Amino acid sequence homology of monoclonal serum free light chain dimers and tissue deposited light chains in AL amyloidosis: a pilot study.

OBJECTIVES: Diagnosis of light chain amyloidosis (AL) requires demonstration of amyloid deposits in a tissue biopsy followed by appropriate typing. Previous studies demonstrated increased dimerization of monoclonal serum free light chains (FLCs) as a pathological feature of AL. To further examine the pathogenicity of FLC, we aimed at testing amino acid sequence homology between circulating and deposited light chains (LCs). METHODS: Matched tissue biopsy and serum of 10 AL patients were subjected to tissue proteomic amyloid typing and nephelometric FLC assay, respectively. Serum FLC monomers (M) and dimers (D) were analyzed by Western blotting (WB) and mass spectrometry (MS). RESULTS: WB of serum FLCs showed predominance of either κ or λ type, in agreement with the nephelometric assay data. Abnormal FLC M-D patterns typical of AL amyloidosis were demonstrated in 8 AL-λ patients and in one of two AL-κ patients: increased levels of monoclonal FLC dimers, high D/M ratio values of involved FLCs, and high ratios of involved to uninvolved dimeric FLCs. MS of serum FLC dimers showed predominant constant domain sequences, in concordance with the tissue proteomic amyloid typing. Most importantly, variable domain sequence homology between circulating and deposited LC species was demonstrated, mainly in AL-λ cases. CONCLUSIONS: This is the first study to demonstrate homology between circulating FLCs and tissue-deposited LCs in AL-λ amyloidosis. The applied methodology can facilitate studying the pathogenicity of circulating FLC dimers in AL amyloidosis. The study also highlights the potential of FLC monomer and dimer analysis as a non-invasive screening tool for this disease.

The Prognostic Value of Anti-PLA2R Antibodies Levels in Primary Membranous Nephropathy.

Anti-PLA2R antibodies (Ab) are a diagnostic and prognostic biomarker in primary membranous nephropathy (PMN). We assessed the relationship between the levels of anti-PLA2R Ab at diagnosis and different variables related to disease activity and prognosis in a western population of PMN patients. Forty-one patients with positive anti-PLA2R Ab from three nephrology departments in Israel were enrolled. Clinical and laboratory data were collected at diagnosis and after one year of follow-up, including serum anti-PLA2R Ab levels (ELISA) and glomerular PLA2R deposits on biopsy. Univariable statistical analysis and permutation-based ANOVA and ANCOVA tests were performed. The median [(interquartile range (IQR)) age of the patients was 63 [50-71], with 28 (68%) males. At the time of diagnosis, 38 (93%) of the patients had nephrotic range proteinuria, and 19 (46%) had heavy proteinuria (≥8 gr/24 h). The median [IQR] level of anti-PLA2R at diagnosis was 78 [35-183] RU/mL. Anti-PLA2R levels at diagnosis were correlated with 24 h proteinuria, hypoalbuminemia and remission after one year (p = 0.017, p = 0.003 and p = 0.034, respectively). The correlations for 24 h proteinuria and hypoalbuminemia remained significant after adjustment for immunosuppressive treatment (p = 0.003 and p = 0.034, respectively). Higher levels of anti-PLA2R Ab at diagnosis in patients with active PMN from a western population are associated with higher proteinuria, lower serum albumin and remission one year after the diagnosis. This finding supports the prognostic value of anti-PLA2R Ab levels and their possible use in stratifying PMN patients.

Diagnostic Challenges and Solutions in Systemic Amyloidosis.

Amyloidosis refers to a clinically heterogeneous group of disorders characterized by the extracellular deposition of amyloid proteins in various tissues of the body. To date, 42 different amyloid proteins that originate from normal precursor proteins and are associated with distinct clinical forms of amyloidosis have been described. Identification of the amyloid type is essential in clinical practice, since prognosis and treatment regimens both vary according to the particular amyloid disease. However, typing of amyloid protein is often challenging, especially in the two most common forms of amyloidosis, i.e., the immunoglobulin light chain amyloidosis and transthyretin amyloidosis. Diagnostic methodology is based on tissue examinations as well as on noninvasive techniques including serological and imaging studies. Tissue examinations vary depending on the tissue preparation mode, i.e., whether it is fresh-frozen or fixed, and they can be carried out by ample methodologies including immunohistochemistry, immunofluorescence, immunoelectron microscopy, Western blotting, and proteomic analysis. In this review, we summarize current methodological approaches used for the diagnosis of amyloidosis and discusses their utility, advantages, and limitations. Special attention is paid to the simplicity of the procedures and their availability in clinical diagnostic laboratories. Finally, we describe new methods recently developed by our team to overcome limitations existing in the standard assays used in common practice.

Treatment of Severe Renal Artery Stenosis with Acute Kidney Injury Requiring Hemodialysis by Percutaneous Transluminal Renal Angioplasty and Stent Implantation.

PURPOSE: To evaluate the outcomes of percutaneous transluminal renal angioplasty with stent implantation (PTRAS) among patients with renal artery stenosis (RAS) who become dialysis-dependent due to acute deterioration of renal function. MATERIALS AND METHODS: This was a single-center retrospective cohort study of all PTRAS procedures performed from 2003 to 2019 in a referral hospital. A total of 109 procedures were performed in 92 patients. Eleven patients (12%) presented with anuric acute kidney injury (AKI) secondary to high-grade RAS (defined as intraluminal stenosis above 70% per angiography) and underwent PTRAS after starting hemodialysis. Data collected included demographic parameters, medical background, creatinine, blood pressure, indication for intervention, procedure characteristics, adverse events, and long-term data including dialysis treatment and mortality. Among the dialysis-dependent AKI group, outcome measures were defined based on the postprocedural improvement in kidney function and discontinuation of dialysis. RESULTS: Following PTRAS, 8 of 11 patients (73%) demonstrated improved kidney function and were able to discontinue dialysis. The median time on dialysis was 18 days (range, 2-35 days) before PTRAS and 4.5 days (range, 1-24 days) to recovery of kidney function after the time of intervention. CONCLUSIONS: Patients with atherosclerotic RAS who develop RAS-related AKI may benefit from PTRAS even after several weeks of anuria and dialysis dependence.

Amyloid storm: acute kidney injury and massive proteinuria, rapidly progressing to end-stage kidney disease in AA amyloidosis of familial Mediterranean fever.

OBJECTIVE: Amyloid A nephropathy of FMF usually progresses over many years to end-stage renal disease (ESRD). We aim to describe an acute condition, termed here 'amyloid storm', typically manifesting with a rapid (≤2 weeks) increase in serum creatinine and urine protein, that has never been characterized in FMF amyloidosis. METHODS: This retrospective analysis features amyloid storm by comparing between FMF amyloidosis patients who have experienced an episode of amyloid storm (study group) and matched patients who have not (control group). The primary outcome was ESRD or death within 1 year from study entry. Featured data were retrieved from hospital files. RESULTS: The study and control groups, each comprising 20 patients, shared most baseline characteristics. However, they differed on the time from FMF onset to reaching serum creatinine of 1.2 mg/dl [26.5 years (s.d. 15.15) vs 41.55 (10.98), P = 0.001] and the time from the onset of proteinuria to study entry [8.8 years (s.d. 6.83) vs 15.75 (13.05), P = 0.04], culminating in younger age at study entry [39.95 years (s.d. 16.81) vs 48.9 (9.98), respectively, P = 0.05] and suggesting an accelerated progression of kidney disease in the study group. Within 1 year from study entry, 16 patients in the study and 3 in the control groups reached the primary endpoint (P = 0.000). The major triggers of amyloid storm were infections, occurring in 17 of 20 patients. CONCLUSION: Amyloid storm is a complication of FMF amyloidosis, induced by infection and associated with poor prognosis and death.

Kidney disease and plasma cell dyscrasias: ambiguous cases solved by serum free light chain dimerization analysis.

BACKGROUND: Plasma cell dyscrasias (PCD) comprise a wide spectrum of disorders, which may adversely affect the kidney. However, in some PCD cases associated with kidney disease, the routine laboratory tests may be incapable to determine precisely the form of PCD, i.e., benign or malignant. Moreover, the kidney biopsy needed for precise diagnosis may be risky or declined. To overcome these limitations, we have developed and reported a new non-invasive technique based on serum free light chains (FLC) monomer (M) and dimer (D) pattern analysis (FLC MDPA), which allowed differentiation between malignant and benign PCD forms. The objective of our retrospective study was to demonstrate the utility of FLC MDPA in solving ten puzzling PCD cases complicated with kidney disease (patients 1-10). METHODS: Ten patients with uncertain form of PCD or with a questionable response to treatment were studied. In addition to routine laboratory tests and clinical evaluation of the PCD patients, our previously developed FLC MDPA in sera and biochemical amyloid typing in biopsy tissues were applied. RESULTS: The FLC MDPA aided the diagnosis of the PCD underlying or accompanying the kidney disease in patients 1-5, and helped to interpret properly the response to treatment in patients 1, 6-10. The FLC MDPA findings were confirmed by a biochemical analysis of tissue amyloid deposits and subsequently by the outcome of these patients. CONCLUSIONS: FLC MDPA is a non-invasive diagnostic test useful in the management of ambiguous cases of PCD associated with kidney disease.

The use of serum free light chain dimerization patterns assist in the diagnosis of AL amyloidosis.

The discrimination between benign and malignant forms of plasma cell dyscrasia (PCD) is often difficult. Free light chain monomer-dimer pattern analysis (FLC-MDPA) may assist in solving this dilemma and distinguish between AL amyloidosis and benign PCD. Serum samples of patients with AL amyloidosis and benign PCD were analysed in a blinded manner. Quantitative Western blotting was performed to estimate dimerization and clonality indices, and thereby determine the source of the tested samples, as derived either from benign or malignant PCD. The findings obtained by the FLC-MDPA were compared with the actual diagnosis. Of 37 samples from patients with active AL amyloidosis, 34 (91·9%) fulfilled dimerization criteria for diagnosis of AL amyloidosis. Of the 45 samples from patients with benign PCD, 10 (21·2%) tested falsely positive or gave an inconclusive result. Thus, the sensitivity of the analysis was 92·5% with a remarkable negative predictive value of 91·9%. In addition, of 20 patients who were in complete or very good partial remission, only one tested positive. By multivariate analysis, FLC-MDPA was the best independent marker predicting AL amyloidosis (odds ratio of 84). The FLC-MDPA offers a highly effective tool in the diagnostic assessment of patients with PCD.

Normal arterial stiffness in familial Mediterranean fever. Evidence for a possible cardiovascular protective role of colchicine.

OBJECTIVES: Familial Mediterranean fever (FMF) is an autoinflammatory disorder with episodic and persistent inflammation, which is only partially suppressed by continuous colchicine treatment. While chronic inflammation is considered an important cardiovascular risk factor in many inflammatory disorders, its impact in FMF is still disputed. We measured arterial stiffness, a marker of atherosclerotic cardiovascular disease, in a group of FMF patients, in order to evaluate the cardiovascular consequences of inflammation in FMF and the role of colchicine in their development. METHODS: Eighty colchicine treated FMF patients, without known traditional cardiovascular risk factors, were randomly enrolled in the study. Demographic, genetic, clinical and laboratory data were retrieved from patient files and examinations. Arterial stiffness was measured using pulse wave velocity (PWV). The recorded values of PWV were compared with those of an age and blood pressure adjusted normal population, using internationally endorsed values. RESULTS: FMF patients displayed normal PWV values, with an even smaller than expected proportion of patients deviating from the 90th percentile of the reference population (5% vs. 10%, p=0.02). The lowest PWV values were recorded in patients receiving the highest dose of colchicine (≥2 mg vs. 0-1 mg, p=0.038), and in patients of North African Jewish origin, whose disease was typically more severe than that of patients of other ethnicities; both observations supporting an ameliorating colchicine effect (p=0.043). CONCLUSIONS: Though subjected to chronic inflammation, colchicine treated FMF patients have normal PWV. Our findings provide direct evidence for a cardiovascular protective role of colchicine in FMF.

Anakinra for Colchicine-Resistant Familial Mediterranean Fever: A Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: Familial Mediterranean fever (FMF) is refractory to colchicine prophylaxis in 10-20% of patients. In a number of patient series, treatment with anakinra, an interleukin-1-blocking agent, prevented FMF attacks in those with colchicine-resistant FMF. This study was undertaken to evaluate the efficacy and safety of anakinra in the treatment of colchicine-resistant FMF, using a randomized controlled trial. METHODS: Patients with colchicine-resistant FMF receiving colchicine (dosage ≥1.5 to ≤3 mg/day) were recruited and randomly assigned to receive anakinra or placebo (vehicle). The treatment duration was 4 months. Primary efficacy outcomes were the number of attacks per month, and the number of patients with a mean of <1 attack per month. Quality of life was assessed using a 0-10-grade visual analog scale (VAS), and safety was assessed according to the number and severity of adverse events. RESULTS: Twenty-five patients with colchicine-resistant FMF (14 women) were enrolled, of whom 12 were randomized to receive anakinra and 13 to receive placebo. The mean ± SD number of attacks per patient per month was 1.7 ± 1.7 in those receiving anakinra and 3.5 ± 1.9 in those receiving placebo (P = 0.037). Six patients in the anakinra group, compared to none in the placebo group, had <1 attack per month (P = 0.005). A beneficial effect of anakinra was noted in the number of attacks in the joints per month in patients receiving anakinra (mean ± SD 0.8 ± 1.6 versus 2.1 ± 1.1 in the placebo group; P = 0.019) and in quality of life (mean ± SD VAS score 7.7 ± 2.3 in the anakinra group versus 4.2 ± 2.9 in the placebo group; P = 0.045). The number of adverse events per patient per month was comparable between the anakinra group and the placebo group (mean ± SD 2.03 ± 1.75 versus 3.34 ± 2.5; P = 0.22). There were no severe adverse events. CONCLUSION: In this randomized controlled trial, anakinra appears to be an effective and safe treatment for colchicine-resistant FMF.

Prevalence of severe mitral regurgitation eligible for edge-to-edge mitral valve repair (MitraClip).

OBJECTIVES: We assessed the prevalence of moderately severe or severe mitral regurgitation (MR) justifying edge-to-edge mitral valve (MV) repair (MitraClip(®)) in patients attending the University Hospital Wuerzburg, a tertiary care centre located in Wuerzburg, Germany. BACKGROUND: Transcatheter edge-to-edge MV repair of advanced MR is a non-surgical treatment option in inoperable and high-risk patients. It is unknown how many patients are potentially eligible for MitraClip(®) since several anatomical prerequisites of the MV apparatus have to be met for optimal treatment results. METHODS: Using a novel clinical data warehouse we searched for all patients attached to our Department of Internal Medicine from 01/2008 to 01/2012 with moderately severe or severe MR and aged ≥18 years. The current status of their treatment regime and eligibility for MitraClip(®) was assessed and re-evaluated according to current guidelines and echocardiographic criteria. RESULTS: The search of electronic medical records amongst 43,690 patients employed an innovative validated text extraction method and identified 331 patients with moderately severe or severe MR who had undergone echocardiographic assessment at our institution. Of these, 125 (38 %) received MV surgery and 206 (62 %) medical therapy only. Most patients not undergoing surgery had secondary MR (70 %). After evaluation of medical and echocardiographic data of medically treated patients (n = 206), 81 (39 %) were potential candidates for MitraClip(®) therapy, and 90 (44 %) died during the median follow-up time of 23 months. CONCLUSION: A large fraction of patients with moderately severe or severe MR but not operated was detected. Medically treated patients had a bad prognosis and about 40 % of them were potential candidates for MitraClip(®) therapy.

Acquired familial Mediterranean fever associated with a somatic MEFV mutation in a patient with JAK2 associated post-polycythemia myelofibrosis.

BACKGROUND: A study was designed to identify the source of fever in a patient with post-polycythemia myelofibrosis, associated with clonal Janus Kinase 2 (JAK2) mutation involving duplication of exon 12. The patient presented with 1-2 day long self-limited periodic episodes of high fever that became more frequent as the hematologic disease progressed. METHODS: After ruling out other causes for recurrent fever, analysis of the pyrin encoding Mediterranean fever gene (MEFV) was carried out by Sanger sequencing in peripheral blood DNA samples obtained 4 years apart, in buccal cells, laser dissected kidney tubular cells, and FACS-sorted CD3-positive or depleted mononucleated blood cells. Hematopoeitc cells results were validated by targeted deep sequencing. A Sanger sequence based screen for pathogenic variants of the autoinflammatory genes NLRP3, TNFRSF1A and MVK was also performed. RESULTS: A rare, c.1955G>A, p.Arg652His MEFV gene variant was identified at negligible levels in an early peripheral blood DNA sample, but affected 46 % of the MEFV alleles and was restricted to JAK2-positive, polymorphonuclear and CD3-depleted mononunuclear DNA samples obtained 4 years later, when the patient experienced fever bouts. The patient was also heterozygous for the germ line, non-pathogenic NLRP3 gene variant, p.Q705K. Upon the administration of colchicine, the gold standard treatment for familial Mediterranean fever (FMF), the fever attacks subsided. CONCLUSIONS: This is the first report of non-transmitted, acquired FMF, associated with a JAK2 driven clonal expansion of a somatic MEFV exon 10 mutation. The non-pathogenic germ line NLRP3 p.Q705K mutation possibly played a modifier role on the disease phenotype.

Familial Mediterranean fever without MEFV mutations: a case-control study.

BACKGROUND: Although familial Mediterranean fever (FMF) was originally defined as an autosomal recessive disorder, approximately 10-20% of FMF patients do not carry any FMF gene (MEFV) mutations. Fine phenotype characterization may facilitate the elucidation of the genetic background of the so called "FMF without MEFV mutations". In this study we clinically and demographically characterize this subset. METHODS: MEFV mutation-negative FMF and control patients were recruited randomly from a cohort followed in a dedicated FMF clinic. The control subjects comprised 2 groups: 1. typical population of FMF, consisting of genetically heterogeneous patients manifesting the classical spectrum of FMF phenotype and 2. a severe phenotype of FMF, consisting of FMF patients homozygous for the p.M694V mutation. RESULTS: Forty-seven genetic-negative, 60 genetically heterogeneous and 57 p.M694V homozygous FMF patients were enrolled to the study. MEFV-mutation negative FMF patients showed a phenotype closely resembling that of the other 2 populations. It differed however from the p.M694V homozygous subset by its milder severity (using Mor et al. scoring method), as determined by the lower proportion of patients with chest and erysipelas like attacks, lower frequency of some of the chronic manifestations, lower colchicine dose and older age of disease onset. CONCLUSIONS: MEFV mutation-negative FMF by virtue of its classical FMF phenotype is probably associated with a genetic defect upstream or downstream to MEFV related metabolic pathway.

Immunoglobulin-free light chain monomer-dimer patterns help to distinguish malignant from premalignant monoclonal gammopathies: a pilot study.

Multiple myeloma (MM) and AL amyloidosis (AL) are two malignant forms of monoclonal gammopathies. For the purposes of prognosis and treatment, it is important to distinguish these diseases from the premalignant forms of monoclonal gammopathies, such as monoclonal gammopathy of unknown significance (MGUS) and smoldering myeloma (SMM). Routine serum/urine tests for monoclonal protein are insufficient for differential diagnosis. Thus, invasive procedures, such as tissue aspiration or biopsy, are applied. In this study, we aimed at characterization of serum-free light chain (FLC) monomer-dimer patterns to distinguish the malignant from the premalignant forms of monoclonal gammopathies. A quantitative Western blotting was applied to estimate the FLC monomer and dimer levels in AL, MM, MGUS, and SMM patients, and in control subjects (healthy individuals and patients with AA amyloidosis). AL and MM patients displayed an abnormally increased dimerization of monoclonal FLC, accompanied by higher clonality values of FLC dimers, as compared to that of monomers. These abnormalities of FLC patterns were not observed in patients with MGUS, SMM, AA amyloidosis, and healthy individuals. Analysis of FLC patterns helped to differentiate AL and MM from MGUS and SMM, a goal difficult to achieve using routine serum tests. Also, our technique might serve as a complimentary diagnostic tool in the cases with suspected AL amyloidosis, where the diagnosis of MM is excluded, while the results of amyloid typing by routine immunohistochemical techniques are inconclusive.

Familial Mediterranean fever (FMF) with proteinuria: clinical features, histology, predictors, and prognosis in a cohort of 25 patients.

OBJECTIVE: Reactive (AA) amyloidosis may complicate familial Mediterranean fever (FMF), the prototype of autoinflammatory diseases. Thus, proteinuria in FMF is commonly viewed as resulting from amyloidosis, and kidney biopsy is deemed superfluous. However, nephropathy other than amyloidosis has been described in FMF, but its rate and distinctive characteristics are unknown. Our aim was to determine the rate and underlying pathology of FMF-related nonamyloidotic proteinuria and compare its clinical course, demographic, and genetic features to those of FMF-amyloid nephropathy. METHODS: This study is a retrospective analysis of data from patients with FMF undergoing kidney biopsy for proteinuria above 0.5 g/24 h, over 10 years (2001-2011). Clinical, laboratory, genetic, and pathology data were abstracted from patient files. Biopsies were viewed by an experienced pathologist, as necessary. RESULTS: Of the 25 patients referred for kidney biopsy, only 15 (60%) were diagnosed with amyloid kidney disease (AKD), and 10 were diagnosed with another nephropathy. The AKD and nonamyloid kidney disease (NAKD) groups were comparable on most variables, but showed distinct characteristics with regard to the degree of proteinuria (6.45 ± 4.3 g vs 2.14 ± 1.6 g, p = 0.006), rate of severe FMF (14 vs 5 patients, p = 0.022), and rate of development of end stage renal disease (73.3% vs 20%, p = 0.015), respectively. CONCLUSION: NAKD is common in FMF and, compared to amyloidosis, it is featured with milder course and better prognosis. Contrary to common practice, it is highly recommended to obtain a kidney biopsy from patients with FMF and proteinuria more than 0.5 g/24 h.

Serum amyloid A levels in kidney-transplanted patients with familial Mediterranean fever-amyloidosis.

BACKGROUND: Amyloidosis of familial Mediterranean fever (FMF) may lead to end-stage renal failure, culminating in kidney transplantation. Since amyloidosis is prompted by high serum amyloid A (SAA) levels, increased SAA is expected to persist after transplantation. However, no data are available to confirm such an assumption. OBJECTIVES: To determine SAA levels in kidney-transplanted FMF-amyloidosis patients and evaluate risk factors for the expected high SAA levels in this patient group. METHODS: SAA, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) values were obtained from 16 kidney-transplanted FMF-amyloidosis patients, 18 FMF patients without amyloidosis and 20 kidney-transplanted patients with non-inflammatory underlying disease. Demographic, clinical and genetic risk factors evaluation was based on data extracted from files, interviews and examination of the patients. RESULTS: SAA level in FMF patients who underwent kidney transplantation due to amyloidosis was elevated with a mean of 21.1 +/- 11.8 mg/L (normal < or = 10 mg/L). It was comparable to that of transplanted patients with non-inflammatory disorders, but tended to be higher than in FMF patients without amyloidosis (7.38 +/- 6.36, P = 0.08). Possible risk factors for the elevated SAA levels in kidney transplant patients that were excluded were ethnic origin, MEFV mutations, gender, age and disease duration. CONCLUSIONS: Kidney-transplanted patients with FMF-amyloidosis and with other non-FMF causes displayed mildly elevated SAA levels, possibly resulting from exposure to foreign tissue rather than from various FMF-related factors.