Complement Activation And Thrombotic Microangiopathy Associated With Monoclonal Gammopathy: A National French Case Series Ⅰ
Dec 29, 2023
Rationale & Objective: Hemolytic uremic syndrome (HUS), a thrombotic microangiopathy (TMA) with kidney involvement, is a rare condition in patients with monoclonal gammopathy. In the absence of known causes of TMA, the role of complement activation in endothelial injury in patients with monoclonal gammopathy remains unknown and was the focus of this investigation.
Study Design: Case series.
Setting & Participants: We studied the 24 patients in the French national registry of HUS between 2000 and 2020 who had monoclonal gammopathy without other causes of secondary TMA. We provide the clinical histories and complement studies of these patients.
Findings: Monoclonal gammopathy–associated TMA with kidney involvement is estimated to be 10 times less frequent than adult atypical HUS (aHUS) in the French national registry. It is characterized by severe clinical features, with 17 of 24 patients requiring dialysis at disease onset, and with median renal survival of only 20 months. TMA-mediated extrarenal manifestations, particularly cutaneous and neurological involvement, were common and associated with poor overall prognosis. Complement studies identified low C3, normal C4, and high soluble C5b-9 levels in 33%, 100%, and 77% of tested patients, respectively, indicating a contribution of the alternative and terminal complement pathways in the pathophysiology of the disease. Genetic abnormalities in complement genes known to be associated with aHUS were found in only 3 of 17 (17%) who were tested.
Limitations: Retrospective study without comparison group; limited number of patients, limited available blood samples.
Conclusions: Within the spectrum of TMA, TMA associated with monoclonal gammopathy represents a distinct subset. Our findings suggest that HUS associated with monoclonal immunoglobulin is a complement-mediated disease akin to aHUS.
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The hemolytic uremic syndrome is a thrombotic microangiopathy (TMA) characterized, in its complete form, by a triad of thrombocytopenia, mechanical hemolytic anemia, and acute kidney injury, which results from thrombi formation within glomerular capillaries, arterioles, and small arteries. Causes of secondary TMA are heterogeneous and reflect the large spectrum of biological mechanisms involved in renal endothelial injury, including Shiga toxin–producing bacteria, cobalamin C defect, and various coexisting diseases or conditions (malignancy, drugs, transplant, systemic disease, infections). Atypical hemolytic uremic syndrome (aHUS) refers to complement-mediated TMA characterized by over-activation of the alternative complement pathway (AP) on endothelial cell surfaces. aHUS mainly affects children and young adults and is most commonly inherited, with 60% of patients having pathogenic variants of AP regulatory proteins such as factor H, factor I, CD46, components of the C3 convertase (C3 or factor B), or, less frequently, thrombomodulin.1 Acquired causes of AP-mediated TMA are less frequent, identified in 5%-15% of cases in European countries, also with predominant childhood onset. In these cases, endothelial AP overactivation is driven by polyclonal anti–factor H antibodies, which are typically associated with homozygous CFHR1-CFHR3 deletion.2 aHUS is associated with low C3 level in 30% of cases.1 Since 2011, treatment of aHUS has been based on eculizumab, a monoclonal antibody against the C5 complement protein that has transformed the renal prognosis of patients.3
Monoclonal gammopathy is defined by the presence of monoclonal immunoglobulin in the serum or urine, which is secreted by benign or malignant clonal plasma cells or B lymphocytes. A high prevalence of monoclonal immunoglobulin has been recently described in 2 small series of adult patients with TMA, including patients with kidney involvement, suggesting a pathogenic link between monoclonal gammopathy and TMA.4,5 Schurder et al reported favorable renal response to eculizumab in a patient with monoclonal gammopathy and TMA,6 indicating that abnormal complement activation may be central to the development of TMA, as already shown in monoclonal Ig–associated C3 glomerulopathy.7 To date, the potential mechanisms of endothelial injury in monoclonal gammopathy–associated TMA remain unknown.
In this retrospective series of cases identified in the French nationwide hemolytic uremic syndrome registry, we aimed to determine epidemiological, clinical, and immunological characteristics of monoclonal gammopathy–associated TMA with kidney involvement.
Methods Study Population
Patients were selected for inclusion based on the following criteria: (i) diagnosis of TMA in 2000-2020, with kidney involvement (see below); (ii) detection of a serum and/or urine monoclonal immunoglobulin by electrophoresis and immunofixation; and (iii) complement studies performed at the Laboratory of Immunology at Hospital Europeen Georges-Pompidou (see below). Patients gave consent for participation in the French national registry and for genetic analysis. The genetic study was approved by the ethics committee of the French National Clinical Research Projects Authority (approval AOM08198).
We excluded patients with other possible causes of TMA, such as thrombotic thrombocytopenic purpura (TTP), cryoglobulinemia, POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes) syndrome, or TMA secondary to drugs, bone marrow transplant, infection, or autoimmune disorders. Patients with positive anti-nuclear, anti–double–stranded DNA, anti-phospholipid, or anti–β2-glycoprotein 1 antibody and those with antineutrophil cytoplasmic antibodies were excluded. Diagnostic criteria for TMA with kidney involvement were defined by the occurrence of the following: thrombocytopenia (platelet count <150 × 109 / L), mechanical hemolytic anemia (at least 2 among hemoglobin level <10 g/dL, increased serum lactate dehydrogenase level, low haptoglobin level, or full blood count showing >1% spherocytosis), acute kidney injury (1.5-fold increase in serum creatinine level or >15% increase from baseline level), and/or a kidney biopsy showing acute and/or chronic lesions of TMA, as previously described.
Demographic clinical and laboratory data were recorded at the time of diagnosis and at the last follow-up. The estimated glomerular filtration rate was calculated using the CKD-EPI creatinine equation.9 Serum and urine monoclonal immunoglobulin were detected and quantified by electrophoresis and characterized by immunofixation. The diagnoses of monoclonal gammopathy of undetermined significance, multiple myeloma, chronic lymphocytic leukemia, and Waldenstrom macroglobulinemia were established according to international criteria.10,11
Pathology Studies
A kidney biopsy was performed in 22 of 24 patients. All kidney biopsy samples were processed for light and immunofluorescence microscopy according to standard methods. For immunofluorescence, 3-μm cryostat sections were stained using polyclonal fluorescein isothiocyanate conjugates specific for α, γ, and μ heavy chain and κ and λ light chain (Dakopatts) and C3, C4, C1q, fibrin, and albumin (Morphosys). The pathology-based diagnosis of acute TMA was assessed in the presence of glomerular ischemic changes, including endothelial swelling and narrowing of the capillary lumens, fibrin/thrombi deposits in capillary lumens or fibrinoid necrosis of larger vessels, and thrombosis and endothelial cell proliferation in small arteries and arterioles. Chronic TMA was diagnosed based on intense wall thickening of small arteries and arteriolar walls, aneurysmal dilation and proliferation of arterioles, double contour appearance of the glomerular basement membranes, swelling and detachment of glomerular endothelial cells, and subendothelial accumulation of proteins and cellular debris.5 Electron microscopy was performed in 9 cases on ultrathin sections processed and examined under a JEM-1010 electron microscope (JEOL).
Assays for Complement Components and Genetic Analysis
All immunological and genetic analyses were performed at the French reference laboratory for the investigation of the complement system (Hospital Europeen GeorgesPompidou). EDTA plasma samples were obtained from all patients at diagnosis of hemolytic uremic syndrome. Plasma concentrations of C3 and C4 were measured by nephelometry. Soluble C5b-9 level was determined using the MicroVue sC5b-9 Plus enzyme immunoassay (Quidel) according to the manufacturer's instructions. Reference values were determined after testing plasma from 100 healthy donors. Anti-CFH antibodies were screened by enzyme-linked immunosorbent assay as previously described.12 Direct sequencing of all CFH, CFI, CD46, C3, CFB, and thrombomodulin exons was performed as previously described.13 Screening for complex genetic alterations affecting CFH, CFHR1, and CFHR3 secondary to nonallelic homologous recombination was undertaken using multiplex ligation–dependent probe amplification (MRC Holland) and homemade probes as previously described.14 In our study, only rare variants (ie, minor allele frequency <0.1% in the European population per the GnomAD database; gnomad.broadinstitute.org) were reported.
To demonstrate the contribution of complement over-activation, we tested the capacity of total purified immunoglobulin G (IgG) from 7 patients with monoclonal gammopathy–associated TMA to enhance complement on endothelial cells compared with control IgG from healthy donors and patients older than 50 years with aHUS without monoclonal immunoglobulin.
Results Epidemiological Data
Between 2000 and 2020, after the exclusion of all secondary causes of TMA, 24 adult patients with a diagnosis of TMA in the setting of monoclonal gammopathy were referred to the laboratory of immunology at Hospital Europeen Georges-Pompidou for complement studies (9 patients in 2000-2010 and 15 patients in 2011-2020, corresponding to a mean incidence of 1.5 cases per year). In comparison, in 2011-2020, there were 199 adult (aged >18 years) patients who were diagnosed with aHUS and referred to the laboratory of immunology for complement studies, including 60 patients older than 50 years. All 199 were tested for monoclonal immunoglobulin and were negative. This suggests that monoclonal gammopathy–associated TMA is 13 and 4 times less frequent than aHUS in adults and in adults older than 50 years, respectively. Observations relating to 3 of these patients have been reported previously.6,15,16
Table 1. Clinical and Laboratory Parameters at Baseline and During Follow-up of 24 Patients with TMA and Monoclonal Gammopathy
Clinical Features and Laboratory Evaluation of Patients at Onset of Kidney Disease
Patients' clinical and laboratory data are detailed in Table 1. At diagnosis, the median age was 63.5 (range, 30-85) years, and the ratio of men to women was 1.4:1. All patients had decreased kidney function characterized by acute kidney injury, with a median serum creatinine level of 363 (range, 124-1,708) μmol/L and median estimated glomerular filtration rate of 15 mL/min/1.73 m2 . At diagnosis, 17 of 24 (71%) patients required dialysis. Median proteinuria was 4 (range, 0.5-17) g/d, and none of the patients had nephrotic syndrome. Median platelet count was 110 (range, 28-277) × 109 /L, with a hemoglobin level of 8.3 (range, 5-15) g/dL. Twenty of 22 patients with available data had laboratory signs of TMA as described in Methods (thrombocytopenia and/or mechanical hemolytic anemia). Sixteen (63%) patients had concomitant TMA-mediated extrarenal manifestations, including skin lesions (n = 7), central nervous system symptoms (n = 9) or peripheral neuropathy (n = 3), gastrointestinal tract involvement (n = 2), heart involvement (n = 1), and pulmonary lesions (n = 1). Monoclonal gammopathy was of IgG (n = 19), IgA (n = 1), or IgM (n = 4) subtype. Light-chain isotype was λ in 14 of 24 (58%) patients. On serum electrophoresis, the median monoclonal immunoglobulin level was 5.5 (range, 2-25) g/L. The hematological diagnosis was consistent with monoclonal gammopathy of undetermined significance (n = 18), symptomatic multiple myeloma (n = 2), chronic lymphocytic leukemia (n = 1), and Waldenstrom macroglobulinemia (n = 3; Table 2).
Table 2. Immunological and Hematological findings at Diagnosis of 24 Patients With TMA and Monoclonal Gammopathy
Complement Study and Genetic Analysis
Eight of 24 (33%) patients had low C3 level, with a median plasma C3 level of 835 (range, 383-1,710) mg/L. C4 level was normal in all but one case. Soluble C5b-9 was increased in 10 of 13 (77%) tested patients (Table 2). Six of 22 tested patients (27%) were positive for anti–factor H antibodies with low titers. In 2 cases, the anti–factor H isotype specificity was IgG3 and did not match the subclass of the serum IgG1 monoclonal gammopathy (patients 1 and 17). Patient 5 had IgA monoclonal gammopathy and anti–factor H IgA antibodies. Of 17 patients who underwent genetic analysis, rare pathogenic variants in complement genes were identified in 3 (17%), including 2 with a C3 variant (a predicted lysine-to-glutamine substitution at amino acid 65 [p.Lys65Gln]; minor allele frequency, 0.006%) and 1 with a CFI variant (a predicted alanine-to-threonine change at amino acid 258 [p.Ala258Thr]; minor allele frequency, 0.04%). Among the 6 patients positive for anti–factor H antibodies, only 1 was found to have a homologous deletion in CFHR1. When endothelial cells were incubated with IgG purified from patients, C3 and C5b-9 deposits were significantly increased in samples from 4 of 7 (57%) patients with monoclonal gammopathy–associated TMA (ie, had a mean value >2 SD greater than that observed in the presence of IgG from 13 healthy donors). A significant increase in complement deposits was observed in the presence of immunoglobulin from 1 of 7 (14%) control patients with aHUS without monoclonal immunoglobulin (Fig S1).
Pathology Data
Kidney biopsies, which were performed in 22 of 24 patients, invariably confirmed the presence of TMA lesions affecting glomeruli and/or extraglomerular small vessels (Table 3). In 18 (82%) cases, TMA lesions were acute, defined by glomerular and/or arteriolar thrombi (Fig 1A and B; Fig S2), mesangiolysis, and/or endothelial swelling. Four patients exhibited predominantly chronic changes (Fig 1D and E; Fig S3) with double-contour appearance of the glomerular capillary walls and arteriolar "onion-skin" lesions. Of these, 3 patients had concomitant clinical evidence of TMA from laboratory studies despite undetectable acute TMA lesions in the kidney. Glomerular extra-capillary proliferation was observed in 2 patients, and 2 others showed mild diffuse endocapillary hypercellularity. Immunofluorescence studies showed segmental glomerular capillary wall or intraluminal staining for fibrin in 7 patients (Fig 1C). In 5 patients, weak C3 staining was positive within capillary walls. In one patient, IgA staining was positive along capillary walls. The remaining 16 biopsy specimens did not show any monoclonal immunoglobulin and/or C3 in the glomeruli or along vessel walls. As reported previously,16 for one patient, monoclonal IgA λ light chain deposition was detected within glomerular thrombi and along capillary walls. Another case, in a subsequent biopsy, performed a few months later, exhibited glomerular linear deposits of monoclonal γ heavy chain suggestive of γ heavy-chain deposition disease. Ultrastructural studies confirmed the presence of TMA lesions in all 7 patients with adequate samples available (Fig 1F-H).
