Free Light Chain Concentrations Research Articles

B-311 Relation of Free Immunoglobulin Ligh Chain Concentration to Overflow Proteinuria

Abstract Background Free immunoglobulin light chains are small proteins that undergo substantial glomerular filtration. When high serum concentrations of free light chains occur in multiple myeloma, the high filtered load of light chains exceeds the uptake capacity of proximal tubules, and overflow proteinuria ensues. Massive excretion of light chains can lead to cast formation and renal injury. The present study sought to determine the relation of serum light chain concentration to overflow proteinuria. Methods Results for patients with plasma cell disorders were reviewed as approved by an institutional review board. A Binding Site Optilite analyzer measured serum free light chain concentrations. Urine and serum protein electrophoresis were performed on Helena agarose gels. A total of 170 paired serum and 24-hour urine specimens were evaluated for 36 patients with increased kappa light chains and 26 patients with increased lambda light chains. Results Overflow proteinuria with either immunoglobulin light chain presented with light chain as the major urinary protein and limited increases of urinary albumin. Light chain excretion exceeded albumin excretion when light chain excretion surpassed 100 mg/d. Similar results were obtained for cases with increased kappa and lambda free light chains. There was a general relation of increased serum kappa and lambda light chains and urinary excretion of the corresponding light chain. Limited urinary excretion of light chains (<120 mg/d) was seen when the serum light chain concentration was < 250 mg/L, representing an approximate threshold for substantial overflow proteinuria. Overflow proteinuria exceeding 2,000 g/d was seen only when serum free light chain concentrations exceeded 800 mg/L. However, a few patients with high serum light chain values (>1,000/L) had low urinary light chain excretion that was discordant from the relation of serum light chain concentration to light chain excretion. Serum light chain values >4,000 mg/L (N = 10) were all associated with overflow proteinuria of > 1,500 mg/d and, in 7 cases, >10,000 mg/d. In a few cases, there was evidence that serum light chain assays overestimated light chain concentrations. In one case, the serum free light chain value was twice the total protein and about 12-fold higher than the serum M-spike, suggestive of substantial overestimation. Conclusions Defining the relation between serum free light chain concentrations and the magnitude of overflow proteinuria helps identify patients at low and high risk of massive overflow proteinuria that can cause renal injury. Limited overflow proteinuria (<120 mg/d) occurred when serum light chain concentration was < 250 mg/L, and substantial overflow proteinuria occurred when serum light chains exceeded 4,000 mg/L Serum free light chain concentrations above 250 mg/L were incompletely predictive of urinary free light chain excretion; a few discordant cases with high measured serum light chain concentrations (>1,000 mg/L) and low excretion of light chains were observed. Further investigation is indicated for cases with possible overestimation of free light chains or discordance between serum light chain values and urinary light chain excretion. Unexpected results in some cases could relate to polymerization of free light chains as previously reported.

B-341 Comparative Study of Free Light Chains in the Framework of the Project Prevalence of Monoclonal Gammopathies in Adult Uruguayan Population

Abstract Background Within the framework of a study to evaluate the Prevalence of Monoclonal Gammopathies in the Adult population of Uruguay through detection monoclonal bands by capillary electrophoresis and free light chains in serum, the aim is evaluating the comparison of quantification of FLC (Free Light Chains) in this group of patients with 2 assays: Freelite Biding Site® versus Sebia® FLC Elisa. Methods Study period: September 2021 to November 2022. Prospective, single-cohort, descriptive study. Approved by the Hospital de Clinicas Ethics committee, Montevideo, Uruguay.A total of 3905 patients were included (≥40 years, without previously known plasma cell disorder).Serum samples were collected from three health centers, and processed in Clinical Chemistry Laboratory at Hospital de Clinicas, following an algorithm designed to perform electrophoretic proteinogram (by capillary electrophoresis); suspicious samples with monoclonal bands were confirmed by immunotyping, and in some cases by immunofixation (difficult interpretation), and quantification FLC by 2 different assays (Sebia FLC: manual sandwich ELISA technique- BIO-TEK wash/reader; Freelite The Binding Site immunoturbidimetric assay : Spa Plus and Optilite instruments. Comparative analysis of FLC is a transversal and descriptive study.For statistical analysis: Spearman test, Passing and Bablok and Bland-Altman plot. Results κFLC, λFLC and ratio κ/ λ were measured in 93 of the 104 positive serum samples for Monoclonal Gammopathies (new diagnoses, prevalence 2.7%), using two groups Sebia FLC and Spa Plus Freelite, and Sebia FLC and Optilite Freelite. During the project, the Spa Plus (n=48) FLC was replaced with Optilite (n=45), since the comparative group was not analytically homogeneous, so the comparison was carried out in two groups. Spearman rank correlation coefficients between first group (N:48) for κFLC, λFLC and ratio κ/ λ were r=0.596, 0,549, 0.709, respectively (p<0.0001); for the second group (N:45) Spearman rank correlation coefficients were r=0.778, 0.714, 0.878, respectively (p<0.0001). The first group showed good Spearman rank correlation for ratio, although less better correlation for kappa and lambda isolated. Bland Altman showed high media differences in assays for values for Sebia. For the second group, there was a better Spearman Correlation for ratio and in kappa and lambda isolated too. Bland-Altman plot revealed, in the second group analysis, that Freelite provides higher values than Sebia in Kappa and ratio, but not in Lambda. Passing Bablock showed differences systematic and proportional in different group of comparatives. Conclusions There is a good agreement; despite this, within high FLC concentrations, there are particularly significant differences between both assays, in both groups. We found in same cases FLC quantifications with Freelite ratios lower than those reported by the Elisa Sebia assay, which in these patients could change the diagnosis and therefore the opportunity for treatment or the time between follow-ups. In other cases, greater amount of ratio FLC assayed by Freelite, which may represent loss of linearity in subsequent dilutions. Falsely high results may occur due to polymerization of light chains.Our results suggest that Sebia FLC is a valid assay alternative, but needs more studies to demonstrate these findings; the prospective follow up of this group promises more.

Concentration of immunoglobulin free light chains in cerebrospinal fluid in the diagnosis of multiple sclerosis

Concentration of immunoglobulin free light chains in cerebrospinal fluid in the diagnosis of multiple sclerosis

Design and Rationale of Prolonged Nightly Fasting for Multiple Myeloma Prevention (PROFAST): Protocol for a Randomized Controlled Pilot Trial.

Obesity is an established, modifiable risk factor of multiple myeloma (MM); yet, no lifestyle interventions are routinely recommended for patients with overweight or obesity with MM precursor conditions. Prolonged nightly fasting is a simple, practical dietary regimen supported by research, suggesting that the synchronization of feeding-fasting timing with sleep-wake cycles favorably affects metabolic pathways implicated in MM. We describe the design and rationale of a randomized controlled pilot trial evaluating the efficacy of a regular, prolonged nighttime fasting schedule among individuals with overweight or obesity at high risk for developing MM or a related lymphoid malignancy. We aim to investigate the effects of 4-month prolonged nightly fasting on body composition and tumor biomarkers among individuals with overweight or obesity with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or smoldering Waldenström macroglobulinemia (SWM). Individuals with MGUS, SMM, or SWM aged ≥18 years and a BMI of ≥25 kg/m2 are randomized to either a 14-hour nighttime fasting intervention or a healthy lifestyle education control group. Participants' baseline diet and lifestyle patterns are characterized through two 24-hour dietary recalls: questionnaires querying demographic, comorbidity, lifestyle, and quality-of-life information; and wrist actigraphy measurements for 7 days. Fasting intervention participants are supported through one-on-one telephone counseling by a health coach and automated SMS text messaging to support fasting goals. Primary end points of body composition, including visceral and subcutaneous fat (by dual-energy x-ray absorptiometry); bone marrow adiposity (by bone marrow histology); and tumor biomarkers, specifically M-proteins and serum free light-chain concentrations (by gel-based and serum free light-chain assays), are assessed at baseline and after the 4-month study period; changes therein from baseline are evaluated using a repeated measures mixed-effects model that accounts for the correlation between baseline and follow-up measures and is generally robust to missing data. Feasibilityis assessed as participant retention (percent dropout in each arm) and percentage of days participants achieved a ≥14-hour fast. The PROlonged nightly FASTing (PROFAST) study was funded in June 2022. Participant recruitment commenced in April 2023. As of July 2023, six participants consented to the study. The study is expected to be completed by April 2024, and data analysis and results are expected to be published in the first quarter of 2025. PROFAST serves as an important first step in exploring the premise that prolonged nightly fasting is a strategy to control obesity and obesity-related mechanisms of myelomagenesis. In evaluating the feasibility and impact of prolonged nightly fasting on body composition, bone marrow adipose tissue, and biomarkers of tumor burden, this pilot study may generate hypotheses regarding metabolic mechanisms underlying MM development and ultimately inform clinical and public health strategies for MM prevention. ClinicalTrials.gov NCT05565638; http://clinicaltrials.gov/ct2/show/NCT05565638. DERR1-10.2196/51368.

High Responses Rates with Single Agent Belantamab Mafodotin in Relapsed Systemic AL Amyloidosis

Introduction: Systemic AL amyloidosis, a rare incurable plasma cell disorder, has no approved treatments at relapse. Belantamab mafodotin is an antibody-drug conjugate which targets BCMA antigen approved for relapsed refractory myeloma. We report our results using belantamab mafodotin monotherapy for the treatment of patients with relapsed refractory AL amyloidosis including those traditionally excluded from clinical trials (eGFR<30 ml/min/1.73m 2 and cardiac Mayo stage IIIb disease). Methods: All patients treated with belantamab mafodotin monotherapy at standard dose (2.5 mg/kg) or dose reduction between April 2021 - July 2023. Standardised assessments including measurement of cardiac biomarkers, clonal parameters and imaging were performed and disease assessment was reported as per International Society of Amyloidosis consensus criteria. Results: Twenty-seven patients were included. The median age at was 65 years (range 41-76) and eight (30%) patients were aged >70 years. Eighteen (67%) had λ AL-type and nine (33%) κ AL-type. A median of two organs were involved at baseline (range 1-4) with renal and cardiac involvement in 20 (74%) and 15 (56%) respectively (3 each for Mayo stage IIIa and stage IIIb). The median time from AL amyloidosis diagnosis to first administration of belantamab mafodotin was 69 (range 5-174) months (~6 years) with a median of 3 prior lines of treatment (range 2-6). Prior drug class exposure included proteasome inhibitors (100%), immunomodulatory drugs (81%) and anti-CD38 antibody (81%) therapy. Ten patients (37%) had undergone prior melphalan-conditioned autologous stem cell transplantation. At the time of first belantamab mafodotin dose, the median involved free light chain concentration was 118mg/l (range 31-1778), eGFR was 40 ml/min (range 7 - 120) and a third (9/27) with an eGFR <30 ml/min/1.73m 2. NT-proBNP was 845 ng/l (range 99-70,000). At data cut-off, a median of 5 (range 1-11) doses of belantamab mafodotin were delivered. Of those with evaluable disease, best haematological overall response rate (partial response or better) was 80% (20/25) at a median time to best haematological response of 2 months (95% CI 1-4, range 1-17). Complete response (CR) or very good partial response (VGPR) was achieved in 64% (16/25). Two patients have only had 1 cycle and too early to perform response assessment. Reasons for treatment discontinuation (n=12) were: inadequate response/progression (n=7), keratopathy (n=1), grade 3 thrombocytopenia (n=1), physician choice (n=3: discontinued due to treatment for unrelated metastatic squamous cell carcinoma but remains in CR; facilitation of renal transplant; prior to BCMA-directed CAR-T in VGPR). At a median follow-up of 13 months follow-up (95% CI 4-18, range 1-27) from belantamab mafodotin initiation, 15 patients (56%) remain on therapy. Two patients died (1 due to progressive disease, 1 unrelated) and seven patients progressed and had subsequent line of therapy at a median time of 6 months (95% CI 2-4) in those that had a next line of therapy. Median treatment-free survival or death was 27 months (10-NR), and 1-year treatment-free survival/death was 69% (95% CI 41-86). Median overall survival (OS) is not reached. 1-year OS is 88% (95% CI 59-97). Bilateral microcystic corneal keratopathy, the most frequent adverse event, was seen in 19 (70%) patients (5%; 1/19 grade 4).Ocular adverse events improved in all patients after treatment delay. Only one patient required treatment cessation due to ocular toxicity (despite achieving PR after one dose). Thrombocytopenia was reported in 3 (11%) (grade 3: 1; grade 2: 1). Only two patients remained on the standard dose of 2.5mg/kg for >3 cycles. Nine patients had eGFR <30ml/min and 6/9 started at a dose of 1.25mg/kg with no unexpected side effects (1 patient had grade 3 keratopathy). One patient post-renal transplantation (on tacrolimus and mycophenolate) had no significant toxicity and achieved a CR after cycle 3. No treatment-related deaths, cardiac or renal toxicities observed in our cohort. Conclusion: Belantamab mafadotin has high efficacy and good tolerability in multiply relapsed AL amyloidosis including efficacy in patients otherwise excluded from clinical trials. Treatment-emergent adverse events (especially keratopathy) do not preclude delivery of drug with appropriate dose reductions.

Outcomes Associated with Clone-Directed Therapies for Monoclonal Gammopathy of Renal Significance

Background: Monoclonal Gammopathy of Renal Significance (MGRS) is a category of diseases not meeting criteria for cancer, in which a clonal population of B-lymphoid or plasma cells produces a nephrotoxic protein leading to kidney injury. Informal consensus has arisen that therapy targeting the causal clone is most likely to be efficacious, and the primary goal of therapy is to prolong renal survival. Light chain (AL) amyloidosis has been extensively studied, yet there is a paucity of data describing outcomes associated with clone-directed therapy for the various subtypes of non-AL MGRS. Methods: This was a retrospective analysis of patients treated at two National Cancer Institute (NCI)-designated cancer centers in the United States. Medical records of adult patients seen between January 2010 and March 2023 were screened for MGRS. Patients were eligible if they had renal biopsy-confirmed histopathologic findings compatible with MGRS. AL amyloidosis was excluded. The primary outcome was renal survival, defined as time from diagnosis of MGRS to requiring renal replacement therapy (RRT) or renal transplant, with patients censored at last follow up or death. Baseline characteristics were described, including 24-hour proteinuria, estimated glomerular filtration rate (eGFR), whether a clonal population of B cells or plasma cells could be identified on histopathologic examination of marrow or lymph node biopsy, concentration of monoclonal protein, and involved serum free light chain concentration. Initial therapy was reported, although differences in outcomes across therapeutic strategies were not compared. Baseline clinical characteristics between patients who required RRT and those who did not were compared. Baseline clinical variables and time to RRT were compared across the two most common MGRS subtypes in the cohort: proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) and light chain deposition disease (LCDD). Continuous variables were compared across subgroups using Wilcoxon rank-sum test. Time-to-event outcomes were analyzed according to the Kaplan-Meier method. All analyses were conducted using R version 3.6.1. Results: In total, 65 patients were included, 23 (35%) of whom ultimately required RRT or transplant. Four patients died, all after RRT. Median baseline proteinuria was 2802 mg/24h (IQR 972.5 - 4492.0) and median eGFR was 33.0 mL/min (IQR 23 - 53) for the entire cohort. In all cases proteinuria was mostly albumin. Median baseline involved light chain concentration was 8.3 mg/dL (IQR 3.1 - 26.4). A majority of patients (n=33, 51%) had no identifiable serum monoclonal protein. The two most common MGRS subtypes were PGNMID (n = 26) followed by LCDD (n = 21). Clonal populations thought to be causing MGRS were identified in 41/64 patients (64%), including 37 (58%) with plasma cell clones, 1 (2%) with a lymphoplasmacytic lymphoma clone, and 3 (4.7%) with other B-cell clones. Clones were not identified in 23 (36%). The most common chemotherapy agents included bortezomib (n=42, 64%) and rituximab (n=14, 22%). Compared to patients not requiring RRT, patients requiring RRT had higher baseline proteinuria (median 3531 vs. 1980 mg/24h, p<0.001) and lower eGFR (median 16 v. 44 mL/min, p<0.001); differences in serum free light chain concentrations were not significant ( Figure 1). A clonal population thought to be causing MGRS was identified on bone marrow biopsy in a majority of LCDD patients (n = 20, 95%), while clones were infrequently found in PGNMID (n = 6, 23%). The vast majority (n=20, 95%) of patients with LCDD received bortezomib-based therapy. Initial therapy in patients with PGNMID was more evenly divided: 9 (35%) received bortezomib-based therapy, 10 (38%) received rituximab-based therapy, and 6 (23%) received other regimens. Despite differences in therapeutic approach, patients with LCDD and in patients with PGNMID had similar risk of RRT (cumulative risk 35% v. 43%, p=0.54; HR 0.83 (95% CI 0.52-1.33); p =0.44) ( Figure 2). Conclusion: Patients with MGRS remain at high risk of requiring RRT even with clone-directed therapy. Despite differences in pathogenic clones and therapeutic strategies, outcomes of patients with PGNMID and LCDD are similar. Because the severity of baseline renal function across MGRS histologies predicts renal survival, early diagnosis and treatment is likely critical to improving renal outcomes.

Novel Assay for Quantification of Free Light Chain (FLC) Dimers in Serum of Patients with Plasma Cell Dyscrasias

Free light chains (FLCs) are a well-established biomarker of plasma cell dyscrasias. FLCs exist as monomers, dimers and oligomers. Lambda FLC is particularly prone to form dimers and oligomers. Under certain pathological conditions, FLC dimerization changes towards the increased dimer formation. For instance, high levels of FLC dimers were found in AL amyloidosis and multiple myeloma (MM) even when the total FLC concentrations were near the normal levels. Thus, FLC dimer quantification can be used as a tool to diagnose and monitor AL amyloidosis and MM. Here we report a novel assay to detect and quantify FLC dimers with mass spectrometry. To demonstrate the power of the assay, we quantified FLC dimers in LC-only MM serum samples and compared the results to the gold standard Freelite assay. FLC dimer quant assay relies on the detection and quantification of constant region dimeric peptides of lambda and kappa LCs in serum with targeted mass spectrometry. The serum sample is enzymatically digested under non-reducing condition for 1 hour, specific disulfide-bridged dimeric peptides are then separated by Evosep One liquid chromatography and analyzed with parallel reaction monitoring (PRM) on Orbitrap Exploris mass spectrometer. FLC dimers can be quantified relative to control serum samples and by absolute quant using stable isotope labelled (SIL) peptides resulting in mg/dL values. The identities of the peptides were confirmed by mass spectrometry analysis of synthetic dimeric peptides and recombinant FLCs. To determine the lower limit of quantification (LLoQ) the control pooled serum was spiked with the increasing amounts of recombinant FLC. The assay demonstrated exceptional linearity from 0.78 to 50 mg/dL of spiked FLC kappa or lambda (R 2=0.999 for both). The assay only requires 10 μL of serum without the need for a diagnostic sample and can be performed at any time point, as frequently as needed. FLC dimer quant assay was compared to Freelite assay for 17 newly diagnosed patients with LC-only MM. All patients underwent autologous stem cell transplant (ASCT) and had banked serum samples at diagnosis and day 100 post-ASCT. 15 patients (88%) reached CR at day 100 post-ASCT. 8 patients had lambda LC MM, while 9 patients had kappa LC MM. At diagnosis the median involved FLC was 250 mg/dL (range: 26.1 - 1440), while at day 100 post-ASCT the median involved FLC was 1.1 mg/dL (range 0 - 2.5). The FLC dimer quant results were reported as a ratio to control, whereby the FLC dimer amount in control pooled serum was set to 1. FLC dimers were detected at all time points in all patients. The median involved FLC dimer ratio to control at diagnosis was 23.3 (range: 4.5 - 445.7), while the median involved FLC dimer ratio to control at day 100 post-ASCT was 1.0 (range: 0.3 - 3.8). Strong correlation (R 2=0.993 for lambda and R 2=0.931 for kappa) was observed between FLC dimer and Freelite assays (Fig.1). To conclude, we developed a novel assay to measure FLC dimer with mass spectrometry. The assay demonstrated a good correlation to Freelite assay. This study establishes an FLC dimer assay as a promising diagnostic and monitoring tool in LC MM patients. Large clinical studies are needed to establish reference ranges for kappa and lambda dimers in healthy population and demonstrate clinical utility in other plasma cell dyscrasias.

Quantification of Free Immunoglobulin Light Chains in Urine.

The serum-free immunoglobulin light chain assay has been recommended as a screening test for monoclonal gammopathy. We evaluated the usefulness of urine free immunoglobulin light concentration for selection of specimens for immunofixation electrophoresis. Using kits from The Binding Site for Freelite ®, we validated examination of urine for measuring free κ and λ light chains. The results of urine free light chain concentrations were evaluated to ascertain if the results could be used to reduce the number of specimens requiring urine protein immunofixation electrophoresis. In the 515 specimens examined, there was no evidence of monoclonal gammopathy or history of monoclonal gammopathy in 331. Monoclonal κ or λ light chains were detectable in 42 and 30 specimens, respectively. There was history of κ or λ chain associated monoclonal gammopathy in 62 and 50 patients, respectively. In the 38 monoclonal κ positive urine specimens, with light chain data, κ/λ ratio was >5.83 in all specimens. In 27 specimens positive for monoclonal λ light chains, with light chain data, the urine λ/κ ratio was > 0.17 in 24 of 27 specimens and > 0.041 in all specimens. In patients without monoclonal gammopathy all specimens had a κ/λ ratio of >5.83 or λ/κ ratio >0.17. The Freelite ® assay from The Binding Site is suitable for quantification of free light chains in urine. In patients with known history of monoclonal gammopathy, urine immunofixation electrophoresis may be omitted in specimens with κ/λ ratio of <5.83 for κ associated lesions and λ/κ ratio of <0.041 for λ associated lesions. However, the results do not support using this test for first-time urine testing for monoclonal light chains as it is not predictive of positive result, nor does it exclude a monoclonal light chain in urine.

Extracorporeal light-chain elimination in myeloma with simple medium cutoff membrane hemodialysis: a retrospective cohort study.

We determined the efficacy of free light chain (FLC) removal by regular dialysis equipment (high-flux filtration) with medium cutoff (MCO) membrane hemodialysis (HD) as an adjuvant treatment to standard chemotherapy for patients with acute kidney injury complicating multiple myeloma (MM) and its impact on further dialysis dependency. Sixty patients with acute dialysis-dependent renal failure secondary to MM were treated with MCO-HD (55 patients) or HCO (high cutoff)-HD (5 patients) as a control. FLC serum concentration, total protein, immunoglobulins, and LDH were measured throughout the dialysis therapy. The kidney function of the patients was followed up for 1 year. The median age was 69 years; 25 female and 35 male patients were enrolled. HD significantly reduced FLC kappa levels in the MCO/HCO group by 58%/84% (MCO/HCO group; p < 0.05) and FLC lambda by 39%/33% (MCO/HCO group; p < 0.05). Single HD data (MCO) showed a relative reduction of 70% in kappa and 37% in lambda FLC concentration, as expected by the different sizes of the light chains. Renal function improved significantly and continuously from starting creatinine 5.7/3.8 mg/dl (MCO/HCO group) before HD to 1.4/2.0 mg/dl (MCO/HCO group; p < 0.001) after 1 year. No significant alteration of total protein, immunoglobulins, and LDH concentrations by HD (HCO and MCO group) was observed. After 1 year, 37 of 60 patients were alive and 34 of them were off dialysis. FLC elimination with MCO-HD is effective, technically easy, and less cost-intensive as compared with HCO-HD. Kidney function recovery in MM patients is achievable.

Freelite and Kloneus assays in free light chain measurements in patients with renal impairment

BackgroundSerum free light chain (FLC) quantification is a diagnostic criterion for monoclonal gammopathy and its values in patients with renal impairment are different from those in healthy subjects. The aim of this study was to evaluate Freelite and Kloneus assays in these patients. MethodsIn this retrospective study, serum samples from 226 patients with chronic kidney disease (CKD) of stages 2–5 were measured with a Freelite assay on the Optilite system and with a Kloneus assay on the AU5800 system and compared with controls without renal impairment. ResultsBoth kappa FLC (K-FLC) and lambda FLC (L-FLC) concentrations increased with Kloneus and Freelite assays with each increment in CKD stage. In patients with CKD, Kloneus detected lower concentrations of K-FLC (median: 20.4 mg/L; 95% range: 9.8–57.2) than Freelite (median: 36.5 mg/L; 95% range: 16.5–137.7) and higher concentrations of L-FLC (median: 32.2 mg/L; 95% range: 14.4–96.7) than Freelite (median: 25.4 mg/L; 95% range: 11.9–86.0). This resulted in significantly different kappa/lambda ratios (K/L-FLC) in patients with CKD for the two tests. The Freelite K/L-FLC in the CKD group (median: 1.50; min–max: 0.66–3.45) was significantly increased compared with healthy controls, and the Kloneus K/L-FLC in the CKD group (median: 0.63; 95 % min–max: 0.34–1.01) was slightly lower. ConclusionsThese findings demonstrate that Freelite and Kloneus assays provide higher but not parallel values when FLCs are measured in patients with CKD, so an increase in K/L-FLC was observed in the case of Freelite, and we found a slight decrease in the case of Kloneus.

Elevated serum polyclonal immunoglobulin free light chains in patients with severe asthma.

Background: Inflammation plays a pivotal role in the pathophysiology of asthma. Free light chains (FLC) can cause inflammation by mast cell antigen-activation. Serum immunoglobulin (Ig) FLC κ, but not λ, were shown elevated in adult males with asthma. We sought to investigate if serum Ig FLC concentrations are affected by asthma severity and their relationships with inflammatory outcomes. Methods: By using immunoassays, we measured serum κ and λ Ig FLCs in 24 severe persistent asthma patients, 15 patients with moderate persistent asthma, 15 steroid-naïve mild persistent asthma patients and 20 healthy control subjects in a cross-sectional observational study. Total and specific serum IgE concentrations, fractional exhaled nitric oxide (FENO), lung function, peripheral blood eosinophils and neutrophils, and C reactive protein (CRP) were also measured. Results: Serum κ FLC concentrations were elevated in severe asthma patients compared mild asthma patients (p < 0.05) and healthy subjects (p < 0.05). Serum λ FLCs were higher in severe asthma patients than in healthy subjects (p < 0.05) and correlated with blood eosinophil counts (percentage, κ: r = 0.51, p = 2.9678-6; λ: r = 0.42, p = 1.7377-4; absolute values, κ: r = 0.45, p = 6.1284-5; λ: r = 0.38, p = 7.8261-4), but not with total or specific serum IgE. In severe asthma patients, serum Ig FLC correlated with serum CRP (κ: r = 0.33; p = 0.003; λ: r = 0.38, p = 8.8305-4) and blood neutrophil cell counts (percentage, κ: r = 0.31; p = 0.008; λ: r = 0.29, p = 0.01; absolute values, κ: r = 0.40; p = 3.9176-4; λ: r = 0.40, p = 4.5479-4), were elevated in subjects with blood eosinophilia (≥300 cells/µL) (n = 13) compared with non-eosinophilic subjects (n = 10) (κ: 19.2 ± 1.2mg/L versus 12.1 ± 1.3mg/L, p < 0.001; λ: 27.2 ± 2.6mg/L versus 16.8 ± 2.5mg/L, p < 0.01), but were similar in atopic (n = 15) versus nonatopic subjects (n = 9) (κ: p = 0.20; λ: p = 0.80). Serum FLC were negatively correlated with lung function tests, including forced expiratory volume in one second (FEV1) (κ: r = -0.33; p = 0.0034; λ: r = -0.33; p = 0.0035), and FEV1/forced vital capacity ratio (κ: r = -0.33; p = 0.0034; λ: r = -0.33; p = 0.0036). Conclusion: Serum Ig FLCs are elevated in severe asthma adults and might represent new surrogate markers of inflammation. The pathophysiological implications of these findings require further research. This study was approved by the ethics committee of the University Hospital Agostino Gemelli Foundation and Catholic University of the Sacred Heart (approval number P/1034/CE2012).

#5130 THE EXPERIENCE OF SELECTIVE PLASMA EXCHANGE THERAPY IN PATIENTS WITH MULTIPLE MYELOMA

Abstract Background and Aims High concentrations of free light chains (FLCs) in multiple myeloma lead to cast-nephropathy and obstructive acute renal failure in 18-56% of patients. The aim of this research is to estimate the efficiency of extracorporeal free light chains of immunoglobulin elimination under this pathological condition during plasma exchange therapy using selective filters. Method Selective plasma exchange therapy (SEPET) was carried out in 11 patients with multiple myeloma. The patients were 61 [47-69] years old. Paraproteinemia level at primary examination varied from 1.6 to 78.8 g/liter. All patients exhibited proteinuria of 0.56 to 12.7 g/day, Bence Jones protein excretion (from 0.4 to 11.2 g/day) was detected in 9 cases. Blood creatinine levels ranged from 272 μmol/L to 534 μmol/L. None of the patients required renal replacement therapy. FLCs levels above 500 mg/L, irrespective of azotemia level, served as an indication for starting the procedure. All patients received VMP induction chemotherapy after sessions of selective plasma exchange therapy. SEPET was carried out on a “Multifiltrate” (Fresenius) hemodialysis machine in hemofiltration mode. «Evaclio 2C20» plasmafilter (Japan) was used. Patients underwent one procedures of SEPET in a dose of 3 volumes of circulating plasma. Blood flow velocity during varied from 40 to 60 ml/min, the duration of each procedure was 5-6 hours. Plasma substitution was performed with «Prismasol K4» solution - 10.0 L and albumin solution 20%-400 ml. Regulated clotting by nonfractionated heparin was calculated individually from the APTT values. No early or delayed complications of extracorporeal hemocorrection were recorded. FLCs, creatinine levels, and coagulogram results of patients were examined before and after the procedure. Results Analysis of serum FLC values before treatment showed a significant increase of κ- or λ-FLC levels, significantly surpassing normal values: 3-21.5 mg/liter κ-FLC and 5-27 mg/liter λ-FLC, with κ/λ-FLC proportion of 0.25-1.65. The primary levels of involved FLC medians in the group were 472 [312;44700] mg/L for κ-FLC and 163 [ 473; 24673] mg/L for λ-FLC, κ/λ-FLC proportions were 2.89. Through the membrane of the plasma filter “Evaclio 2C20” are sieved and removed substances with a molecular weight less than the molecular weight of albumin (65 kD). Kappa FLCs circulate as monomers of 22 kDa but may assemble into dimers of 45 kDa, particularly lambda FLCs. There is a partial loss of albumin. High molecular weight substances do not pass through this membrane. During SEPET the κ-FLC level in the plasmafiltrate was 272 mg/L, the λ-FLC level was 94 mg/L. After SEPET median serum κ-FLC was 231 mg/L, median serum λ-FLC was 96 mg/L. Compared with baseline data, serum FLC levels decreased after 24 hours, so that median serum κ-FLC was 281 mg/L, median serum λ-FLC was 101 mg/L. Clinical tolerance of extracorporeal detoxification was satisfactory and hemodynamically stable in all patients; and there were no were traumas of blood elements or decrease of serum albumin. Renal function recovered in 3 patients in the course of therapy, improved in 5 patients with renal insufficiency, and remained unchanged in 3 patients. Conclusion The Selective plasma exchange therapy directly removes FLCs excess from circulating blood. Rapid reduction of FLCs level in the course of selective extracorporeal elimination can prevent the development of irreversible renal failure and make it possible to carry out adequate antitumor therapy.

#6760 EFFICACY OF HEMOSORPTION IN REMOVAL OF HIGH MOLECULAR WEIGHT UREMIC TOXINS IN CHRONIC HEMODIALYSIS PATIENTS

Abstract Background and Aims Symptoms associated with the accumulation of large uremic toxins (the development of chronic inflammation, accelerated progression of cardiovascular diseases and an increase in the incidence of protein-energy malnutrition) are common for patients who get chronic hemodialysis (HD) for a long time. HD effectively removes small and medium molecules (up to 15 kDa), while convection techniques, which are not always available, are used to remove substances of higher molecular weight. An alternative strategy for removing uremic toxins, especially large medium molecules, is the use of sorption techniques. The aim of the study was to compare the removal efficiency of substances with a molecular weight from 11.8 to 45 kDa using HD on a high-flux membrane and a combination of a high-flux membrane with hemosorption. Method The study included patients with a duration of hemodialysis therapy for more than 5 years. Blood sampling was carried out before and after the procedures, in the average RRT session per week. The following indicators were determined: β-2-microglobulin, leptin, free light chains (FLC) κ and λ, Il-6. The first HD session was performed on a Fresenius Fx-80 high-flux membrane. The second procedure (one week after) was carried out using the same filter, but with the connection of a Jafron HA130 sorption column (HD+HP). The duration of the both procedures was 4 hours. Blood flow did not exceed 300 ml/min. Results Total 10 patients were included in the study. Mean duration of renal replacement therapy was 12±5 years. The ratio of men and women was equal. Mean age 54±12 years. All patients used AV fistulas as vascular access. When determining and comparing the concentrations of β-2-microglobulin, leptin, Il-6, a comparable significant decrease in concentrations was noted after both procedures without significant statistical differences between the methods. At the same time, the efficiency of FLC removal on HD and HD+GP was convincingly different: - average kappa-FLC concentration change after HD was +1.8±9.1 mcg/ml, after HD+GP: -9.4±7.5 mcg/ml, p = 0.04. - average lambda -FLC concentration change after HD/HD+GP was +3.2±16.3 and -12.9±4.4 mcg/ml, respectively. p = 0.02. Conclusion The use of sorption techniques as an adjunct to standard HD therapy can increase the excretion of medium-large molecular substances, such as FLC kappa/lambda, which may help reduce the severity of symptoms associated with the accumulation of these uremic toxins. Additional studies are required to evaluate the clinical effectiveness of the new sorption method in chronic HD.

Free Light Chains κ and λ as New Biomarkers of Selected Diseases.

Diagnostic and prognostic markers are necessary to help in patient diagnosis and the prediction of future clinical events or disease progression. As promising biomarkers of selected diseases, the free light chains (FLCs) κ and λ were considered. Measurements of FLCs are currently used in routine diagnostics of, for example, multiple myeloma, and the usefulness of FLCs as biomarkers of monoclonal gammopathies is well understood. Therefore, this review focuses on the studies concerning FLCs as new potential biomarkers of other disorders in which an inflammatory background has been observed. We performed a bibliometric review of studies indexed in MEDLINE to assess the clinical significance of FLCs. Altered levels of FLCs were observed both in diseases strongly connected with inflammation such as viral infections, tick-borne diseases or rheumatic disorders, and disorders that are moderately associated with immune system reactions, e.g., multiple sclerosis, diabetes, cardiovascular disorders and cancers. Increased concentrations of FLCs appear to be a useful prognostic marker in patients with multiple sclerosis or tick-borne encephalitis. Intensive synthesis of FLCs may also reflect the production of specific antibodies against pathogens such as SARS-CoV-2. Moreover, abnormal FLC concentrations might predict the development of diabetic kidney disease in patients with type 2 diabetes. Markedly elevated levels are also associated with increased risk of hospitalization and death in patients with cardiovascular disorders. Additionally, FLCs have been found to be increased in rheumatic diseases and have been related to disease activity. Furthermore, it has been suggested that inhibition of FLCs would reduce the progression of tumorigenesis in breast cancer or colitis-associated colon carcinogenesis. In conclusion, abnormal levels of κ and λ FLCs, as well as the ratio of κ:λ, are usually the result of disturbances in the synthesis of immunoglobulins as an effect of overactive inflammatory reactions. Therefore, it seems that κ and λ FLCs may be significant diagnostic and prognostic biomarkers of selected diseases. Moreover, the inhibition of FLCs appears to be a promising therapeutical target for the treatment of various disorders where inflammation plays an important role in the development or progression of the disease.

The Clinical Significance of Serum Free Light Chains in Bladder Cancer.

This research aimed to assess the clinical usefulness of serum kappa (κ) and lambda (λ) free light chains (FLCs) in patients with bladder cancer (BC). One hundred samples were collected and analysed from healthy volunteers (C) and bladder cancer patients. Cancer patients were divided into two subgroups: low-grade (LG) and high-grade cancer (HG). Concentrations of FLCs, CEA, CA19-9, creatinine and urea were measured per manufacturers' guidelines. The concentrations of κ and λ FLCs and CEA were significantly higher in BC patients in comparison to the control group. Moreover, the concentrations of κ and λ FLCs and CEA were significantly higher in both low-grade as well as high-grade cancer in comparison to the controls. The levels of κ and λ FLCs differed between tumour grades, with patients presenting higher concentrations in high-grade compared to low-grade cancer. In the total study group, κFLC correlated with λFLC, the κ:λ ratio, CRP, CEA, CA19-9, creatinine and urea. There was also a correlation between λFLC and κFLC, CRP, CEA, creatinine and urea. The λFLC showed a higher ability (sensitivity and PPV) to detect bladder cancer in comparison to κFLC and CEA. In addition, λFLC had a higher ability to exclude BC (specificity and NPV) than κFLC and CEA. λFLC also showed the highest accuracy in the detection of bladder cancer. In conclusion, the revealed differences in the concentrations of both κ and λ FLCs suggest their potential participation in bladder cancer development. Increased concentrations of free light chains in bladder cancer patients and the association with the tumour grade suggest that κ and λ FLC measurements may be useful in the diagnosis and prognosis of bladder cancer. This is the first research that evaluates the concentration of FLCs in bladder cancer, so further studies are necessary to confirm their usefulness as tumour markers of this malignancy.

Diagnostic value of the saliva immunoglobulin free light chains concentrations measured in primary Sjцgren's syndrome

Proliferation and hyperactivation of B-lymphocytes in the salivary glands is a feature of primary Sjцgren's syndrome (pSS). Detection in saliva of proteins synthesized by B-lymphocytes may be important in the diagnosis of this disease.Objective: to evaluate the diagnostic value of measuring the concentration of immunoglobulin free light chains (FLC) in saliva in patients with pSS.Material and methods. The cross-sectional study included 24 patients with pSS over the age of 18 years. PSS was diagnosed according to the 2016 ACR/EULAR classification criteria. The control group consisted of 11 healthy volunteers. Blood-salivary glands histohematic barrier permeability ratio for albumin, FLC was measured. Quantitative determination of FLC and in blood and saliva was performed by enzyme immunoassay. An immunohistochemical study of biopsies of minor salivary glands (MSG) was carried out with a quantitative assessment of CD3+, CD4+, CD8+, CD20+, CD21+, CD68+, CD138+ cells. The Mann–Whitney U-test was used to compare quantitative traits. Identification of diagnostic thresholds for the concentration of FLC in saliva for the diagnosis of pSS was carried out using the ROC analysis method. An operating characteristic curve was plotted, the area under the curve, indicators of diagnostic specificity, diagnostic sensitivity, and diagnostic accuracy were calculated.Results and discussion. The obtained values corresponded to the low permeability of the histohematic barrier of the salivary glands for albumin and FLC in patients with pSS and healthy individuals. The median concentrations of FLC ê and ë in the saliva of patients with pSS and healthy volunteers were 1.08 [0.58; 1.91], 1.038 [0.55; 2.03] mg/l and 0.36 [0.32; 0.54], 0.35 [0.21; 0.52] mg/l, respectively. The concentration of FLC in the saliva of patients with pSS was statistically significantly higher than in the control group (p&lt;0.01). The amount of FLC ê and ë in saliva correlated with the rate of unstimulated saliva flow: rs=-0.483 (p=0.02), rs=-0.491 (p=0.017), respectively.A relationship was found between the concentration of ê-chains in saliva and the specific number of CD138+ cells: rs=0.733 (p=0.025). Statistically significant correlations between the concentration of ë-chains and the number of mononuclear cells in the MSG have not been established.Based on the results of ROC analysis, diagnostic thresholds for FLC concentrations in the saliva of patients with pSS were determined. Concentrations of ê- and ë-type FLC in saliva of 0.56 and 0.68 mg/l correspond to area under the curve values of 0.84 (95% confidence interval, CI 0.69–0.98) and 0.83 (95% CI 0.71–0.97), sensitivity 79.2% (95% CI 59.5–90.8) and 75% (95% CI 55.1–88), specificity 81.8% (95% CI 52.3–96.8) and 90.9% (95% CI 62.3–99.5), respectively.Salivary FLC concentrations were compared in patients with pSS receiving and not receiving glucocorticoids (GC). The groups did not differ in a statistically significant way in terms of clinical and laboratory parameters. The median daily dose of GC was 10 [5; 10] mg in prednisolone equivalent. There were no significant differences between the concentrations of saliva FLC in patients of these groups.Conclusion. Salivary-fixed FLCs are most likely produced by cells localized in the stroma of the salivary glands. Determination of the concentration of FLC in saliva can be proposed as a diagnostic test for the pSS. The concentration of free ê-chains in saliva can be considered as a surrogate marker of benign B-cell proliferation in the MSG. Therapy with low and medium doses of GC in pSS does not affect the concentration of FLC in saliva.

116 A CASE REPORT OF ISOLATED CARDIAC LIGHT CHAIN AMYLOIDOSIS WITHOUT HEART FAILURE: AN UNDER-RECOGNISED PRESENTATION

Abstract Background Cardiac involvement in light-chain amyloidosis (AL) usually represents a brick in the wall of a multi-system disease. The presence of cardiac deposition of free light chains (FLCs) is the main determinant of survival. Isolated cardiac AL is un uncommon scenario characterised by a challenging diagnostic and therapeutic work-up. Case Summary A 57-year-old asymptomatic man presented for an incidental finding of myocardial necrosis at the ECG performed for newly-diagnosed arterial hypertension. Alongside signs of previous myocardial infarction, transthoracic echocardiography showed a severely increased left ventricular (LV) wall thickness not consistent with ECG voltages, segmental akinesia with normal LV systolic function with ‘apical sparing’ pattern. Laboratory assessment showed an unexpectedly high level of natriuretic peptide and persistently abnormal troponin in the absence of symptoms or signs of heart failure or ongoing ischemia. Coronary angiogram confirmed the coronary artery disease. Before revascularization, a complete diagnostic work-up was carried. Serum electrophoresis detected a monoclonal gammopathy that was further investigated by serum immunofixation, revealing high lambda FLCs concentration. Fat pad, bone marrow and salivary glands biopsies resulted negative for amyloid deposition. Finally, endomyocardial biopsy was consistent with AL amyloidosis. Urgent percutaneous revascularization was performed and the patients was timely started on chemotherapy. Discussion The diagnosis of isolated cardiac AL amyloidosis is challenging and carries important therapeutic implications. As the short-term prognosis might be severely compromised, an accurate diagnostic flow-chart have to be systematically pursued to obtain a precise diagnosis and address the optimal, tailored management.

Free Light Chains, High Mobility Group Box 1, and Mortality in Hemodialysis Patients.

Background: Uremic toxins are associated with immune dysfunction and inflammation. The inadequate removal by hemodialysis (HD) of serum free light chains (FLCs) determines their accumulation. This study evaluated FLCs in HD patients, analyzing their relations with other biomarkers, such as serum high mobility group box 1 (HMGB1). Methods: FLC and HMGB1 were evaluated in a cohort of 119 HD patients. κFLC and λFLC were summated to give a combined (c) FLC concentration. Patients were followed prospectively until the end of the observation period of four years, or until the endpoint: the patient’s death. Results: cFLC values in HD patients were 244.4 (197.9−273.5) mg/L. We detected a significant reduction in CD8+ cells and a decreased CD4+/CD8+ ratio. HMGB1 levels were 94.5 (55−302) pg/mL. After multivariate analysis, cFLCs correlated with β2-microglobulin and the CD4+/CD8+ ratio. Subjects with cFLC values above 263 mg/L and with sHMGB1 values < 80 pg/mL experienced a significantly faster evolution to the endpoint (mean follow-up time to progression of 27.5 and 28.5 months, respectively; p < 0.001). After an adjusted multivariate Cox analysis, cFLCs were associated with 11% increased risk of death, whereas low sHMGB1 increased this risk by 5%. Conclusions: cFLCs and HMGB1 reflect the inflammation and immune dysfunction in HD patients representing two strong and independent risk markers of mortality.

Infectious Complications and Their Relationship with Mortality in Patients Newly Diagnosed with Multiple Myeloma

INTRODUCTION. Early mortality after diagnosis of Multiple Myeloma (MM) is common and frequently associated with infections. A Danish study reported a mortality of 22% among the first 180 days with infections being responsible for 50.9% of deaths. Treatment for MM has been related with infections in some studies, but this data is not consistent. Infections typically occur during the first months after MM diagnosis and initiation of treatment. In Latin America there is a lack of information about infections in patients diagnosed with MM, with only one study reporting a prevalence of 35.9% within the first 6 months, and most of them, 67.3%, during the first 3 months. To describe infections and to evaluate potential risk factors associated with them could help to improve the care of patients with MM. METHODS We conducted a retrospective cohort from a tertiary referral center in Mexico City. We included patients diagnosed with MM according to the International Myeloma Working Group criteria, between January 2017 and December 2020. Baseline clinical characteristics, disease parameters and infections at or after the MM diagnosis were collected, as well as the etiologic agents and their outcomes. The primary outcome was to describe the infectious complications of patients with MM in the first 6 months after diagnosis. The secondary outcome was to identify risk factors for early mortality defined as death during the first 6 months after diagnosis. Baseline characteristics and infections were described using mean and standard deviation or median and interquartile ranges. For the secondary outcome, patients were analysed in two groups depending in the presence of infections or not. Comparisons between groups were made by chi square, Fisher's exact test, t-tests, and rank-sum test; multivariate analysis by means of a logistic regression was performed. Two-sided p-values of <0.05 were considered statistically significant. All analysis were performed using STATA version 15.1. RESULTS Eighty-two patients were diagnosed and included in the study in the specific period. Median age was 66.5 and 56% were male. Median follow-up time was 28.6 months; 49/82 (60%) developed > 1 infections, of which 33/49 (67%) were in the first 4 months after MM diagnosis and 16/33 patients (48%) experienced two or more episodes. Distribution of infections was as follows: lower respiratory tract (45%), urinary tract (24%), gastrointestinal tract (9%) and primary bacteriemia (6%). Bacteria were the most common etiologic agents. There were 3 cases of Clostridium difficile infection documented during the second and third episodes of infection. The mortality associated with this infection was very high (66%). High plasma cell infiltration in bone marrow aspirate (p=0.03), increased kappa free light chain concentration (p=0.02), lower hemoglobin concentration (p=0.003), decreased serum albumin (p=0.004) and high serum globulin concentration (p=0.04) were associated with infection. Alkylating agents were the only treatment associated with increased risk of infection (p=0.05). Overall mortality during the first 6 months was 11%. There was a significantly higher cumulative risk (46.4%) of early death in patients who developed infections, compared to those who did not. CONCLUSIONS. Sixty percent newly diagnosed MM developed infections during the first 6 months after diagnosis. The most common affected sites were lower respiratory tract, urinary tract, gastrointestinal tract and primary bacteriemia. Sixty-seven percent of the infectious episodes were developed among the first four months after the diagnosis. The most common etiologic agents were bacteria. There were two episodes of infections caused by candida SPP and three caused by influenza virus. Gram-negative bacteria were the most common bacteria isolated during the second and third episodes, with 3 cases of Clostridium difficile infection. Infections were responsible of 46.4% more early deaths in newly diagnosed MM patients compared to those that did not have infections. High tumor burden characteristics were associated with greater risk of infections. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Defining new reference intervals for serum free light chains in individuals with chronic kidney disease: Results of the iStopMM study

Serum free light chain (FLC) concentration is greatly affected by kidney function. Using a large prospective population-based cohort, we aimed to establish a reference interval for FLCs in persons with chronic kidney disease (CKD). A total of 75422 participants of the iStopMM study were screened with serum FLC, serum protein electrophoresis and immunofixation. Estimated glomerular filtration rate (eGFR) was calculated from serum creatinine. Central 99% reference intervals were determined, and 95% confidence intervals calculated. Included were 6461 (12%) participants with measured FLCs, eGFR < 60 mL/min/1.73 m2, not receiving renal replacement therapy, and without evidence of monoclonality. Using current reference intervals, 60% and 21% had kappa and lambda FLC values outside the normal range. The FLC ratio was outside standard reference interval (0.26–1.65) in 9% of participants and outside current kidney reference interval (0.37–3.10) in 0.7%. New reference intervals for FLC and FLC ratio were established. New reference intervals for the FLC ratio were 0.46–2.62, 0.48–3.38, and 0.54–3.30 for eGFR 45–59, 30–44, and < 30 mL/min/1.73 m2 groups, respectively. The crude prevalence of LC-MGUS in CKD patients was 0.5%. We conclude that current reference intervals for FLC and FLC ratio are inaccurate in CKD patients and propose new eGFR based reference intervals to be implemented.