Method comparison of three serum free light chain assays on the Roche Cobas 6000 c501 chemistry analyzer

Objectives Free light chains (FLC) are important in the diagnosis, prognosis and monitoring of therapy response of patients with monoclonal gammopathies. In this study, we performed a method comparison of three FLC assays on the Cobas 6000 c501 chemistry analyzer of Roche Diagnostics. Methods Samples of 119 patients with various monoclonal gammopathies and 26 control patients were measured with the Freelite (The Binding Site), Diazyme (Diazyme Laboratories) and KLoneus (Trimero Diagnostics) FLC assays. A method comparison was performed and reference intervals of the three assays were validated. Results The analysis of the Bland-Altman agreement showed bias between the three FLC assays, ranging from −62.7 to 5.1% for κFLC and between −29.2 to 80.5% for λFLC. The Freelite and Diazyme assays have the highest agreement. The concordance of the FLC-ratio ranges from 41 to 75%, with the highest concordance between the Freelite and KLoneus assays. The FLC-ratio in 25 sera from healthy controls were within the reference ranges of the Freelite and KLoneus assays. The FLC-ratio was elevated in all 25 samples tested with the Diazyme assay. Conclusions The agreement for the free light chains is highest between the Freelite and the Diazyme assay and fair for the KLoneus assay. However, concordance of the FLC-ratio is highest when the Freelite and KLoneus assays were compared. Our data suggest that concordance for the Diazyme assay could be improved by recalibration. Because of absolute differences between the three methods in individual patients, none of the three FLC assays can be used interchangeably.

Determination of MYD88L265P mutation fraction in IgM monoclonal gammopathies

We here describe a novel method for the detection of MYD88L265P mutation using a competitive allele specific PCR (Cast-PCR) assay. This assay has a sensitivity of 1x10-3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg) and allowed the detection of MYD88L265P somatic mutation in both tumor derived DNA (tDNA) and cell free DNA (cfDNA). In addition, using Cast-PCR assay we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with IgM-MGUS and 110 with WM, of which 54 were asymptomatic and 56 symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared to asymptomatic WM and in those with asymptomatic WM compared to IgM-MGUS patients. In addition, the evaluation of sequential samples showed that MAF decreased after treatment while increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly-sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tumor and cell free DNA samples which also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

Free Light-Chain Levels in Patients with Transthyretin Amyloid Cardiomyopathy in Attr-ACT

Introduction: In cardiac amyloidosis (CA), immunoglobulin-derived light chains (AL) and transthyretin (TTR) are the most common amyloidogenic proteins infiltrating the heart. 1 Identification of the specific precursor protein leading to amyloid deposition is needed to establish the correct therapeutic approach. 2 In both AL- and TTR-related CA, an early and accurate diagnosis is critical to achieving the best treatment outcomes. TTR amyloid cardiomyopathy (ATTR-CM) is often misdiagnosed as other causes of heart failure (HF), including AL CA. 3 While almost all patients with AL amyloidosis have elevated serum free light-chain (sFLC) levels and abnormal free kappa:lambda (κ:λ) ratios, 4 patients with ATTR-CM can also have abnormal sFLC levels due to either an unrelated monoclonal gammopathy or relative κ-predominance in renal dysfunction. 2,5 Because ATTR-CM most often occurs in elderly adults and is commonly accompanied by renal comorbidity, we theorized that patients with ATTR-CM may have κ:λ ratios that approach, or exceed, the upper limit of the normal reference range (0.26-1.65 6). High light-chain ratios in these patients have the potential to increase the likelihood of misdiagnosis of a monoclonal plasma cell disorder and may lead to unnecessary referrals to hematologists and/or inappropriate treatments. To explore this theory, we evaluated κ:λ ratios in patients with biopsy-proven ATTR-CM who were enrolled in the Tafamidis in Transthyretin Cardiomyopathy Clinical Trial (ATTR-ACT). 7 Methods: In ATTR-ACT, a double-blind, placebo-controlled, randomized study, patients with biopsy-proven ATTR-CM, and without light-chain amyloidosis, received tafamidis or placebo for 30 months. In the current analysis, sFLC levels and κ:λ ratios were assessed in the intent-to-treat population (N=441), excluding patients with a prior diagnosis of monoclonal gammopathies (n=13; defined by MedDRA preferred terms: 'monoclonal gammopathy', 'plasma cell myeloma', 'plasma cell disorder', and 'hypergammaglobulinemia benign monoclonal'). Subgroup analyses were performed by estimated glomerular filtration rate (eGFR) category (≥60 vs ≥40 to <60 vs <40 mL/min/1.73 m 2). Findings were summarized using descriptive statistics (min/max, mean, median, and interquartile range [IQR]). Results: In 422 patients with ATTR-CM and available sFLC data, and without a prior diagnosis of monoclonal gammopathies, the mean κ:λ ratio was 1.45 (median, 1.20 [IQR, 0.98, 1.47]) (Figure). The ratio increased with declining renal function: eGFR ≥60 mL/min/1.73 m 2, mean, 1.25 (median [IQR], 1.11 [0.94, 1.38]); ≥40 to <60 mL/min/1.73 m 2, 1.52 (1.22 [0.99, 1.49]); and <40 mL/min/1.73 m 2, 1.62 (1.30 [1.05, 1.68)]. Conclusions: In patients with biopsy-proven ATTR-CM without known monoclonal gammopathies who were enrolled in ATTR-ACT, the mean κ:λ ratio showed a κ-predominance, often exceeding the upper range of normal in patients with more advanced kidney dysfunction. The findings suggest that individuals with ATTR-CM can have higher sFLC levels than those normally seen in the general population, and such elevations do not necessarily indicate the presence of monoclonal gammopathy of undetermined significance or AL CA. In an era when most patients with ATTR-CM and without monoclonal gammopathies are diagnosed noninvasively using bone scintigraphy, age- and renal-function-based sFLC norms are critical to ensure appropriate use of diagnostic testing modalities. References: 1. Maleszewski JJ. Cardiovasc Pathol. 2015;24:343-50. 2. Donnelly JP, et al. Cleve Clin J Med. 2017;84:12-26. 3. Witteles RM, et al. JACC Heart Fail. 2019;7:709-716. 4. Falk RH, et al. J Am Coll Cardiol. 2016;68:1323-41. 5. Geller HI, et al. Mayo Clin Proc. 2017;92:1800-5. 6. Katzmann JA, et al. Clin Chem. 2002;48:1437-44. 7. Maurer MS, et al. N Engl J Med. 2018;379:1007-16. Figure 1 Figure 1. Disclosures Hoffman: BMS, Celgene: Honoraria. Sultan: Pfizer: Current Employment, Current equity holder in publicly-traded company. Gundapaneni: Pfizer: Current Employment, Current equity holder in publicly-traded company. Witteles: Pfizer: Honoraria, Research Funding; Alnylam: Honoraria, Research Funding; Eidos: Research Funding.

Acute Kidney Injury in Monoclonal Gammopathies

Monoclonal gammopathies (MG) encompass a variety of disorders related to clonal expansion and/or malignant transformation of B lymphocytes. Deposition of free immunoglobulin (Ig) components (light or heavy chains, LC/HC) within the kidney during MG may result over time in multiple types and degrees of injury, including acute kidney injury (AKI). AKI is generally a consequence of tubular obstruction by luminal aggregates of LC, a pattern known as “cast nephropathy”. Monoclonal Ig LC can also be found as intracellular crystals in glomerular podocytes or proximal tubular cells. Proliferative glomerulonephritis with monoclonal Ig deposits is another, less frequent form of kidney injury with a sizable impact on renal function. Hypercalcemia (in turn related to bone reabsorption triggered by proliferating plasmacytoid B cells) may lead to AKI via functional mechanisms. Pharmacologic treatment of MG may also result in additional renal injury due to local toxicity or the tumor lysis syndrome. The present review focuses on AKI complicating MG, evaluating predictors, risk factors, mechanisms of damage, prognosis, and options for treatment.

Laboratory Detection and Initial Diagnosis of Monoclonal Gammopathies: Guideline From the College of American Pathologists in Collaboration With the American Association for Clinical Chemistry and the American Society for Clinical Pathology

Context.— The process for identifying patients with monoclonal gammopathies is complex. Initial detection of a monoclonal immunoglobulin protein (M protein) in the serum or urine often requires compilation of analytical data from several areas of the laboratory. The detection of M proteins depends on adequacy of the sample provided, available clinical information, and the laboratory tests used. Objective.— To develop an evidence-based guideline for the initial laboratory detection of M proteins. Design.— To develop evidence-based recommendations, the College of American Pathologists convened a panel of experts in the diagnosis and treatment of monoclonal gammopathies and the laboratory procedures used for the initial detection of M proteins. The panel conducted a systematic literature review to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, recommendations were created based on the available evidence, strength of that evidence, and key judgements as defined in the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework. Results.— Nine guideline statements were established to optimize sample selection and testing for the initial detection and quantitative measurement of M proteins used to diagnose monoclonal gammopathies. Conclusions.— This guideline was constructed to harmonize and strengthen the initial detection of an M protein in patients displaying symptoms or laboratory features of a monoclonal gammopathy. It endorses more comprehensive initial testing when there is suspicion of amyloid light chain amyloidosis or neuropathies, such as POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes) syndrome, associated with an M protein.

Clinical Presentation, Renal Histopathological Findings, and Outcome in Patients with Monoclonal Gammopathy and Kidney Disease

Monoclonal gammopathies are associated with acute and chronic kidney injury. Nephrotoxicity of the secreted monoclonal (M)-protein is related to its biological properties and blood concentration. Little is known about epidemiology, clinical manifestations, and outcome of monoclonal gammopathies in patients with kidney disease. We retrospectively collected data about demographics, clinical manifestations, and renal histological lesions of all patients (n = 1334) who underwent kidney biopsy between January 2000 and March 2017. Monoclonal gammopathy was detected in 174 (13%) patients with a mean age of 66.4 ± 13.1 years. The spectrum of monoclonal gammopathies comprised monoclonal gammopathy of undetermined significate (MGUS) (52.8%), multiple myeloma (MM) (25.2%), primary amyloidosis (AL) (9.1%), smoldering MM (SMM) (4%), non-Hodgkin lymphoma (NHL) (6.8%), and Hodgkin lymphoma (HL) (1.7%). Monoclonal gammopathy of renal significance (MGRS) accounted for 6.5% in patients with MGUS and 14.2% in patients with SMM. Evaluation of kidney biopsy revealed that M-protein was directly involved in causing kidney injury in MM (93.1%). MM was the only gammopathy significantly associated with an increased risk of kidney injury (odds ratio [OR] = 47.5, CI 95%, 13.7–164.9; P ≤ 0.001 ). While there were no significant differences in the progression toward end-stage renal disease or dialysis P = 0.776 , monoclonal gammopathies were associated with a different risk of death P = 0.047 at the end of the follow-up. In conclusion, monoclonal gammopathy was a frequent finding (13%) in patients who underwent kidney biopsy. M-protein was secreted by both premalignant (56.8%) and malignant (43.2%) lymphoproliferative clones. Kidney biopsy had a key role in identifying MGRS in patients with MGUS (6.5%) and SMM (14.2%). Among monoclonal gammopathies, only MM was significantly associated with biopsy-proven kidney injury. The rate of end-stage renal disease or dialysis was similar among monoclonal gammopathies, whereas NHL, MM, and SMM showed a higher rate of deaths.