADAMTS13 activity > 67.9%; BB Complement, 244–961 ng/ml; C3, 87–200 mg/dl; C4, 18–52 mg/dl; C2, 25–47 U/ml; CH50, 30–75 U/ml; SC5b-9, 34–248 ng/ml; C5a, 19–48 ng/dl; C4d, 279–1846 ng/ml.
CA Cassol et al.: Eculizumab deposits in TMA
Age
TMA-associated conditions/ precipitating factors
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Table 1 | (Continued) Clinical characteristics
Interval between last eculizumab Biopsy dose and Biopsy type biopsy, d N
10
2
A
1
3
N
695
4a
A
Pre-eculizumab
4b
A
7
5
N
162
6
N
155
7
A
5
8 9a 9b
A A A
1 Pre-eculizumab 5
10a
A
Pre-eculizumab
10
A
13
11 12a 12b
A N N
14 Pre-eculizumab 14
13
N
13
TMA þ possible C3GN No rejection, no TMA Advanced chronic kidney injury, no TMA Mixed antibody and cell-mediated rejection, no TMA Ongoing mixed antibody and cellmediated rejection, no TMA Chronic TMA þ class II LN Ongoing TMA primarily affecting glomeruli ATN, no rejection, no TMA TMA TMA, no rejection ATN, no rejection, no TMA TMA ATN, no rejection, no TMA No rejection, no TMA TMA Ongoing TMA involving primarily the glomeruli FSGS and chronic TMA
C5b-9 arteriolar C5b-9 glomerular IgG2 glomerular
IgG2 arteriolar wall
IgG4 glomerular
IgG4 arteriolar wall
k glomerular
k arteriolar
3þ
3þ mesangial
1þ mesangial
1þ
2þ mesangial
2þ
1þ mesangial
2þ
3þ
1þ mesangial
1þ
1þ mesangial
1 to 2þ
Negative
2þ
3þ
2þ mesangial and GBM 1þ mesangial
Negative
Negative
Negative
Negative
Negative
3þ
3þ
2þ mesangial
Negative
Negative
Negative
Negative
Negative
Negative
3þ
1þ mesangial and GBM
1þ mesangiala
2þ
Trace mesangiala
2þ
Negative
1þ
2þ
1þ mesangial and GBM 2þ mesangial and GBM
1þ mesangial
1þ
Negative
1þ
Negative
Negative
Negative
3þ
Negative
3þ
Trace mesangial
3þ
3þ
3þ mesangial
Trace mesangial
1þ
1þ mesangial
2þ
1þ mesangial
2þ
2þ 3þ 2þ
Trace mesangial Trace mesangial 1þ mesangial
Negative Negative Trace mesangiala
1þ Negative 1 to 2þ
Negative Negative Trace mesangiala
1þ Negative 1 to 2þ
Negative Negative Trace mesangial
2þ Negative 2þ
2þ
No glomeruli present Trace mesangiala
Negative
No glomeruli present Trace mesangiala
Negative
No glomeruli present Trace mesangial
Negative
3þ
No glomeruli present 1þ mesangial
3þ 3þ 3þ
1þ mesangial 1þ mesangial Trace mesangial
1þ Negative 3þ
3þ mesangial
Negativea Negative 2þ segmental mesangial (vascular pole)a Negative
1þ Negative 2þ
3þ
Negativea Negative 1þ segmental mesangial (vascular pole)a Negative
3þ
1þ
2þ
2þ
2þ
Negative Negative 1þ segmental mesangial (vascular pole)a Negative
A, allograft; C3GN, C3 glomerulonephritis; FSGS, focal segmental glomerulosclerosis; GBM, glomerular basement membrane; LN, lupus nephritis; N, native kidney; TMA, TMA, thrombotic microangiopathy. a Focal Bowman’s capsule and tubular basement membrane deposits were also noted in these cases.
2þ 1þ Negative 3þ
2þ
5
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Diagnosis
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Table 2 | Pathologic findings
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CA Cassol et al.: Eculizumab deposits in TMA
Figure 1 | Illustrative case (patient 12) showing positive arteriolar staining for IgG, C5b-9, and k, with no l staining, mimicking monoclonal Ig deposition (top row). IgG subclasses reveals IgG2 and IgG4 restricted positivity within vessel walls, consistent with eculizumab deposition. Immunofluorescence: top row 400, bottom row 200. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
treated for TMA, a disease state that, unlike C3 glomerulopathy, is not associated with immune-type electron-dense deposits. Deposition occurred primarily in arterioles, with IgG2, IgG4, and k positivity by IF detected as early as 1 day after infusion and after a single dose of the drug and lasting as long as 5 months after the last eculizumab infusion, with complete disappearance 24 months after therapy. Less prominent mesangial deposition also was observed in a few cases, as well as focal positivity within Bowman’s capsule and tubular basement membranes. During the development of eculizumab, tissue cross-reactivity studies showed no nonspecific or unexpected binding of eculizumab to human tissues,1 which supports the supposition that the positivity detected in our cases indeed reflects specific binding of the drug to C5, present in the vascular wall, as opposed to nonspecific staining. The absence of IgG2 and IgG4 deposits in one biopsy taken before initiation of therapy and their presence in a biopsy specimen from the same patient taken 10 days later also supports this interpretation. In a previous article describing renal biopsy findings of patients with C3GN who were treated with eculizumab, deposits corresponding to this hybrid monoclonal antibody (IgG2, IgG4, and k) were noted in all patients within glomeruli, tubular basement membranes, and vessel walls after a year of treatment.16 Deposits also were noted by electron microscopy, occasionally with a granular powdery appearance reminiscent of monoclonal Ig deposition disease, and were postulated to represent binding of the drug to preexisting deposits.16 The current series adds to this knowledge by detailing the pathologic findings after eculizumab therapy in native and transplant biopsy specimens from patients with TMA, taken at different time points during eculizumab treatment. Unlike in C3 glomerulopathy, in biopsy specimens with TMA, glomerular electron-dense deposits are not seen, except in cases with other concomitant immune-complex or complement6
mediated (C3GN) diseases, where the expected glomerular electron-dense deposits are evident. In our series, a more restricted tissue distribution of IF staining was observed, with positivity most prominently noted in vessel walls and only mild mesangial deposition in a minority of cases (including one with possible concomitant C3GN and one with class II lupus nephropathy). As opposed to previously described in biopsies from patients with C3GN after eculizumab therapy,16 we were unable to detect any granular punctate powdery deposits similar to monoclonal Ig deposition disease in our cases. One possible explanation for this discrepancy is that TMA does not course with preexisting discrete electron-dense immune-type deposits. In their article, Herlitz et al.16 postulate that the powdery appearance of deposits observed could in fact represent a transformation of the appearance of pre-existing deposits as a result of binding of eculizumab. Alternatively, given the frequent association of C3GN with monoclonal gammopathy, it is possible that the deposits observed in the C3GN cases could represent early monoclonal Ig deposition disease. Although our case numbers may be too small to draw definitive conclusions, it appears that the intensity of IgG2/ IgG4 and k IF staining (and thus eculizumab deposition) is brighter in cases with active ongoing TMA, as opposed to cases with no morphologic evidence of TMA (cases in remission after treatment). In contrast, C5b-9 staining intensity and distribution showed no correlation with disease activity, which is similar to what was observed by Herlitz et al.16 in C3GN biopsies 1 year after eculizumab therapy, as well as in biopsy specimens of persons with lupus nephritis,18 in which similar C5b-9 staining was observed in both active and inactive disease cases. These findings could either represent slow tissue clearance as a result of a prolonged half-life of C5b-9 when bound to extracellular matrix or could indicate incomplete complement inhibition by eculizumab.19 Kidney International (2019) -, -–-
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CA Cassol et al.: Eculizumab deposits in TMA
Clinically, the role of measuring complement biomarkers such as soluble C5b-9 and C5a in patients with TMA is also controversial. When compared with patients who had idiopathic thrombocytopenic purpura, patients with aHUS had significantly higher levels of C5a and SC5b-9, helping to differentiate aHUS from thrombocytopenic purpura.20 However, Noris et al.21 found that a substantial fraction of aHUS did not have elevated levels of C3, SC5b-9, and C5a during the acute phase of disease. These data emphasize the fact that aHUS is a disorder characterized by localized, endothelial cell complement activation rather than fluid phase complement activation and are in keeping with observation by us and others16 of prolonged tissue deposition of C5b-9 and hence eculizumab at sites of complement activation. Therefore, although elevated biomarkers of the alternative and terminal complement pathways may be used to confirm a clinical diagnosis of aHUS, normal levels cannot be used to exclude the diagnosis and withhold complement inhibition therapy. The increasing recognition of the role of complement system in a large variety of systemic and local disorders, together with the development and clinically verified therapeutic success of the terminal pathway complement inhibitor, eculizumab, has reignited clinical and research interest in the complement system and its components as therapeutic targets. Within the kidney, complement activation is known to play an important role in the pathogenesis not only of atypical HUS and C3 glomerulopathy but also of immune complex-mediated diseases such as lupus nephritis22 and IgA nephropathy23; and even non–immune-complex–mediated diseases such as antineutrophilic cytoplasmic autoantibody–associated vasculitis24 and diabetic nephropathy.25 Therefore, it is reasonable to expect that in the future, more and more diseases will be found to benefit from complement-targeted therapies, including eculizumab. Eculizumab not only efficiently binds to C5 and prevents its proteolytic cleavage but also has been engineered to not elicit proinflammatory responses. The tissue deposition of eculizumab in the vascular wall may be beneficial in patients with TMA by controlling local complement activation in arteries/arterioles that otherwise would lead to endothelialvascular wall damage, causing TMA. This highly desirable property, however, may create diagnostic challenges for pathologists, because the drug remains in tissue for prolonged periods and can yield positive IF results resembling monoclonal gammopathy-related renal disease (IgG and k restricted IF positivity), unless IgG subclass staining is performed to reveal the typical IgG2 and IgG4 positivity characteristic of tissue eculizumab deposits. Conclusions
In this study, we have demonstrated that IgG2, IgG4, and k positive deposits corresponding to eculizumab in renal arterioles can be detected as early as 1 day after treatment initiation and can remain detectable in the tissue for as long as 5 months after therapy discontinuation in patients who received this drug for TMA treatment or prevention of TMA recurrence in the renal allograft. Knowledge of this finding is Kidney International (2019) -, -–-
important because the detection of Ig-positive deposits in patients with clinically suspected TMA (which does not usually course with immune-type deposits) could lead to misdiagnoses, especially if therapy was discontinued a while prior to the biopsy and the pathologist was not informed of antecedent eculizumab use and if IgG subclass staining is not performed. METHODS Study design and participants Institution approval was obtained from The Ohio State University Office of Responsible Research Practices. This retrospective cohort study included patients who received eculizumab for the treatment or prevention of recurrence in the allograft of TMA between January 1, 2011, and October 18, 2018, at the Wexner Medical Center at The Ohio State University. Cases were identified through a natural language search in Copath using the term “eculizumab” in the clinical history field of pathologic reports. Reports were reviewed to ensure that eculizumab therapy indication was treatment or prevention of recurrence of TMA. Patients who received eculizumab for the treatment of C3GN were not included.16 Only cases with material available for IgG subclasses staining by immunofluorescence were included. Tissue processing and immunostaining Native and allograft renal biopsies were processed according to standard techniques for light microscopy, IF, and electron microscopy. Direct IF was performed on frozen sections with fluorescein isothiocyanate-conjugated polyclonal rabbit antibodies to IgG1, IgG2, IgG3, and IgG4 (Binding Site, Inc., San Diego, CA) as described previously.26 To confirm that eculizumab deposition was occurring at sites of complement activation, staining for C5b-9 was performed using a mouse anti-human C5b-9 antibody (clone ae11, M0777, Agilent Technologies, Santa Clara, CA) at 1:25 dilution for 1 hour, followed by fluorescein isothiocyanate–conjugated polyclonal rabbit anti-mouse (F0261, Agilent) for 1 hour. Additional Data Sources Clinical data were obtained from electronic medical records or faxed clinical notes sent from outside nephrologists. Definitions Intensity of IF staining was graded semiquantitatively on a scale of 0 to 3þ: 0, absent; 1þ, weak; 2þ, moderate; and 3þ, strong. DISCLOSURE SC has been a consultant for Alexion Pharmaceuticals since 2018 and has received consulting and lecture fees. All the other authors declared no competing interests.
ACKNOWLEDGMENTS
We thank Dr. Natalia Maroz for providing clinical details on 5 patients, Cherri Bott and Kyle Ware for technical assistance in performing IgG subclass immunofluorescence, and Shawn Scully, Senior Systems Consultant at the Department of Pathology from the Ohio State University, for assistance with figure preparation. SUPPLEMENTARY MATERIAL Figure S1. Patient 4 underwent a biopsy on the same day as but prior to infusion of eculizumab. The specimen shows only tubular casts and background staining for IgG1 to IgG4 (top row; biopsy 4a in Table 2). A subsequent biopsy specimen 6 days later showed moderate IgG2 7
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and IgG4 deposition within arteriolar walls (bottom row; biopsy 4b in Table 2). Immunofluorescence 200. Supplementary material is linked to the online version of the paper at www.kidney-international.org. REFERENCES 1. Rother RP, Rollins SA, Mojcik CF, et al. Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria. Nat Biotech. 2007;25:1256. 2. Hillmen P, Young NS, Schubert J, et al. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2006;355:1233–1243. 3. Legendre CM, Licht C, Muus P, et al. Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome. N Engl J Med. 2013;368:2169–2181. 4. George JN, Nester CM. Syndromes of thrombotic microangiopathy. N Engl J Med. 2014;371:654–666. 5. Brocklebank V, Wood KM, Kavanagh D. Thrombotic microangiopathy and the kidney. Clin J Am Soc Nephrol. 2018;13:300–317. 6. Tsai HM. Atypical hemolytic uremic syndrome: beyond hemolysis and uremia. Am J Med. 2019;132:161–167. 7. Goodship TH, Cook HT, Fakhouri F, et al. Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference. Kidney Int. 2017;91:539–551. 8. Zuber J, Fakhouri F, Roumenina LT, et al. Use of eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies. Nat Rev Nephrol. 2012;8:643–657. 9. Cavero T, Rabasco C, Lopez A, et al. Eculizumab in secondary atypical haemolytic uraemic syndrome. Nephrol Dial Transplant. 2017;32:466–474. 10. Dhakal P, Giri S, Pathak R, Bhatt VR. Eculizumab in transplant-associated thrombotic microangiopathy 2017;. 2017;23:175–180. 11. Matsumura D, Tanaka A, Nakamura T, et al. Coexistence of atypical hemolytic uremic syndrome and crescentic IgA nephropathy treated with eculizumab: a case report. Clin Nephrol Case Stud. 2016;4:24–28. 12. El-Husseini A, Hannan S, Awad A, et al. Thrombotic microangiopathy in systemic lupus erythematosus: efficacy of eculizumab. Am J Kidney Dis. 2015;65:127–130.
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