NELL1 is a target antigen in malignancy-associated membranous nephropathy
Authors: Tiffany N. Caza, Samar I. Hassen, Zeljko Dvanajscak, Michael Kuperman, Rick Edmondson, Christian Herzog, Aaron Storey, John Arthur, L. Nicholas Cossey, Shree G. Sharma, Daniel J. Kenan, Christopher P. Larsen
Journal: Kidney International (2020)

Patients with membranous nephropathy have an increased risk of malignancy compared to the general population, but the target antigen for malignancy-associated membranous nephropathy is unknown. To explore this, we utilized mass spectrometry for antigen discovery in malignancy-associated membranous nephropathy examining immune complexes eluted from frozen kidney biopsy tissue using protein G bead immunoglobulin capture. Antigen discovery was performed comparing cases of membranous nephropathy of unknown and known type. Mass spectrophotometric analysis revealed that nerve epidermal growth factor-like 1 (NELL1) immune complexes were uniquely present within the biopsy tissue in membranous nephropathy. Additional NELL1-positive cases were subsequently identified by immunofluorescence. In a consecutive series, 3.8% of PLA2R- and THSD7A-negative cases were NELL1-positive. These NELL1-positive cases had segmental to incomplete IgG capillary loop staining (93.4%) and dominant or co-dominant IgG1-subclass staining (95.5%). The mean age of patients with NELL1-positive membranous nephropathy was 66.8 years, with a slight male predominance (58.2%) and 33% had concurrent malignancy. Compared with PLA2R- and THSD7A-positive cases of membranous nephropathy, there was a greater proportion of cases with malignancies in the NELL1-associated group. Thus, NELL1-associated membranous nephropathy has a unique histopathology characterized by incomplete capillary loop staining, IgG1-predominance, and is more often associated with malignancy than other known types of membranous nephropathy. Patients with membranous nephropathy have an increased risk of malignancy compared to the general population, but the target antigen for malignancy-associated membranous nephropathy is unknown. To explore this, we utilized mass spectrometry for antigen discovery in malignancy-associated membranous nephropathy examining immune complexes eluted from frozen kidney biopsy tissue using protein G bead immunoglobulin capture. Antigen discovery was performed comparing cases of membranous nephropathy of unknown and known type. Mass spectrophotometric analysis revealed that nerve epidermal growth factor-like 1 (NELL1) immune complexes were uniquely present within the biopsy tissue in membranous nephropathy. Additional NELL1-positive cases were subsequently identified by immunofluorescence. In a consecutive series, 3.8% of PLA2R- and THSD7A-negative cases were NELL1-positive. These NELL1-positive cases had segmental to incomplete IgG capillary loop staining (93.4%) and dominant or co-dominant IgG1-subclass staining (95.5%). The mean age of patients with NELL1-positive membranous nephropathy was 66.8 years, with a slight male predominance (58.2%) and 33% had concurrent malignancy. Compared with PLA2R- and THSD7A-positive cases of membranous nephropathy, there was a greater proportion of cases with malignancies in the NELL1-associated group. Thus, NELL1-associated membranous nephropathy has a unique histopathology characterized by incomplete capillary loop staining, IgG1-predominance, and is more often associated with malignancy than other known types of membranous nephropathy. Membranous nephropathy (MN) is the most common cause of idiopathic nephrotic syndrome in adults. It is a disease caused by the deposition of immune complexes in a glomerular distribution. It can be caused by antibodies directed against podocyte antigens (primary MN) or through secondary deposition of circulating immune complexes (or alternatively in situ immune complex formation) in glomeruli. The most common causes of secondary MN are autoimmune diseases, of which systemic lupus erythematosus accounts for the majority of cases (membranous lupus nephritis). The second most common secondary etiology is malignancy. Other causes include infections (such as viral hepatitis), graft-versus-host disease, allergic responses (such as against bovine serum albumin), and drug reactions. MN is the most common glomerular disease occurring coincidently with neoplasms. It can be the first sign of an occult malignancy in up to 20% of cases.1Beck J.L. Membranous nephropathy and malignancy.Semin Nephrol. 2010; 30: 635-644Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar This association was first reported by Lee et al. in 1966, where 11% of patients with nephrotic syndrome had an underlying carcinoma, and enriched in patients with MN (73%).2Lee J.C. Yamauchi H. Hopper Jr., J. The association of cancer and the nephrotic syndrome.Ann Intern Med. 1966; 64: 41-51Crossref PubMed Scopus (298) Google Scholar The risk of cancer in patients with MN is 2- to 3-fold higher than the age-matched general population up to 4 years after the diagnosis of MN.3Birkeland S.A. Storm H.H. Glomerulonephritis and malignancy: a population-based analysis.Kidney Int. 2003; 63: 716-721Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Although malignant neoplasms are a known "secondary" association of MN, determining who should undergo a thorough workup to exclude a coexistent cancer is a subject of debate. Risk factors for concurrent malignancy include older age (>65 years), a ≥20-pack-year smoking history, use of chronic immunosuppression, or presence of thrombotic complications (such as renal vein thrombosis, deep venous thrombosis, or pulmonary embolism).4Pani A. Porta C. Cosmai L. et al.Glomerular diseases and cancer: evaluation of underlying malignancy.J Nephrol. 2016; 29: 143-152Crossref PubMed Scopus (30) Google Scholar In a study by Ronco, as many as 22% of patients with MN who were 60 years of age or older had malignancy, which was 10-fold higher than that in age-matched controls.5Ronco P.M. Paraneoplastic glomerulopathies: new insights into an old entity.Kidney Int. 1999; 56: 355-377Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar Other factors characteristic of malignancy-associated MN include phospholipase A2 receptor (PLA2R) negativity and IgG1- and/or IgG2-restricted subclass predominance.6Lonnbro-Widgren J. Ebefors K. Molne J. et al.Glomerular IgG subclasses in idiopathic and malignancy-associated membranous nephropathy.Clin Kidney J. 2015; 8: 433-439Crossref PubMed Scopus (24) Google Scholar,7Ohtani H. Wakui H. Komatsuda A. et al.Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy.Nephrol Dial Transplant. 2004; 19: 574-579Crossref PubMed Scopus (144) Google Scholar Neural epidermal growth factor–like 1 (NELL1) is a recently described antigen in MN.8Sethi S. Debiec H. Madden B. et al.Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.Kidney Int. 2020; 97: 163-174Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Our findings suggest that NELL1-associated MN is commonly associated with malignancy and has unique histopathologic findings that may aid in the identification of cases in everyday clinical practice. Protein G was used to isolate IgG-containing immune complexes from frozen tissue remnants from each of the 59 renal biopsies. These affinity-purified immune complexes were then analyzed by mass spectrometry (MS) to determine their proteomic composition. Two groups were compared: the first group contained MN cases of known antigen types, including phospholipase A2 receptor (PLA2R, n = 4), thrombospondin type-1 domain-containing 7A (THSD7A, n = 3), and exostosin 1/2 (EXT1/2, n = 4). Diabetic glomerulopathy and arterionephrosclerosis samples with no positive immunofluorescence staining were used as negative controls (n = 7). The second group contained cases negative for PLA2R, THSD7A, and EXT1/2 (n = 41). By comparing proteomes from the known with the unknown MN types, it was possible to discern unique proteins. This technique was highly sensitive and specific for the identification of antigenic targets in MN of known type. PLA2R was the only protein that immunoprecipitated with IgG in 4 of 4 PLA2R-positive MN biopsies and 0 of 55 remaining biopsies (Supplementary Figure S1A). THSD7A was the only protein identified in 3 of 3 THSD7A-positive MN cases and 0 of 56 remaining biopsies (Supplementary Figure S1B). EXT was identified in 4 of 4 EXT-positive MN cases and 0 of 55 remaining biopsies (Supplementary Figure S1C). There were no proteins that uniquely immunoprecipitated with IgG in diabetic nephropathy or arterionephrosclerosis samples. Thus, as a proof of principle, this approach specifically identified the autoantigen in cases with MN of known type. When the same analysis was applied to cases of MN of unknown antigenic type, there were 2 of 41 cases in which NELL1 was identified (Figure 1). No membranous lupus nephritis cases (which included 16 of 41 PLA2R-negative, THSD7A-negative, and EXT1/2-negative MN cases) submitted for MS were NELL1 positive. This analysis confirms that IgG is bound to NELL1 in a subset of MN cases, which were also confirmed to be positive by tissue immunofluorescence staining. Dual NELL1-Rhodamine Red-X (Jackson Immunoresearch, West Grove, PA) and IgG-fluorescein isothiocyanate (FITC) labeling showed near-complete colocalization of NELL1 with IgG by confocal microscopy (86.7% ± 9.4%) (Supplementary Figure S2, top panel). There was no significant colocalization of NELL1 and IgG in PLA2R-positive MN cases used as controls (25.1% ± 14.4%) (Supplementary Figure S2, bottom panel). A graphical representation of the colocalization data is provided in Supplementary Figure S3. There was a significant increase in NELL1/IgG colocalization in NELL1-positive MN cases compared with PLA2R-positive MN controls (P < .0001). To determine the frequency of NELL1 positivity in MN, we stained 349 consecutive MN cases with tissue available for PLA2R, THSD7A, EXT2, and NELL1. This series included 83 pure class V membranous lupus nephritis (no proliferation) and 266 primary MN cases. Of the 349 total cases, 181 were PLA2R positive (51.9%), 10 were THSD7A positive (2.9%), 33 were EXT2 positive (9.5%), and 6 were NELL1 positive (1.7%). Of the 266 primary MN cases only, 180 were PLA2R positive (67.7%), 7 were THSD7A positive (2.6%), 6 were EXT2 positive (2.3%), and 5 were NELL1 positive (1.9%). Of the 83 class V membranous lupus nephritis cases, 1 was PLA2R positive (1.2%), 3 were THSD7A positive (3.6%), 27 were EXT2 positive (32.5%), and 1 was NELL1 positive (1.2%). As there were too small of a number of cases from the 9-month consecutive case series to identify potential clinical or histologic associations of NELL1-associated MN, NELL1-associated MN cases were identified from over the course of 5 years of MN cases. In total, 91 NELL1-associated MN cases were identified. On light microscopy, NELL1-associated MN cases showed prominent glomerular capillary loops (Figure 2a). Forty-one of 91 cases (45.1%) showed the formation of spikes or holes on silver stains (Figure 2b), often in a segmental distribution in glomeruli. A segmental (involvement of <50% of glomerular tuft) to incomplete global (segmental loops uninvolved by staining) capillary loop staining pattern was seen in 93.4% of cases of NELL1-associated MN (Figure 2c and d) and in no cases of PLA2R-associated (0 of 181) or THSD7A-associated (0 of 10) MN. Complete (100% tuft involvement) capillary loop staining was seen in 6 of 91 NELL1-associated MN (6.6%) compared with 100% of PLA2R-positive MN and 100% of THSD7A-positive MN control cases (Supplementary Figure S4). The segmental pattern of immune deposits was also seen by the distribution of electron-dense immune-type deposits by electron microscopy, showing sparing of some glomerular capillary loops (Figure 2e), with other loops containing electron-dense immune-type deposits (Figure 2f). Biopsies of patients with NELL1-associated MN showed no significant difference in IgA positivity (7.7% NELL1 vs. 10% PLA2R; P = .52), IgM positivity (9.9% NELL1 vs. 15.5% PLA2R; P = .26), and C1q positivity (0% NELL1 vs. 2% PLA2R; P = .30) and lacked a full house immunofluorescence pattern with the expression of IgA, IgG, IgM, C3, and C1q (0% NELL1 vs. <1% PLA2R; P = 1.0) as compared with PLA2R-positive MN (Table 1). C3 positivity was less common in NELL1-associated MN than in PLA2R-positive MN (78.0% NELL1 vs. 91% PLA2R; Fisher exact test, P = .002). There was no significant extraglomerular staining in cases of NELL1-associated MN, with no cases demonstrating deposits along tubular basement membranes, Bowman's capsule, or vessels. EXT2-positive MN cases had a higher frequency of IgA, IgM, C3, and C1q, full house immunofluorescence, and the presence of mesangial electron-dense deposits than did NELL1-associated, PLA2R-positive, or THSD7A-positive MN cases. A summary of the histopathologic features is given in Table 1.Table 1Histopathologic parameters of kidney biopsies with NELL1-associated membranous nephropathy compared with PLA2R-associated, THSD7A-associated, and EXT2-associated membranous nephropathyParameterNELL1-positive (n = 91)PLA2R-positive (n = 181)THSD7A-positive (n = 10)EXT2-positive (n = 33)P (Fisher exact test NELL1 vs. PLA2R)Incomplete global capillary loop (>50%)44 of 91 (48.3)0 of 181 (0)0 of 10 (0)0 of 33 (0)<0.0001Pure segmental (<50%)41 of 91 (45.0)0 of 181 (0)0 of 10 (0)0 of 33 (0)<0.0001Segmental or incomplete capillary loop staining85 of 91 (93.4)0 of 181 (0)0 of 10 (0)0 of 33 (0)<0.0001IgA positivity7 of 91 (7.7)19 of 181 (10)4 of 10 (40)12 of 33 (36)0.52IgG positivity91 of 91 (100)181 of 181 (100)10 of 10 (100)33 of 33 (100)N/AIgM positivity9 of 91 (9.9)28 of 181 (15.5)1 of 10 (10)12 of 33 (36)0.26C3 positivity71 of 91 (78.0)166 of 181 (91)6 of 10 (80)31 of 33 (94)0.002C1q positivity0 of 91 (0)4 of 181 (2)1 of 10 (10)12 of 33 (36)0.30Full-house immunofluorescence0 of 91 (0)1 of 181 (<1)0 of 10 (0)5 of 33 (15)1.00Mesangial deposits21 of 88aMesangial and subendothelial electron-dense deposits are assessed from 88 total cases instead of 91 total cases, as 3 of 91 cases did not have a glomerulus available for evaluation by electron microscopy. (23.9)19 of 181 (10)4 of 10 (40)26 of 33 (79)0.01Subendothelial deposits0 of 88aMesangial and subendothelial electron-dense deposits are assessed from 88 total cases instead of 91 total cases, as 3 of 91 cases did not have a glomerulus available for evaluation by electron microscopy. (0)2 of 181 (1)1 of 10 (10)6 of 33 (18)1.00EXT2, exostosin-2; N/A, not applicable; NELL1, nerve epidermal growth factor-like 1; PLA2R, phospholipase A2 receptor; THSD7A, thrombospondin type-1 domain-containing 7A.Data include frequency (% total), unless otherwise noted.a Mesangial and subendothelial electron-dense deposits are assessed from 88 total cases instead of 91 total cases, as 3 of 91 cases did not have a glomerulus available for evaluation by electron microscopy. Open table in a new tab EXT2, exostosin-2; N/A, not applicable; NELL1, nerve epidermal growth factor-like 1; PLA2R, phospholipase A2 receptor; THSD7A, thrombospondin type-1 domain-containing 7A. Data include frequency (% total), unless otherwise noted. IgG subclass staining was performed on 67 cases with residual frozen tissue available for testing (Table 2). There was IgG1 staining in all cases of NELL1-associated MN (67 of 67 cases) (Figure 3; Supplementary Figure S5), and 49 of 67 cases were IgG1 dominant (73.1%). Eighteen of 67 cases (26.9%) had IgG1 restriction. Concurrent IgG2 staining was present in 25 of 67 cases (37.3%), and concurrent IgG4 staining was present in 36 of 67 cases (53.7%). Three of 67 cases (4.5%) were IgG1 and IgG2 codominant, and 12 of 67 cases (17.9%) were IgG1 and IgG4 codominant. Therefore, 95.5% of NELL1-associated MN cases had dominant or codominant IgG1 subclass staining (Figure 3). There were 3 of 67 cases with IgG1, IgG2, and IgG3 staining (4.5%) without IgG4, a pattern seen in autoimmune disease.Table 2IgG subclass staining in NELL1-associated MNaStaining intensity trace or greater is included.IgG subclassNumber of casesPercentage of totalIgG167 of 67100IgG225 of 6737.3IgG310 of 6714.9IgG436 of 6753.7IgG1-dominant49 of 6773.1IgG2-dominant0 of 670IgG3-dominant0 of 670IgG4-dominant3 of 674.5IgG1/IgG2-codominant3 of 674.5IgG1/IgG4-codominant12 of 6717.9IgG1/IgG2/IgG3-positive3 of 674.5IgG1 restriction18 of 6726.9MN, membranous nephropathy; NELL1, neural epidermal growth factor–like 1.a Staining intensity trace or greater is included. Open table in a new tab MN, membranous nephropathy; NELL1, neural epidermal growth factor–like 1. Electron microscopy was available for 88 of 91 cases of NELL1-associated MN. A majority of cases showed severe podocyte foot process effacement (>50%) (69 of 88 cases [78.4%]), with 11 of 88 cases (12.5%) showing moderate podocyte foot process effacement (25%–50%), and 8 of 88 cases (9.1%) showing mild podocyte foot process effacement (<25%). NELL1-associated MN cases had a higher incidence of mesangial deposits (21 of 88 cases for NELL1 [23.9%] vs. 19 of 181 cases for PLA2R [10%]; Fisher exact test, P = .01) and no subendothelial deposits (0 of 88 cases [0%] vs. 2 of 181 cases [1%]; P = 1.0) (Table 1). Patients with NELL1-associated MN were older than patients with PLA2R-positive MN (mean age, 66.8 ± 10.8 years NELL1 vs. 56.4 ± 13.9 years PLA2R), patients with THSD7A-associated MN (mean age, 45.1 ± 16.3 years), and EXT-associated MN (mean age, 39.6 ± 16.1 years). Patients with NELL1-associated MN with malignancy were significantly older than patients without malignancy (71.0 ± 8.6 years vs. 65.0 ± 10.5 years; Student t test, P = .01). There was a slight male predominance (58.2%). Approximately a quarter of NELL1-associated MN cases had a history of diabetes mellitus (22 of 91 [24.2%]); however, only 9% had changes in diabetic nephropathy on biopsy. Twenty of 91 patients with NELL1-associated MN (22.0%) had acute tubular injury on biopsy, and 2 of 91 (2.2%) showed concurrent acute interstitial nephritis. Additional disease associations comparing PLA2R-, THSD7A-, EXT2-, and NELL1-positive MN are provided in Supplementary Table S1. Two cases of NELL1-associated MN had a history of Crohn's disease. To further explore this, we stained all cases of PLA2R-negative and THSD7A-negative MN in patients with a history of Crohn's disease, ulcerative colitis, or inflammatory bowel disease from our database and found that 2 of 8 cases (25%) were NELL1 positive. Although NELL1 was identified as a gene of interest in Crohn's disease in a genome-wide association study,9Franke A. Hampe J. Rosenstiel P. et al.Systematic association mapping identifies NELL1 as a novel IBD disease gene.PLoS One. 2007; 2: e691Crossref PubMed Scopus (103) Google Scholar whether this represents a potential secondary etiology for the development of NELL1-associated MN in a subset of patients is unclear. Five years of idiopathic MN biopsies from our database were studied to identify malignancy-associated MN cases. One hundred eleven malignancy-associated MN cases were identified, of which 35 were PLA2R positive, 4 were THSD7A positive, and 30 were NELL1 positive (Table 3). Given the much higher prevalence of PLA2R-positive MN than of NELL1-positive MN, patients with cancer are more likely to have PLA2R-positive MN. However, the prevalence of malignancy is much higher in patients with NELL1-positive MN than in those with other types of MN. For example, 33% of NELL1-positive patients had malignancy (30 of 91) whereas only 4.2% of PLA2R-positive patients (35 of 829) and 10.8% of THSD7A-positive patients had concurrent malignancy (4 of 37). Two THSD7A-positive patients and 4 NELL1-positive cases had 2 concurrent primary tumors (Table 3). The associated antigens for malignancy-associated MN were unknown in 42 patients, of whom 3 had 2 primary tumors (Supplementary Figure S6).Table 3Increased prevalence of malignancy in NELL1-associated MN compared with PLA2R-associated MN, THSD7A-associated MN, and MN due to unknown antigensaThe types and frequencies of tumors seen in patients with malignancy-associated MN are shown.Type of neoplasmPLA2R-positive (n = 35 of 829)THSD7A-positive (n = 4 of 37)NELL1-positive (n = 30 of 91)Unknown antigen (n = 42 of 421)Prostate adenocarcinoma103610Breast carcinoma12158Gastric carcinoma0012Soft-tissue tumor0010Glioma0010Colon adenocarcinoma1016Lung carcinoma1034Bladder carcinoma5020Renal cell carcinoma2135Pancreatic carcinoma0001Hepatocellular carcinoma0001Thyroid carcinoma0012Ovarian carcinoma0010Uterine carcinoma0001Cervical carcinoma1010Testicular carcinoma1000Skin (melanoma, BCC, SCC)2041SCC (head/neck)0112Laryngeal carcinoma0001Nasopharyngeal carcinoma0010Thymoma0010Lymphoma0011Total356bAlthough 4 patients in the THSD7A-positive cohort had a history of malignancy, the number of tumors is >4 because of 2 patients with 2 primary tumors.34cAlthough 30 patients in the NELL1-positive cohort had a history of malignancy, the number of tumors is >30 because of 4 patients with 2 primary tumors.45dForty-two patients with MN of unknown type had a history of malignancy, and the number of tumors is >42 because of 3 patients with 2 primary tumors.BCC, basal cell carcinoma; MN, membranous nephropathy; NELL1, neural epidermal growth factor–like 1; PLA2R, phospholipase A2 receptor; SCC, squamous cell carcinoma; THSD7A, thrombospondin type-1 domain-containing 7A.a The types and frequencies of tumors seen in patients with malignancy-associated MN are shown.b Although 4 patients in the THSD7A-positive cohort had a history of malignancy, the number of tumors is >4 because of 2 patients with 2 primary tumors.c Although 30 patients in the NELL1-positive cohort had a history of malignancy, the number of tumors is >30 because of 4 patients with 2 primary tumors.d Forty-two patients with MN of unknown type had a history of malignancy, and the number of tumors is >42 because of 3 patients with 2 primary tumors. Open table in a new tab BCC, basal cell carcinoma; MN, membranous nephropathy; NELL1, neural epidermal growth factor–like 1; PLA2R, phospholipase A2 receptor; SCC, squamous cell carcinoma; THSD7A, thrombospondin type-1 domain-containing 7A. Of note, the epidemiology of MN changed within this 5-year time period, where PLA2R-positive MN made up 87% of cases of MN at our institution in 2014, but only 42% in 2019. We attribute this to serum-based approaches to the diagnosis of primary MN,10De Vriese A.S. Glassock R.J. Nath K.A. et al.A proposal for a serology-based approach to membranous nephropathy.J Am Soc Nephrol. 2016; 28: 421-430Crossref PubMed Scopus (147) Google Scholar with anti-PLA2R and anti-THSD7A assays now widely available for screening of patients presenting with nephrotic syndrome. A majority of patients with NELL1-associated MN with a history of malignancy had concurrent proteinuria and ongoing malignancy (n = 19 of 30); 8 patients had the cancer identified before MN; 1 patient had malignancy identified in a workup for causes of secondary MN; and in 2 patients, the underlying malignancy preceded the diagnosis of MN but the time of diagnosis was unknown (Supplementary Table S2). Seven of these patients had metastatic disease at the time of diagnosis. We excluded patients in whom the diagnosis of malignancy was remote compared with the diagnosis of MN or in complete remission (n = 4 of 34 NELL1-positive patients had a history of malignancy; 30 patients in our cohort had active malignancy). The timing between the diagnosis of cancer and MN in our cohort (when known) is summarized in Supplementary Table S2. Serum creatinine at the time of biopsy and quantitative 24-hour proteinuria values were similar in patients with NELL1-associated MN and those with PLA2R-, THSD7A-, or EXT-associated MN (serum creatinine: PLA2R 1.93 ± 2.19 mg/dl, THSD7A 1.85 ± 2.34 mg/dl, NELL1 1.33 ± 0.86 mg/dl [data available for 73 of 91 patients]; 24-hour proteinuria: PLA2R 8.03 ± 5.09 g, THSD7A 6.68 ± 4.76 g, NELL1 6.6 ± 4.1 g [for which data were available for 59 of 91 patients]). A majority of patients in all groups had normal renal function at the time of biopsy, with the variation due to outliers with concurrent acute kidney injury. A majority of patients with NELL1-associated MN (71.2%) had nephrotic range proteinuria (≥3.5 g) at the time of biopsy (corresponding to 42 of 59 patients with available quantitative proteinuria data). Depressed albumin levels were seen in 74.5% of patients (38 of 51 patients with serum albumin values available with <3.5 g/dl albumin; mean albumin, 2.84 ± 0.95 g/dl). Tissue of the associated tumor was available from 2 patients with NELL1-associated MN. In 1 patient with NELL1-associated MN and concurrent invasive ductal carcinoma of the breast, NELL1 positivity by immunofluorescence and immunohistochemical staining was seen in the patient's breast biopsy specimen (Supplementary Figure S7A and B) and mastectomy specimen (Supplementary Figure S7C and D). A patient with active follicular lymphoma also demonstrated NELL1 staining on an excisional lymph node biopsy (Supplementary Figure S7E and F). Twenty-eight patients with NELL1-associated MN had blood samples available for testing serum reactivity against the NELL1 antigen. Twenty of 28 patients with NELL1-associated MN (71.4%) had serum reactivity to NELL1 recombinant protein under nonreducing conditions (representing Western blots in Supplementary Figure S8). No control sera from PLA2R-positive MN cases showed reactivity (0 of 28 patients). Of note, 3 of 8 NELL1-positive patients without serum reactivity were in clinical remission, with a >50% reduction in proteinuria at the time of serum collection compared with the time of biopsy. Clinical follow-up with treatment response (evaluating remission of MN) and laboratory data were available from 59 of 91 patients (64.8%). In addition to these 59 patients, 8 of 91 patients in our cohort were not on active treatment at follow-up as they were deceased or on dialysis (8 of 91 [12%]) and 6 of 91 (6.6%) did not return for clinical follow-up after their biopsy. The mean clinical follow-up period was 10.4 ± 13.5 months. The median proteinuria (± interquartile range) at follow-up was 1.9 ± 4.1 g. The median change in proteinuria from the time of biopsy was 5.4 ± 6.6 g. Twenty of 59 patients (34%) were in complete remission, with <300 mg of proteinuria per day. Sixteen of 59 patients (27%) were in partial remission, with 300 to 3500 mg of proteinuria per day or a >50% reduction in proteinuria from the time of biopsy. Twenty-three of 59 patients (39%) had no remission, with <50% reduction in proteinuria and >3.5 g of proteinuria per day. A majority of patients had stable renal function at follow-up (median creatinine, 1.1 ± 0.56 mg/dl). Comparisons of the effects of various treatments were between patients with available data (n = 59), and sample medians were provided. For patients on renin-angiotensin system blockade (with angiotensin-converting enzyme inhibitor and/or angiotensin receptor blockade therapy; n = 32), the median proteinuria at the time of biopsy was 4.9 ± 4.7 g, the median proteinuria at follow-up was 1.9 ± 4.0 g, and the median follow-up creatinine was 1.1 ± 0.4 mg/dl. In calcineurin inhibitor–treated patients (n = 8), the median proteinuria at the time of biopsy was 10 ± 9.1 g, the median proteinuria at follow-up was 6.4 ± 10.4 g, and the median follow-up creatinine was 1.8 ± 3.0 mg/dl. In patients treated with cyclophosphamide (n = 3), the median proteinuria at the time of biopsy was 9.6 g, the median proteinuria at follow-up was 3.9 g, and the median follow-up creatinine was 1.4 mg/dl (interquartile range not provided because of low n). In patients treated with mycophenolate and steroid therapy (n = 3), the median proteinuria at the time of biopsy was 8 g, the median proteinuria at follow-up was 0.1 g, and the median creatinine at follow-up was 0.9 mg/dl (interquartile range not provided because of low n). One patient was treated with rituximab, who presented with 10 g of proteinuria and had >10 g of proteinuria at follow-up and 1.0 mg/dl of creatinine at follow-up. For patients undergoing active treatment of an underlying malignancy and not on concurrent immunosuppressive therapy (n = 12), the median proteinuria at presentation was 5 ± 7.4 g, the median proteinuria at follow-up was 0.1 ± 2.1 g, and the median follow-up creatinine was 1.0 ± 0.9 mg/dl. Because of the small number of patients on immunosuppressive therapy (n = 15), an optimal treatment of NELL1-associated MN could not be ascertained; however, patients with successful treatment of an underlying malignancy showed good response to therapy (with 9 of 12 patients treated for malignancy without concurrent immunosuppression in complete or partial remission at follow-up). The treatment and follow-up data after therapy is included in Supplementary Table S3. Our study has several limitations. Complete clinical follow-up was not available for all patients. As patients with malignancy-associated MN can have cancer identified during a workup after the diagnosis of their glomerular disease up to 2 to 4 years after MN and we did not have longitudinal follow-up of all patients for ≥4 years, our data likely underestimate the true frequency of malignancy in patients with MN. NELL1 is a 140-kDa modular glycoprotein, which is expressed in low levels in adult tissues, but is active during development where it is essential for intramembranous and endochondral ossification. It shows sequence similarity to thrombospondin proteins, and its structure consists of 1 N-terminal laminin G–like domain, 3 von Willebrand factor type C (VWF-C) domains, 1 thrombospondin type-1 domain, and 5 tandem epidermal growth factor domains.11Kuroda S. Oyasu M. Kawakami M. et al.Biochemical characterization and expression analysis of neural thrombospondin-1-li