Characterization and outcomes of renal leukocyte chemotactic factor 2-associated amyloidosis

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1 & 2014 International Society of Nephrology see commentary on page 229 Characterization and outcomes of renal leukocyte chemotactic factor 2-associated amyloidosis Samar M. Said 1, Sanjeev Sethi 1, Anthony M. Valeri 2, Anthony Chang 3, Cynthia C. Nast 4, Leslie Krahl 5, Peter Molloy 5, Marc Barry 6, Mary E. Fidler 1, Lynn D. Cornell 1, Nelson Leung 7, Julie A. Vrana 1, Jason D. Theis 1, Ahmet Dogan 1 and Samih H. Nasr 1 1 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA; 2 Division of Nephrology, Columbia University, College of Physicians and Surgeons, New York, New York, USA; 3 Department of Pathology, University of Chicago Medical Center, Chicago, Illinois, USA; 4 Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, California, USA; 5 Department of Pathology, Banner Good Samaritan Medical Center, Phoenix, Arizona, USA; 6 Department of Pathology, University of New Mexico, Albuquerque, New Mexico, USA and 7 Division of Nephrology, Mayo Clinic, Rochester, Minnesota, USA Amyloidosis derived from leukocyte chemotactic factor 2 (ALECT2) is a recently described disease. Here, we report the characteristics and outcome of 72 patients with renal ALECT2, which included 19 who had another kidney disease on biopsy. Ninety-two percent of patients were Hispanics and over half were elderly. Three had other organ, but not cardiac, amyloidosis involvement. All patients without concurrent disease, except three, presented with chronic renal insufficiency. Proteinuria was variable and absent in a third, whereas nephrotic syndrome and hematuria were rare. After a median follow-up of 26 months, one-third developed end-stage renal disease (ESRD). The median renal survival was 62 months. Independent predictors of renal survival were serum creatinine at diagnosis, with a value of 2.0 mg/dl being the best cutoff for predicting ESRD, percentage global glomerulosclerosis, and presence of diabetes. Only four patients died and four had received chemotherapy for an erroneous diagnosis of immunoglobulin light chain-derived amyloidosis. Five patients underwent kidney transplantation; none had graft loss but one had disease recurrence. Patient survival is superior to renal immunoglobulin light chainderived amyloidosis and reactive amyloidosis largely due to the absence of cardiac involvement. Thus, renal ALECT2 mainly affects elderly Hispanics who typically present with chronic renal insufficiency and bland urine sediment, with or without proteinuria. Kidney International (2014) 86, ; doi: /ki ; published online 22 January 2014 KEYWORDS: mass spectrometry; nephrology; proteinuria; renal biopsy; transplant outcomes Correspondence: Samih H. Nasr, Division of Anatomic Pathology, Mayo Clinic, Hilton First Street, SW, Rochester, Minnesota 55905, USA. nasr.samih@mayo.edu Received 30 September 2013; revised 11 November 2013; accepted 21 November 2013; published online 22 January 2014 Amyloidosis is an uncommon group of diseases characterized by extracellular deposition of insoluble fibrils, which results from abnormal folding of proteins. Amyloidosis can either be localized or systemic and may affect any organ. Amyloid deposits characteristically stain Congo-red-positive and show apple-green birefringence when viewed under polarized light. The kidney is the most commonly affected organ in systemic amyloidosis. The two most common types of renal amyloidosis are immunoglobulin light chain-derived amyloidosis (AL) and reactive (secondary) amyloidosis (AA), but several of the rare hereditary forms of amyloidosis, such as those derived from fibrinogen A, apolipoprotein AI, apolipoprotein AIV, transthyretin, gelsolin, and lysozyme, may also affect the kidney. In 2008, Benson et al. 1 discovered a new form of amyloidosis derived from leukocyte chemotactic factor 2 (ALECT2) in a patient who presented with nephrotic syndrome and progressed to end-stage renal disease (ESRD) 7 years later. On the basis of the findings of two recent large renal biopsy series, ALECT2 is now the third most common type of renal amyloidosis in the United States following AL and AA, accounting for % of cases. 2,3 The pathogenesis of ALECT2 is still unknown. Histologically, the disease is characterized by diffuse interstitial amyloid deposits. 3 Murphy et al. 4,5 recently described a series of 10 patients with renal ALECT2; seven of them were Mexican Americans, with a mean age of 68 years at diagnosis, who presented with renal insufficiency and, with the exception of three, minimal proteinuria. Of the seven patients with available follow-up, one progressed to ESRD and one died. Aside from the information provided in the above study, there are no existing data on patient survival and renal survival in this disease. Furthermore, there are no available data on the disease recurrence and outcome after kidney transplantation. In this multicenter study, we analyzed the clinical renal characteristics, systemic characteristics, pathology, outcome, and poor prognostic indicators in a series of 72 patients with renal ALECT Kidney International (2014) 86,

2 SM Said et al.: Renal leukocyte chemotactic factor 2-associated amyloidosis RESULTS Clinical features The 72 patients with renal ALECT2 included 19 (26.4%) patients who had an additional kidney disease on biopsy. Table 1 details the demographics, clinical characteristics, and laboratory findings in all 72 patients and in the 53 without concurrent renal disease on biopsy. Most patients were elderly individuals with a median age of 65.5 years; 39 (54.2%) were X65 years of age and only 2 (2.8%) were below the age of 50 years at diagnosis. Sixty-six (91.7%) patients were Hispanic (of Mexican origin in all 34 patients who disclosed their origin). Out of the remaining six patients, three were from the Middle East (two were Arabs and one was Israeli), one was Native American, one was African, and one was white American of Asian descent. Two-thirds of the patients had a history of hypertension and 26.4% had diabetes. Nine (12.5%) patients had carcinoma (of the kidney in five, bladder in one, prostate in one, uterine in one, and breast in one), including four in whom ALECT2 was discovered in the non-neoplastic portion of nephrectomy specimen performed for kidney tumor resection. Five (6.9%) patients had a history of autoimmune disease (ANCA vasculitis in two, rheumatoid arthritis in one, mixed connective tissue disease in one, and Sjogren s syndrome in one). One patient had hepatic cirrhosis. A history of recent infection was present in four (5.6%) patients (pneumonia in two, skin infection in one, and urinary tract infection in one). Only three (4.2%) patients had histologically proven other-organ involvement by amyloidosis (one patient each showed duodenal, adrenal, and liver involvement). None of the patients had clinical evidence of cardiac involvement. One patient had peripheral neuropathy and one had carpal tunnel syndrome. One kidney biopsy performed for elevated serum creatinine was from a 5-month post-engraftment renal transplant in a patient with ESRD secondary to immunoglobulin (Ig)A nephropathy. On reexamination of the deceased donor s pre-transplant biopsies (from a 65-year-old Hispanic male), it was found that there was ALECT2 in both kidneys and the liver, which were inadvertently transplanted to two different recipients. None of the 72 patients had a known family history of amyloidosis. Five (9.6%) of 52 patients tested had a monoclonal protein on serum immunofixation (IgG kappa in three, IgM kappa in one, IgG kappa and IgG lambda in one). Bone marrow biopsy was performed in six patients, including three of those with positive serum immunofixation, which was positive for plasma cell dyscrasia without amyloid in two, positive for amyloid without plasma cell dyscrasia in one, and negative for both in three patients. Serum-free light chain ratio tested in 23 patients was normal in 20 and mildly elevated in three patients. The clinical renal characteristics in ALECT2 patients with or without a concurrent renal disease on biopsy were not statistically different (data not shown), except for higher proteinuria (P ¼ 0.02) and lower serum albumin (P ¼ 0.005) in those with a concurrent disease. The median serum Table 1 Demographics and clinical characteristics of renal ALECT2 Characteristics All patients (n ¼ 72) ALECT2 patients without a concurrent disease on biopsy (n ¼ 53) Age, median (range) 65.5 (43 88) 66.0 (43 88) Gender (M/F) 37 (51.4%)/35 (48.6%) 26 (49.1%)/27 (50.9%) Hispanic origin 66 (91.7%) 49 (92.5%) Hypertension 49 (68.1%) 33 (62.3%) S. creatinine in mg/dl, median (range) 2.3 ( ) 2.3 ( ) egfr (MDRD study equation) 26.9 (3 68) 25.5 (3 68) Renal insufficiency (S. creatinine 41.2 mg/dl) 64/70 (91.4%) 49/52 (94.2%) Dialysis at biopsy 7 (9.7%) 4 (7.5%) 24-h urine protein, median (range) 1.0 (0 20.0) 0.9 (0 8.9) Proteinuria 52/66 (78.8%) 34/49 (69.4%) Nephrotic range proteinuria 22/66 (33.3%) 15/49 (30.6%) Serum albumin, median (range) 3.7 ( ) 4.0 ( ) Edema 18/65 (27.7%) 13/49 (26.5%) Full nephrotic syndrome 7/69 (10.1%) 5/52 (9.6%) Microscopic hematuria 10/62 (16.1%) 7/46 (15.2%) Malignancy 9 (12.5%) 8 (15.1%) Diabetes mellitus 19 (26.4%) 9 (17.0%) Median duration of follow-up in mos (range) 21.5 (2 117) 26.0 (2 117) Outcome Stable renal function 19/64 (29.7%) 14/47 (29.8%) Progressive renal dysfunction 20/64 (31.3%) 18/47 (38.3%) Progression to ESRD 25/64 (39.1%) 15/47 (31.9%) Median time to ESRD in mos (range) 11 (0 73) 16 (0 73) Median follow-up S. creatinine in mg/dl (range) in patients not 2.3 ( ) 2.4 ( ) reaching ESRD Patients died 4/64 (6.3%) 3/47 (6.4%) Abbreviations: ALECT2, amyloidosis derived from leukocyte chemotactic factor 2; egfr, estimated glomerular filtration rate; F, female; M, male; MDRD, Modification of Diet in Renal Disease; mos, months. Kidney International (2014) 86,

3 SM Said et al.: Renal leukocyte chemotactic factor 2-associated amyloidosis creatinine at diagnosis was 2.3 mg/dl (range ). Serum creatinine did not correlate with the total renal amyloid load or with the degree of amyloid in glomeruli, interstitium, or vessels, but correlated with percent global glomerulosclerosis (P ¼ 0.002), tubular atrophy/interstitial fibrosis (TA/IF) (P ¼ 0.008), and arteriosclerosis (P ¼ 0.01). Proteinuria was not universal; it was absent in 30.6% of patients without a concurrent disease on biopsy, although it was within the nephrotic range in 30.6% of patients. The median 24-h urine protein was 0.9 g (range, g). Proteinuria did not correlate with the total renal or glomerular amyloid load. Hypoalbuminemia, peripheral edema, and full nephrotic syndrome were present in 35.5%, 26.5%, and 9.6% of patients without a concurrent disease on biopsy, respectively (Table 1). Microhematuria was documented in only seven (15.2%) of the patients, which included two who also had transitional cell carcinoma of the urinary tract. Prognosis Clinical follow-up was available in 64 (88.9%) patients, including 47 (88.7%) of those without a concurrent disease on biopsy. After a median follow-up of 26 months (range 2 117) in patients without a concurrent disease on biopsy, 29.8% had stable renal function, 38.3% had progressive renal dysfunction, and 31.9% developed ESRD (Table 1). The median final follow-up serum creatinine in patients not reaching ESRD was 2.4 mg/dl (range ). The median renal survival from diagnosis by the Kaplan Meier survival estimates was 62 months (Figure 1). In the 15 patients who reached ESRD, the median time from diagnosis to ESRD was 16 months (range 0 73). The patient with the longest follow-up in this cohort was diagnosed with renal amyloidosis, unclassified type, in Subsequent laser microdissection/mass spectrometry (LMD/MS) performed on the original paraffin block in 2011 confirmed ALECT2. His latest serum creatinine in June 2013 was 3.2 mg/dl and he has not needed dialysis yet. Only four (6.3%) patients died, one of sepsis and coagulopathy, one of pulmonary failure in the setting of pulmonary involvement by ANCA vasculitis, and two of unknown causes. By Cox regression for continuous variables, the predictors of ESRD by univariate analysis for all patients were serum creatinine at diagnosis (Po0.001), egfr at diagnosis ALECT2 only Creatinine 2 Creatinine < 2 Figure 1 ESRD-free survival in renal amyloidosis derived from leukocyte chemotactic factor 2 (ALECT2) without concurrent disease on biopsy and split by creatinine. (Po0.001), percent global glomerulosclerosis (P ¼ 0.01), TA/IF (P ¼ 0.03), and arteriosclerosis (Po0.04). For those patients who were without a concurrent disease on biopsy, the predictors were serum creatinine at diagnosis (Po0.001), egfr at diagnosis (Po0.001), and percent global glomerulosclerosis (P ¼ 0.02). By the Kaplan Meier survival estimates for categorical variables, predictors of reaching ESRD on univariate analysis for all patients were dialysis at diagnosis (Po0.001), presence of comorbid conditions (P ¼ 0.02), presence of diabetes (P ¼ 0.02), and presence of a concurrent disease on biopsy (P ¼ 0.02). For those patients who were without a concurrent disease on biopsy, only dialysis at diagnosis (Po0.001) was a predictor. Presence of diabetes was marginally significant (P ¼ 0.09). A value of 2.0 mg/dl for serum creatinine at diagnosis was the best cutoff for predicting ESRD for all patients and for those without a concurrent disease on biopsy (AUC on the ROC for ESRD 0.806, P ¼ 0.001, with a sensitivity of and specificity of 0.613). None of the patients with a serum creatinine o2.0 mg/dl progressed to ESRD (Figure 1). As expected, a serum creatinine o2.0 mg/dl correlated with less TA/IF (P ¼ 0.001) and less percent global glomerulosclerosis (P ¼ 0.003). However, it did not correlate with a younger age (P ¼ 0.4), which argues against the possibility of a lead time bias. By using a Cox proportional hazards model that did not include egfr, the independent predictor of the rate of progression to ESRD on multivariate analysis for all patients was serum creatinine at diagnosis (Po0.001; hazard ratio (HR), 1.582; 95% confidence interval (CI), ). Percent global glomerulosclerosis was marginally significant (P ¼ 0.05, HR 1.022, 95% CI ). For those patients who were without a concurrent disease on biopsy, the independent predictors were serum creatinine at diagnosis (P ¼ 0.001, HR 2.002, 95% CI ), percent global glomerulosclerosis (P ¼ 0.006, HR 1.049, 95% CI ), and the presence of diabetes (P ¼ 0.03, HR 7.196, 95% CI ). Four (6.3%) patients had received chemotherapy for an erroneous diagnosis of AL (only one of whom had a monoclonal protein in the blood). One received lenalidomide and dexamethasone followed by stem cell transplant and he progressed to ESRD. One received dexamethasone alone and progressed to ESRD. One received bortezomib and dexamethasone and he had progressive renal dysfunction. The fourth patient who had presented with full nephrotic syndrome and renal insufficiency (serum creatinine 1.5 mg/dl) and had concurrent mild IgA nephropathy on biopsy was treated with cyclophosphamide, bortezomib, and dexamethasone for 4 months, which led to complete remission of nephrotic syndrome and a decline in serum creatinine to 1 mg/dl. Five patients received kidney transplants from living donors (related in three and unrelated in two), which were preemptive in all but one patient. After a mean post-transplant follow-up of 20 months (range 4 34), none had graft loss, 372 Kidney International (2014) 86,

4 SM Said et al.: Renal leukocyte chemotactic factor 2-associated amyloidosis with a final serum creatinine of 1.2 mg/dl (range ). A 6-month post-transplant biopsy in one patient performed for allograft dysfunction showed recurrent disease in the interstitium, interstitial capillaritis, and borderline cellular rejection. Another patient had a 1-month post-transplant biopsy, which did not show recurrence. A third patient had 1-month, 4-month, 1-year, and 2-year post-transplant biopsies, none of which showed recurrence. The remaining two patients have not undergone post-transplant biopsies. The patient with donor-transmitted ALECT2 had a serum creatinine of 1.5 mg/dl 33 months post-transplant (down from 1.8 mg/dl at diagnostic biopsy) without proteinuria. A repeat allograft biopsy to determine whether the amyloid deposits have regressed has not been performed. Pathologic characteristics The pathologic characteristics of renal ALECT2 are summarized in Table 2. The most striking feature of ALECT2 is its preferential involvement of the cortical interstitium, which was present in all cases and was diffuse in 91.5% (Figure 2a d). Although medullary interstitial involvement was seen in most cases in which medulla was sampled, it was minimal in the majority (Figure 2c). Glomerular involvement was seen in 91.7% of cases but was minimal in one-third (Figure 2d) of the patients. Vascular involvement was present in 84.3% of cases but was minimal in 38.6%, with more frequent involvement of arterioles than interlobular arteries (Figure 2a d). There was also mild involvement of small capsular arteries in 2 (8.7%) of 23 cases in which the renal capsule was present in the sample but, when present, there was no involvement of perirenal adipose tissue. In all cases, amyloid deposits were Congo-red-positive with an apple-green birefringence under polarized light (Figure 2a d) and generally stained periodic acid-schiff-weak or -negative and Jones methenamine silvernegative. Compared with patients without a concurrent disease on biopsy, those with a concurrent disease had lower glomerular (P ¼ 0.002), interstitial (Po0.001), and overall (P ¼ 0.003) amyloid load, consistent with ALECT2 diagnosis at an earlier stage. The amyloid deposits were positive for an immunohistochemical stain for LECT2 in all 27 cases tested. Positive staining for SAA was detected in 3 of 38 (8.8%) cases tested, which was performed by the immunoperoxidase technique on paraffin tissue in two cases and by immunofluorescence on frozen tissue in one case. The intensity of staining was weak in two of these cases and strong in one. As SAA staining in these three cases was performed and reported in outside pathology laboratories, we cannot exclude the possibility that the false-positive staining is due to technical problems. False-positive staining of interstitial amyloid deposits for immunoglobulin heavy and/or light chains by immunofluorescence was seen in seven (10.6%) cases (detailed in Table 3); however, no case showed light chain restriction. Amyloid fibrils on electron microscopy appeared randomly oriented, and when measured had a mean diameter ranging from 7 to 12 nm, similar to other forms of amyloidosis (Figures 2e and 1f). Concurrent renal disease on biopsy was present in just over a quarter of the cases (Table 2); the most frequent concurrent disease was diabetic glomerulosclerosis (13.9%) followed by IgA nephropathy (6.9%). The median percent global glomerulosclerosis was 33%. Some degree of TA/IF was present in every case and most cases showed mild to moderate arteriosclerosis (Table 2). DISCUSSION In a recent series of 474 cases of renal amyloidosis processed by the Renal Biopsy Laboratory at Mayo Clinic, the four most common forms were Ig-derived (85.9%), AA (7.0%), ALECT2 (2.7%), and amyloidosis derived from fibrinogen A (1.3%). 3 In the 238 elderly patients (X65 years) included in the above study, ALECT2 was only slightly less common than AA (4.2% vs 5.9%). The mean age at diagnosis for patients with Table 2 Pathologic characteristics Amyloid load Cortical interstitial grade: 0/1 þ /2 þ /3 þ (%) 0/1/5/65 (0/1.4/7.0/91.5) a Glomerular grade: 0/1 þ /2 þ /3 þ (%) 6/25/13/28 (8.3/34.7/18.1/38.9) Vascular grade: 0/1 þ /2 þ /3 þ (%) 11/27/25/7 (15.7/38.6/35.7/10.0) b Overall (composite) grade: 0 3/4 6/7 9 (%) 2/35/32 (2.9/50.7/46.4) Number of glomeruli sampled: median (range) 17 (2 76) c Percent of globally sclerotic glomeruli: median (range) 33 (0 100) c Tubular atrophy and interstitial fibrosis: none/mild/moderate/severe (%) 0/19/24/27 (0/27.1/34.3/38.6) d Arteriosclerosis and arteriolar hyalinosis: none/mild/moderate/severe (%) 6/19/33/12 (8.6/27.1/47.1/17.1) b Concurrent renal disease on biopsy: number (%) 19 (26.4) Diabetic glomerulosclerosis 10 (13.9) IgA nephropathy 5(6.9) Membranous glomerulopathy 2(2.8) ANCA necrotizing and crescentic glomerulonephritis 1(1.4) Acute interstitial nephritis 1(1.4) Acute tubular necrosis 1(1.4) Abbreviation: Ig, immunoglobulin. a Scoring of interstitial amyloid could not be performed in one case due to the scant tissue on the Congo-red slide. b Vessels were not sampled in two cases. c Not including the four nephrectomy specimens. d Scoring could not be performed in two cases due to the scant tissue. Kidney International (2014) 86,

5 SM Said et al.: Renal leukocyte chemotactic factor 2-associated amyloidosis a b c d e f Figure 2 Renal pathology in renal amyloidosis derived from leukocyte chemotactic factor 2 (ALECT2). (a) The case depicted shows extensive interstitial, glomerular, and vascular amyloid deposition (Congo-red stain, 100). (b) Same field as in (a). The congophilic amyloid deposits exhibit a diagnostic apple-green birefringence under polarized light. (c) This case shows diffuse involvement of cortical interstitium and glomeruli. The medullary interstitium (upper left) is spared (Congo-red stain, 400). (d) In this case, there are extensive interstitial and focal arteriolar amyloid deposits. Glomeruli and interlobular arteries are not affected (Congo-red stain, 100). (e) A low-power electron microscopic image shows multiple interstitial collections of amyloid deposits ( 6800). (f) A higher magnification image reveals the fibrillar nature of deposits ( 49,000). ALECT2 in the current report was 65 years, which is comparable to renal AL but is higher than renal AA.3,6 It is likely that ALECT2 is an underdiagnosed disease. Most patients are older individuals with a history of hypertension (present in two-thirds of them in our series) who present with chronic renal insufficiency and bland urinary sediment. Proteinuria is variable, lacking altogether in a third of the patients, and extrarenal involvement is rare. Therefore, these patients may not undergo a renal biopsy, which is needed for diagnosis. Furthermore, in cases in which the deposits are restricted to the interstitium, amyloid could be missed 374 histologically unless Congo red staining is routinely performed on all native kidney biopsies. Over a quarter of our patients had diabetes, which likely reflects their advanced age and the higher prevalence of diabetes in Hispanics compared with non-hispanic whites in the United States.7,8 ALECT2 clearly has an ethnic predisposition, as 92% of our patients and most previously reported patients were Hispanics,5,9 which is strikingly different from all other forms of amyloidosis. Apart from Hispanics, Punjabis and Arabs appear to have a higher incidence of ALECT2 than Caucasians.9 Interestingly, all 34 Hispanic patients in our Kidney International (2014) 86,

6 SM Said et al.: Renal leukocyte chemotactic factor 2-associated amyloidosis Table 3 Immunofluorescence findings in seven renal ALECT2 cases with false-positive staining colocalizing with amyloid Case no. False-positive immune reactants Serum electrophoresis/immunofixation 1 IgG (3þ ), IgM (1 þ ), IgA (1 þ ), k (2 þ ) a Not performed 2 IgG (1þ ), IgM ( þ / ), l (1 þ ) Negative for paraprotein 3 IgG, k, l (all weak) Negative for paraprotein 4 IgG, k, l (all bright) a Not performed 5 IgG (2þ ), IgA (1 2 þ ), IgM (1 2 þ ), k (1 2 þ ), l (1 2 þ ) a Negative for paraprotein 6 IgG (1þ ), k (1 þ ), l (1 þ ) Negative for paraprotein 7 IgG, k, l (all bright); IgA, IgM (both weak) Negative for paraprotein Abbreviations: ALECT2, amyloidosis derived from leukocyte chemotactic factor 2; Ig, immunoglobulin. a Immunofluorescence results in these cases are based on outside renal biopsy reports. study who disclosed their origin were of Mexican descent. As Mexican Americans constitute the largest group of US Hispanics (63%) (US Census Bureau: National Population Estimates available at: briefs/c2010br-04.pdf), it is unclear whether they are more prone to develop ALECT2 compared with US Hispanics originating from other countries. The outcome and prognostic indicators in renal ALECT2 have not been studied previously. On the basis of our findings, ALECT2 has a variable short-term prognosis; within 2 years, roughly one-third of patients remain stable, onethird have a progressive course without reaching ESRD, and one-third develop ESRD. The median renal survival was 62 months. Not surprisingly, serum creatinine at diagnosis was an independent predictor of renal survival, with a value of 2.0 mg/dl being the best cutoff for predicting ESRD. Degree of proteinuria and amyloid load in the kidney did not predict outcome. Only 6% of our patients died after a median follow-up of 22 months. In comparison, in the study by Bergesio et al. 10 of 290 patients with renal amyloidosis, 62% of patients with AL died after a median follow-up of 25 months and 24% of patients with AA died after a median follow-up of 30 months. The better patient survival in renal ALECT2 compared with AL and AA is largely due to the absence of clinical cardiac involvement in ALECT2 patients. Clinical cardiac involvement is present in 33 50% of patients with renal AL and in 14% of patients with renal AA 10,11 and is an independent negative predictor of patient survival in both In contrast, none of our 72 patients with renal ALECT2 had clinical evidence of cardiac amyloidosis. Furthermore, during the study period, we analyzed 1517 cardiac clinical specimens by LMD/MS for amyloid typing, and none showed ALECT2. There is no specific therapy for ALECT2. Four patients in our cohort received AL chemotherapy: three patients without nephrotic syndrome had a progressive course, including two who reached ESRD, and one patient with nephrotic syndrome had a complete remission of nephrotic syndrome and normalization of serum creatinine. Whether ALECT2 patients presenting with nephrotic syndrome may benefit from chemotherapy requires a larger study. As the treatment and care for kidney transplantation patients in the past two decades has steadily improved, renal transplantation appears to be a good therapeutic option for ALECT2 patients with advanced disease. Despite disease recurrence in one of the five patients transplanted in our cohort, the short-term outcome overall was good: after a mean post-transplant followup of 20 months, none had graft loss and two patients had normal final serum creatinine. Additional larger studies with longer post-transplant follow-up are needed to determine the rate of ALECT2 recurrence and its impact on long-term graft survival. Histologically, in line with the findings in previously reported cases, 2,3,9 we show that ALECT2 exhibits preferential interstitial involvement, which is distinct from AL, AA, and fibrinogen A. 3,13,14 Glomerular and vascular involvement was not always present and was minimal in a third of cases. In contrast to renal amyloidosis derived from apolipoprotein A1 and apolipoprotein AIV, 3,15,16 the cortical interstitium was more frequently and more extensively involved than the medullary interstitium (Figure 2c). An observation in our study important for pathologic diagnosis, treatment, and outcome is the high frequency (25%) of concurrent glomerular disease, with diabetic nephropathy and IgA nephropathy being the most common. It is worth noting that ALECT2 can be an incidental pathologic finding; it was discovered in nephrectomy specimens performed for resection of renal cell carcinoma in four of our patients, none of whom had proteinuria and two of whom had normal serum creatinine at diagnosis. This report illustrates the importance of performing amyloid tissue typing in every patient before AL chemotherapy is initiated. Ten percent of our ALECT2 patients had evidence of plasma cell dyscrasia and 6% received chemotherapy for an erroneous presumed diagnosis of AL. The best available technique to diagnose ALECT2 and the other rare forms of amyloidosis is LMD/MS, for reasons we have reported previously. 3,17 A commercially available antibody for LECT2 immunohistochemical staining appears to be very sensitive as it was positive in all 27 cases tested in this series, although its specificity has not been studied yet. In 11% of our ALECT2 patients, the deposits showed false-positive staining for IgG with/without staining for IgM, IgA, kappa, and lambda; however, none showed staining for only kappa or lambda. This suggests that ALECT2 is unlikely to be confused with AL by immunofluorescence, but may be misdiagnosed as heavy þ light chain amyloidosis or heavy chain amyloidosis; this also may occur in cases of AA. 3,6 Kidney International (2014) 86,

7 SM Said et al.: Renal leukocyte chemotactic factor 2-associated amyloidosis LMD/MS is recommended in cases that show preferential interstitial involvement and/or positive staining for Ig heavy chains by immunofluorescence. LECT2, first isolated by Yamagoe et al. in 1996, 18 is a multifunctional factor involved in chemotaxis, inflammation, immunomodulation, and the damage/repair process It is mainly synthesized by hepatocytes 21 but is also expressed in a variety of other cells in many organs, including vascular endothelial cells, smooth muscle cells, adipocytes, and epithelial cells such as renal tubular epithelial cells. 22 The pathogenesis of ALECT2 remains to be elucidated. None of our 72 patients had a family history of amyloidosis and no mutations in the LECT2 gene have been found in the 16 patients analyzed at the molecular level, 1,4,9 which argues against a genetic basis for the disease. However, the observations that most ALECT2 patients are homozygous for the G allele, 1,4,9 and the strong ethnic bias, raise the possibility of genetic predisposition. It is unlikely that ALECT2 is a complication of malignancy, autoimmune disease, or liver disease, as the prevalence of these conditions in our large cohort was comparable to older individuals in general. Our observations that ALECT2 recurs in the transplant and that it may affect other organs (liver, spleen, prostate, and the GI tract, in decreasing incidence) provide evidence against the theory that the disease may be secondary to renal interstitial inflammation leading to increased local synthesis of an amyloidogenic LECT2. An autopsy-based study is needed to determine whether ALECT2 is a systemic form of amyloidosis as in AL and AA, or, as in amyloidosis derived from fibrinogen A, 14 an amyloidosis type with dominant renal involvement and infrequent and clinically insignificant extrarenal involvement. Of note, serum concentrations of LECT2 tested previously in three patients with ALECT2 were not increased; 4 therefore, the disease does not appear to be due to consistently elevated serum levels of LECT2. As hypothesized by Benson 23 and Murphy et al., 4 the disease could be due to interference, possibly due to a genetic defect, in the LECT2 catabolic pathway or LECT2 transport, which may result in an increased local tissue concentration of LECT2, ultimately leading to amyloid fibril formation. Further studies are clearly needed to elucidate the underlying mechanisms of this intriguing disease. A noteworthy limitation of our study is that we only included ALECT2 cases that were diagnosed by LMD/MS at Mayo Clinic; therefore, we cannot exclude type 1 error in the statistical analysis. In summary, renal ALECT2 primarily affects older Hispanic patients who present with chronic renal insufficiency and a bland urinary sediment. Proteinuria is variable and is absent altogether in a third of patients, although full nephrotic syndrome is rare. Histologically, ALECT2 exhibits preferential renal cortical interstitial involvement. Glomerular and vascular involvement is present in most cases but is minimal in up to one-third of them. Renal prognosis is variable with a median survival of 62 months. Patient survival is excellent, likely due to the lack of concurrent cardiac involvement. Renal transplantation is a good treatment option for those with severe renal failure, but the disease may recur. MATERIALS AND METHODS We identified 72 cases of renal ALECT2 by a retrospective review of LMD/MS data of pathology specimens received from 2007 (since we started using LMD/MS for clinical amyloid typing) through 2012 in the Department of Laboratory Medicine and Pathology, Mayo Clinic. The diagnosis of ALECT2 was based on the finding of large number of spectra of LECT2 protein, with 495% probability. 17 The methods for LMD/MS have previously been described in detail. 17,24 During the study period, 48 non-renal cases of ALECT2 from (different patients than renal ALECT2 patients) were diagnosed using LMD/MS (36 liver specimens, five spleen, three prostate, and one each from gallbladder, pancreas, small bowel, and parathyroid gland). Of the 72 specimens of renal ALECT2, 24 biopsies were initially sent to the Mayo Clinic Renal Biopsy Laboratory for full renal pathology evaluation, and LMD/MS was performed for amyloid typing. In the remaining 48 cases, the biopsy processing and diagnosis of renal amyloidosis were performed in other institutions (going back as early as 2004) and the paraffin blocks were subsequently sent to Mayo Clinic for amyloid typing by LMD/MS. The 72 renal ALECT2 specimens included 68 needle biopsies performed for renal dysfunction and four nephrectomy specimens performed for tumor resection. Light microscopic evaluation of renal biopsies included staining with hematoxylin and eosin, periodic acid-schiff, Masson s trichrome, Jones methenamine silver, and Congo-red. Electron microscopy was performed on 60 (83.3%) cases. Standard immunofluorescence staining for IgG, IgM, IgA, C3, C1q, albumin, fibrinogen, kappa (k), and lambda (l) was performed on 66 (91.6%) cases. The pathology and clinical renal characteristics (but not the outcome) of 13 cases included in this study were recently reported. 3,25 The details of LMD/MS data on 26 cases included here were recently reported. 17 Clinical data, including demographic information, presenting clinical and laboratory findings, medical history, and follow-up, were obtained from referral forms and clinical notes submitted at the time of biopsy, patients medical records, and telephone interviews with the referring and managing nephrologists and hematologists. The following clinical definitions were used: proteinuria, 4150 mg/day; nephrotic-range proteinuria X3.0 g/day; hypoalbuminemia, serum albumin o3.5 g/dl; renal insufficiency, serum creatinine 41.2 mg/dl; nephrotic syndrome, nephrotic-range proteinuria, hypoalbuminemia, and peripheral edema; and hypertension, systolic blood pressure 4140 mm Hg, diastolic blood pressure 490 mm Hg, or ongoing treatment with antihypertensive medications. For outcome analysis, stable renal function was defined as final serum creatinine o125% of serum creatinine at diagnosis; progressive renal dysfunction as final serum creatinine X125% of serum creatinine at diagnosis but not reaching ESRD; and ESRD as requiring renal replacement therapy or undergoing pre-emptive renal transplantation. The extent of amyloid deposits in the kidney, TA/IF, and arteriosclerosis was evaluated by reviewing the Congo red and hematoxylin and eosin slides routinely prepared at the Mayo Clinic for specimens received for LMD/MS. Glomerular amyloid deposits were assessed according to a semiquantitative scale: 0, absent; 1 þ, amyloid deposits affecting, on an average,o25% of the glomerulus; 2 þ, 25 50% of the glomerulus; 3 þ, 450% of the glomerulus. The extent of amyloid deposits in cortical interstitium was scored as: 0, 376 Kidney International (2014) 86,

8 SM Said et al.: Renal leukocyte chemotactic factor 2-associated amyloidosis absent; 1 þ, o25% of cortical parenchyma involved; 2 þ, 25 50%; 3 þ, 450%. The extent of amyloid deposits in vessels was scored as: 0, absent, 1 þ, mild, 2 þ, moderate, and 3 þ, severe. The renal amyloid load was defined by the sum of glomerular, vascular, and interstitial deposits (minimal composite score 0, maximal composite score 9). TA/IF was graded on a semiquantitative scale on the basis of an estimate of the percentage of renal cortex affected and recorded as: 0 (none), 1 25% (mild), 26 50% (moderate), or 450% (severe). Statistical analysis was performed using PASW(R), version 18.0 (formerly, SPSS(R), Chicago, IL) Continuous variables were tested using the 1-sample Kolmogorov Smirov test. All of the tested variables were significantly different from a normal distribution. Thus, analysis was performed using nonparametric exact statistical methods. Univariate analysis was performed using the Mann Whitney test, the Fisher s exact test, and the Kruskal Wallis test, as appropriate for variable type. Multivariate analysis was performed using binary and ordinal logistic regression analysis. Survival analysis for progression to ESRD was performed by the methods of Kaplan Meier for categorical variables and Cox regression for continuous variables for univariate analysis and the Cox proportional hazards model for multivariate analysis. Statistical significance was assumed at Po0.05. The study was approved by the institutional review board of the Mayo Clinic Foundation. DISCLOSURE All the authors declared no competing interest. ACKNOWLEDGMENTS We are grateful to the following physicians for providing clinical information on their patients: Mohamed G. Atta, Deborah Martinez Banos, Aviv Hever, Charles Lassman, Edgar V. Lerma, Glen S. Markowitz, Cliff Miles, Michael Stanek, Michael B. Stokes, and Luan D. Truong. REFERENCES 1. 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