Lambda Free Light Chains Research Articles

Free Immunoglobulin Light Chains in Patients With Myocarditis: a New Biomarker of Inflammation and Heart Failure

To study the concentration of immunoglobulin free light chains (FLCs) in patients with myocarditis in comparison with non-inflammatory heart diseases, their relationship with inflammatory markers and the severity of chronic heart failure (CHF). This study included 77 patients (31 women, mean age 54.1±13.3 years): 41 patients with myocarditis verified by myocardial biopsy (n=18) or using a noninvasive diagnostic algorithm, 31 patients with noninflammatory CHF (comparison group), and 5 patients with monoclonal gammopathy identified during the study (4 of them were diagnosed with AL amyloidosis with heart damage). In the myocarditis group, CHF was diagnosed in 29 patients, mean stage IIA, functional class (FC) 2-3, with a mean left ventricular ejection fraction 43%. In the comparison group, patients had predominantly IIA stage, FC 2-3 CHF without systolic dysfunction. The blood concentration of kappa and lambda FLC types was measured with Cloneus S-FLC-K TIA Kit and Cloneus S-FLC-L TIA Kit. Concentrations were considered normal at FLC-kappa 4.84-14.20 mg/l, FLC-lambda 7.03-22.50 mg/l, and the FLC-kappa/lambda ratio 0.426-1.050. Increased FLC concentrations were found in 58% of patients with myocarditis and in 77% of patients in the comparison group. The FLC-lambda concentration was significantly higher in the comparison group; there were no significant differences between the groups in FLC-kappa and their ratio. The closest significant correlations in both groups and the entire cohort were noted between FLCs of either type and CHF, as well as the requirement for loop diuretics (correlation coefficients, 0.60-0.90), independent on the severity of systolic dysfunction. Myocarditis patients also showed correlations of FLCs with the titer of antibodies to cardiomyocyte nuclear antigens, levels of C-reactive protein, leukocytes, neutrophils, erythrocyte sedimentation rate, and the concentration of N-terminal fragment of brain natriuretic peptide. In a subgroup of 10 myocarditis patients who were treated with immunosuppressants, FLCs of both types were significantly lower than in the comparison group; only with the persistence of severe CHF was an increase in FLCs noted. An increased FLC concentration can be considered as an important pathogenesis component that reflects both the specific mechanisms of myocarditis and the severity of CHF. In the absence of a statistically significant increase in general inflammatory markers in the blood of myocarditis patients, the measurement of FLCs can be used as an additional diagnostic marker and predictor of the decompensated variant of the course of myocarditis. However, the diagnostic and prognostic significance of FLC concentration in patients without CHF requires a further study.

Monoclonal gammapathies: Epidemiological, immunochemical and etiological analysis of a series of 50 cases

During the period from September 2022 to January 2023, 50 cases of monoclonal gammopathy (GM) were collected at the Avicenne military hospital in Marrakech. The average age of the patients was 62 years. Thirty patients were male and 20 were female. The etiological diagnosis could be established for the 50 observations: 5 were classified as GM of undetermined significance (GMSI), 42 were classified as myeloma, two case of Waldenström's disease and one case of AL amyloidosis. The clinical symptomatology was dominated by bone pain in 78% of patients with a deterioration in general condition in 43%. The most frequently found biological abnormalities were anemia in 62% of patients, renal failure in 48% of patients and 19% had hypercalcemia. A monoclonal peak was found in the electrophoreses of 45 patients (90%), of whom 6 migrated to the beta zone and 39 migrated to the gamma zone. The IgG Kappa isotype was the most frequent with a rate of 56%, followed by IgG Lambda (27%), IgA Kappa (8%), IgA Lambda (3%), IgM Lambda (3%) and lambda free light chains (3%).

Clonality Determination by Detecting Unmodified Monoclonal Serum Free Light Chains Using On-Probe Extraction Coupled with Liquid Chromatography-High-Resolution Mass Spectrometry.

Serum free light chains (FLCs) are an essential clinical biomarker for the diagnosis and monitoring of patients with plasma cell neoplasms. The current widely used immunoassay methods quantify total serum FLCs, which include monoclonal FLCs as well as FLCs in the polyclonal background. Patients with chronic diseases, inflammatory disorders, or renal dysfunction can have elevated total FLCs that lead to ambiguous results. These patients may benefit from a direct measurement of monoclonal FLCs. The purpose of this study was to develop a method that couples on-probe extraction (OPEX) with liquid chromatography-high-resolution mass spectrometry (LC-HR-MS), abbreviated to OPEX-MS, to directly determine the clonality of FLCs. OPEX immunocapture was performed using microprobes loaded with anti-kappa or anti-lambda light chain antibodies. Captured proteins were separated by reversed-phase LC and analyzed by HR-MS. Four cohorts of samples from unique patients were tested based on immunoassay FLC results. The LC-HR-MS analysis in the OPEX-MS method provides both a unique retention time along with deconvoluted masses of FLC monomers and dimers for each clone. The study found that 16 out of 49 (33%) kappa FLC elevated samples as well as 83 out of 100 (83%) dual kappa and lambda FLC elevated samples did not have monoclonal FLCs, which is consistent with the knowledge that there is often no clonal population in samples with mildly elevated FLC immunoassay results. The OPEX-MS method can serve as a complementary approach to directly determine clonality in patients with difficult-to-interpret FLC immunoassay results.

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-319 Kappa and Lambda Free Light Chains Quantification in Serum Samples with 3 or 4 Monoclonal Components Detected by Serum Immunofixation

Abstract Background Serum free light chain (FLC) quantification is a diagnostic criterion for monoclonal gammopathy. Oligoclonal expansion is defined when two or more small bands are detected in the gamma region of serum protein electrophoresis (SPEP) or immunofixation (SIFE) within the context of infectious diseases, autoimmune diseases, or some lymphoproliferative processes. This study aims to investigate the frequency of FLC disturbances in samples with oligoclonal pattern of 3 or 4 monoclonal bands observable in SIFE. Methods We have analyzed 7553 consecutive samples during the period of January to December 2023 in which both determination of FLC and SIFE were ordered in a large central laboratory at São Paulo, Brazil. FLC quantification and SIFE analysis were performed with FreeLite assay and Optilite system (The Binding Site, UK) and SPIFE 3000 (Helena Laboratories, USA), respectively. Results In our analysis, 119 samples (50 males, 69 females, median age 69 years (range 37-90)), with 3 or 4 monoclonal bands were identified, constituting 1.57% of the overall study population. These samples were then categorized into two groups. The first group had normal FLC Ratio (range 0.26 to 1.65) and the second group had altered FLC Ratio (outside 0.26-1.65 range). Seventy-seven samples (65%) had altered FLC ratio, with kappa elevation being the most common alteration (51 samples). Within these samples, median FLC ratio was 2.99 (range 1.72-719.05). Lambda elevation was present in 26 samples, with median FLC ratio 0.04 (range 0.005-0.16). Conclusions Presence of 3 or more oligoclonal bands in SIFE analysis may be indicative of various pathological processes (benign and malignant) and also be observed in post-transplantation. In this study, we have observed that most of these samples present altered FLC ratio, which indicate monoclonal disturbances in the production of free light chains. Those disturbances may be associated with a lymphoproliferative disease (e.g. multiple myeloma) or may be transient. Clinicians should be aware of the need for follow-up of these cases.

Hemato-Renal Profile of Proliferative Glomerulonephritis with Monoclonal Immunoglobulin Deposits

Background Proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) is a rare entity classified under the umbrella of monoclonal gammopathy of renal significance. The clinical implications of circulating monoclonal immunoglobulin (MIg), light chain restriction on immunofluorescence (IF) microscopy, histopathological pattern, and type of therapy on renal outcomes are not clearly defined. Materials and Methods Sixteen patients of PGNMID diagnosed between 2013 and 2020 were included from a biopsy registry of 11,459 patients at a single center. Follow-up data was collected from electronic medical records until June 2021. Results The mean age of the cohort was 41.7 ± 13.5 years. Forty-four (7/16) percent showed monoclonal protein on serum or urine electrophoresis, 25% (3/12) had IgG kappa by serum immunofixation electrophoresis (IFE) and 38% (5/13) had abnormal kappa: lambda free light chain (FLC) ratio. The predominant light microscopy pattern, membranoproliferative glomerulonephritis (MPGN) was seen in 7/16 (43.7%) patients. The predominant heavy chain detected by IF microscopy was IgG (13/16, 81.3%). Kappa and lambda light chain restriction were seen in 56.3 (9/16) and 43.8 (7/16) percent of patients respectively. Circulating monoclonal kappa light chains were detected in 50 and 29% of kappa-PGNMID patients by IFE and FLC assay respectively. None of the lambda-PGNMID patients had detectable circulating monoclonal lambda light chains. Patients with circulating MIg had more proteinuria, lower estimated glomerular filtration rate, and a higher percentage of plasma cells on bone marrow biopsy. Thirty-eight percent of our cohort (5/13) progressed to kidney failure over a median (range) period of 3 (IQR, 1-7) months. Of these, 4/5 received immunosuppression, and 1/5 were treated with plasma cell-targeted chemotherapy. Conclusion PGNMID is a rare disease with a biopsy incidence of 0.1%. Only a quarter of patients with PGNMID have circulating MIg. Presence of circulating MIg, type of monoclonal light chain restriction in kidney biopsy, and type of therapy did not predict renal outcomes. Patients with MPGN pattern had favorable renal outcomes despite a higher degree of proteinuria at presentation.

Comparison of 2 Serum Free Light Chain Assays with Creatinine Normal and Abnormal Populations Demonstrates the Need for Standardization.

The objective of this study was to compare The Binding Site's Freelite on Optilite and Diazyme's Kappa/Lambda free light chains (K/L FLC) on Abbott Architect c8000 with healthy and renal insufficient populations and to evaluate their respective reference intervals for serum free light chains (sFLCs). Two hundred sixty serum samples were measured for creatinine and sFLCs by both assays and a subset by immunofixation electrophoresis. Verification of manufacturer-defined reference intervals was assessed. Kappa free light chains (KFLC) showed excellent correlation of 0.998 R2 with a slope of 0.73. For Lambda free light chains (LFLC), an acceptable correlation of 0.953 R2 was found with a slope of 1.50 as well as a skewness-based difference with a -12.70 intercept. Healthy estimated glomerular filtration rate (eGFR) ≥60 reference interval verification of central 95% could not be confirmed for either Freelite or Diazyme although LFLC was much closer than KFLC for both assays with Freelite KFLC recovering only 37% of values within reference interval claims. The K/L FLC ratio did not meet 100% claim for both Freelite (91%) and Diazyme (95%) among those with eGFR ≥60. Samples with eGFR ≤59 had increasingly higher levels of KFLC and LFLC for both assays. When comparing worsening eGFR status, Freelite recovered increasingly higher ratios while Diazyme recovered increasingly lower ratios. Healthy reference intervals could not be verified for either Freelite or Diazyme. Renal reference intervals for Freelite are currently warranted while they are not recommended for Diazyme. The differences between these 2 assays can be minimized by standardization efforts such as recalibration.

Evaluation of Sarcopenia in Patients with Monoclonal Gammopathy of Undetermined Significance.

Background: We aimed to determine the prevalence of sarcopenia in patients with monoclonal gammopathy of undetermined significance (MGUS) and to evaluate the links between MGUS and sarcopenia. Methods: Eighty-two patients with a diagnosis of MGUS were enrolled in the study. Muscle strength was measured using the handgrip dynamometer. Physical performance was assessed by assessing gait speed over a 6-minute walking test. Muscle mass was determined using a bioelectrical impedance analyzer. Results: Sarcopenia was confirmed in 34.15% of patients. Male predominance was demonstrated in MGUS subjects with sarcopenia, particularly patients with low hand grip strength, low appendicular skeletal muscle mass (ASMM), or low ASMM index (p < 0.001, 0.013, and 0.001, respectively). Higher age and lower serum free light-chain Lambda levels were shown in MGUS patients with low muscle function scores compared to normal scores (p < 0.001, and 0.014, respectively). In addition, having a low ASMM score was related to low body mass index and high-risk group (p = 0.020, 0.033, respectively). Conclusions: We demonstrated that the frequency of sarcopenia is high in patients with MGUS. Whether sarcopenia has a possible role as a factor contributing to the pathogenesis of MGUS should be supported by further studies containing longitudinal data.

AL–AMYLOIDOSIS RELATED ADVANCED HEART FAILURE DIAGNOSED THROUGH F–18–FLUTEMETANOL PET SCAN

Abstract Background Systemic amyloidosis (SA) is a group of rare diseases in which abnormal misfolded proteins, amyloid fibrils, accumulate in tissue. The gold standard for diagnosis is tissue biopsy of the affected organ, but it is invasive and needs specific proteomic or immunohistochemistry analyses to characterize the involved protein. We report a case of a complex diagnosis of SA in an advanced heart failure (HF) patient. Methods and Results A 68–year–old woman with history of recurrent uveitis started to complain dyspnea in 2022, after Covid19. Since her symptoms worsened, in June 2023 she was diagnosed in another center with restrictive cardiomyopathy with preserved left ventricular (LV) ejection fraction. She underwent coronary angiography (unremarkable), cardiac MRI (aspecific fibrosis), and a FDG–PET scan which showed non–homogeneous LV fixation. The patient was treated with steroids for the suspicion of an immunologic–inflammatory disease, without benefit. Fourteen months after the initial presentation, she was admitted to our centre for advanced inotrope–dependent HF. She had mildly increased LV hypertrophy and severe systolic dysfunction with restrictive physiology and, notably, peri–orbital subcutaneous hemorrhages (Figure 1). Hematological screening for amyloidosis showed elevated serum free lambda light chains. Repeated cardiac MRI scan was consistent with infiltrative disease. A total–body PET scan with dual tracer was performed. The control session with F–18–FDG showed mild aspecific cardiac uptake in the basal septum and lateral wall (Figure 2), while off–label use of F–18–Flutemetanol, an Alzheimer‘s disease approved experimental tracer that binds to amyloid fibrils beta sheets with very high sensitivity and specificity, showed intense cardiac, hepatic, pulmonary and muscular uptake, with wash–out–timing diagnostic for AL–SA (Figure 3). Periumbilical fat biopsy confirmed the clinical suspicion of SA. Hematological investigations led to the diagnosis of stage I–A lambda micromolecular multiple myeloma, with systemic amyloidosis and a IIIb cardiac prognostic score. Conclusions Endomyocardial biopsy is an invasive procedure and periumbilical fat biopsy discriminates the amyloid protein type only by proteomic analysis, which is available in very few Italian centres. The off–label use of Alzheimers’ PET tracers could reduce the delay of cardiac AL–SA diagnosis, allowing early disease identification and prompting timely hematological therapy.

Effects of aerobic and resistance exercise for 9 months on serum free light chains in type 2 diabetes.

Background and aims: Serum polyclonal free light chains (FLCs) levels are associated with overall survival in the general population, reflecting their utility as a biomarker of underlying immune activation and inflammation. Regular exercise is known to ameliorate low-grade inflammation in chronic diseases such as type 2 diabetes; however, the effects of different exercise training modalities on FLCs in adults with type 2 diabetes is unknown. This study investigated the effects of 9-month of aerobic, resistance or combined supervised exercise on serum FLCs in 164 patients with type 2 diabetes (age 58 ± 8years; 63% female). Methods: 164 participants from the Health Benefits of Aerobic and Resistance Training in individuals with type 2 diabetes trial (HART-D) were randomly assigned to no exercise (n = 27), aerobic exercise alone (n = 41), resistance exercise alone (n = 49), or a combination of aerobic and resistance exercise (n = 47). Fasting serum samples were collected before and after completion of the intervention to quantify changes in kappa and lambda FLCs, and serum creatinine, using commercially-available ELISAs. Results: At baseline, combined kappa and lambda FLCs (FLC sum; calculated as kappa + lambda FLCs) were positively correlated with high-sensitive C-reactive protein (hs-CRP) (r = 0.237, p < 0.05) and fat mass (r = 0.162, p < 0.05), and negatively associated with aerobic fitness (r = -0.238, p < 0.05). While non-exercise controls exhibited an increase in FLCs over the 9-month study, exercise training blunted this increase (Δ FLC sum control arm: 3.25 ± 5.07mg∙L-1 vs. all exercise arms: -0.252 ± 6.60mg∙L-1, p < 0.05), regardless of exercise modality. Conclusion: Serum FLCs were associated with physical fitness and body composition in patients with type 2 diabetes. 9-month of exercise training prevented the accumulation of FLCs, regardless of exercise modality. Unlike hs-CRP-which did not change during the trial-serum FLCs may serve as a more sensitive biomarker of chronic low-grade inflammation in this population.

Association of APOE genotype with blood-brain barrier permeability in neurodegenerative disorders

Apolipoprotein E (APOE) is recognized for its role in modulating blood-brain barrier (BBB) permeability in vitro, which may have significant implications for the pathogenesis and progression of neurodegenerative disorders. However, evidence in vivo is contrasting. This study explores the impact of APOE genotypes on BBB integrity among 230 participants experiencing cognitive impairment, encompassing cases of Alzheimer's disease (AD) as well as various non-AD neurodegenerative conditions. To assess BBB integrity, we utilized cerebrospinal fluid (CSF)/serum albumin ratios and CSF/serum kappa and lambda free light chains (FLCs) as indirect markers. Our findings show a dose-dependent increase in BBB permeability in individuals carrying the APOE ε4 allele, marked by elevated CSF/serum albumin and FLCs ratios, with this trend being especially pronounced in AD patients. These results highlight the association of APOE ε4 with BBB permeability, providing valuable insights into the pathophysiology of neurodegenerative diseases.

Concurrent Challenges: Multiple Myeloma and Cardiac Amyloidosis

We report a case of 56-year-old male with known case of Hypertension and Hypothyroidism presented with complaints of generalised weakness and weight loss since 8 months (approx. 40kg), tightening in and around the mouth and hoarseness of voice with dysphagia since 6 months, swelling in feet since 3 months, cough and constipation on and off since one month. The laboratory results indicated that the serum calcium (S.Ca2+) level was 14.1 mg/dl and the ionized calcium (Ca2+) level was 1.62 mmol/l. Immunoglobulins were low {IgA- 17 mg/dl (50-410 mg/dl), IgG- 386 mg/dl (650-1500 mg/dl), IgM- 20 mg/dl (60-280 mg/dl); Immunofixation electrophoresis (IFE) revealed positive result for lambda free light chain (Figure 1) and further quantitative value lambda free light chain was high (Lambda free - 2054.89 mg/l). Whole Body FDG PET Scan revealed non-FDG avid lytic lesions at multiple bone sites. In view of suspicion of Multiple Myeloma, bone marrow aspiration and trephine biopsy were done which confirmed the diagnosis of Multiple Myeloma. ECG showed low voltage QRS complexes and 2D echo revealed- Global LVEF 55-60%, Dilated LA, concentric LVH with altered myocardial echotexture, no RWMA, Grade II diastolic dysfunction present (raised LVEDP), RVSP-39mmhg (moderate PAH), findings S/O- cardiac amyloid; which was later confirmed with Cardiac MRI. Abdominal fat pad biopsy revealed area of necrosis but negative for congo red stain (amyloidosis). The patient underwent VCD (bortezomib; cyclophosphamide; dexamethasone) chemotherapy with Daratumumab. By the time of publication, the patient had received autologous stem cell transplantation and after 10 courses of VCD with Daratumumab, repeat bone marrow aspiration and histo-biopsy revealed complete remission of myeloma. The uncommon Multiple Myeloma complication in this clinical case was associated with restricted cardiomyopathy as a result of secondary cardiac amyloidosis. This is true for both Multiple Myeloma and cardiac amyloidosis. When one of these diagnoses is initially established, more investigation should be done to rule out the other illness, keeping in mind the necessity of meeting standard diagnostic criteria and the challenge of conducting an effective diagnostic workup in occasionally complex clinical situations.

Prospective Multicenter Study on Early Proximal Tubular Injury in COVID-19–Related Acute Respiratory Distress Syndrome

IntroductionDuring COVID-19, renal impairment is the most frequent after lung impairment and is associated with poor prognosis particularly in intensive care unit (ICU). We aimed to assess the existence and incidence of early renal dysfunction and its prognostic value in patients with COVID-19-related acute respiratory distress syndrome (ARDS). MethodsIn this prospective multicenter study, patients aged over 18 years with invasive mechanical ventilation for ARDS were enrolled in 3 ICUs. Precise evaluation of renal dysfunction markers including urinary proteins electrophoresis (UPE) and quantification, was performed within 24 hours after mechanical ventilation onset. ResultsFrom March 2020 to December 2021, 135 patients were enrolled: 100 COVID-19 ARDS and 35 non-COVID-19 ARDS. UPE found more tubular dysfunction in COVID-19 patients (68% vs. 21.4%, p<0.0001) and more normal profiles in non-COVID-19 patients (65.0% vs. 11.2%, p=0.0003). COVID-19 patients significantly displayed early urinary leakage of tubular proteins like beta-2-microglobulin and free-light chains, tended to display more frequently acute kidney injury (AKI) (51.0% vs 34.3%, p=0.088), had longer mechanical ventilation (20 vs. 9 days, p<0.0001) and longer ICU stay (26 vs. 15 days, p<0.0001). In COVID-19 ARDS, leakage of free lambda light chain was associated with the onset of KDIGO ≥2 AKI (OR: 1.014, 95%CI [1.003-1.025], p=0.011). ConclusionsPatients with COVID-19-related ARDS display a proximal tubular dysfunction, prior to the onset of AKI, which predicts AKI. Proximal tubular damage seems an important mechanism of COVID-19-induced nephropathy. Analysis of urinary proteins is a reliable non-invasive tool to assess proximal tubular dysfunction in the ICU.

Isoelectric focusing followed by affinity immunoblotting to detect monoclonal free light chains in monoclonal gammopathies: Comparison with immunofixation electrophoresis and free light chain ratio.

Isoelectric focusing (IEF) is a method with an exquisite resolution, and coupled with affinity immunoblotting (AIB), it can provide superior sensitivity to detect monoclonal free light chains (FLC). We tested the hypothesis that IEF/AIB is more sensitive and specific for monoclonal FLC detection in serum and urine samples than conventional methods, that is, electrophoresis (ELP), immunofixation (IF) and serum FLC ratio assessment. Investigation included 107 samples of 68 patients, among which 21 multiple myeloma patients were recently tested for minimal residual disease and 18 patients with AL amyloidosis. Monoclonal FLC were detected by IEF/AIB in 37% of serum samples negative for monoclonal FLC on ELP/IF. As for urine samples, significant advantage of the IEF/AIB over ELP/IF was not demonstrated. Considering both serum and urine results, IEF/AIB definitely revealed monoclonal FLC in 20/83 (24%) of ELP/IF-negative samples. FLC ratio was abnormally high (>1.65) in all 11 patients definitely positive for monoclonal FLC kappa by IEF/AIB but also in 16/47 (34%) IEF/AIB-negative samples. Abnormally low values (<0.26) were found only in 10/28 samples (36%) positive for monoclonal FLC lambda. Appropriate use of renal FLC ratio reference range reduced the number of presumably false positives (6/47, i.e. 13%) but not false negatives (17/28, i.e. 61%). The IEF/AIB method is more sensitive than IF and might be used in patients with negative IF results before deciding whether to proceed to minimal residual disease testing.

Clinical specificity of two assays for immunoglobulin kappa and lambda free light chains.

Free light chain (FLC) assays and the ratio of κ/λ are recommended for diagnosis, prognosis and monitoring of plasma cell dyscrasias (PCD). Limited data exists on FLC clinical specificity in patients diagnosed with other conditions. We assessed the κ, λ, and κ/λ FLC ratio using the FreeLite assay and the Sebia FLC ELISA assay in 176 patients with clinical presentations of fatigue, anemia, polyclonal hypergammaglobulinemia, joint disorders, kidney disease and non PCD-cancers with no monoclonal protein observed on serum protein electrophoresis or MASS-FIX immunoglobulin isotyping. Manufacturer defined reference intervals (RI) and glomerular filtration rate (GFR) specific RI (renal RI) were utilized. For the κ/λ ratio, 68.7 % (121/176) of specimens on the FreeLite and 87.5 % (154/176) of specimens on the Sebia assay were within RI. For κ, 68.2 % (120/176) and 72.2 % (127/176) of results were outside RI for FreeLite and Sebia respectively. For λ, 37.5 % (66/176) and 84.1 % (148/176) of FreeLite and Sebia results were outside RI. With FreeLite and Sebia, patients with kidney disease (n=25) had the highest κ/λ ratios. 44 patients (25.0 %) had GFR <60 mL/min/BSA. When renal RI were applied, 13.6 % had a FLCr outside the renal RI with FreeLite, and 4.5 % with Sebia. In a cohort of patients with signs and symptoms suggestive of PCDs, but ultimately diagnosed with other conditions, Sebia FLC had improved clinical specificity relative to FreeLite, if one was using an abnormal κ/λ ratio as a surrogate for monoclonality.

M-Protein Analysis Test: Effects of Combining Serum Protein Electrophoresis and Free Light Chain Assay Tests into One Order

Background: Approximately 10-15% of clonal plasma cell disorders (PCDs, such as monoclonal gammopathy of undetermined significance or MGUS, smoldering myeloma or SMM, and multiple myeloma, MM) secrete light chains only, which are often missed on a serum protein electrophoresis (SPEP) and only detected on a serum free light chain (sFLC) or 24 hour urine (UPEP) assay. For this reason, the IMWG has recommended that screening tests for PCDs should include both an SPEP and sFLC. In 2020, clinicians at our institution were instructed to order an sFLC analysis in addition to an SPEP (with reflex immunofixation electrophoresis) when assessing for PCDs. This did not, however, appear to improve adherence to IMWG guidelines or alter screening practice. In 2021 separate orders for SPEP and sFLC were removed from the EMR and a new, combined order, called an “M-protein analysis”, was implemented. We now evaluate the effect of this change in test order by comparing the data for the 12 months preceding to the 12 months after the change. Methods: All 1 st time SPEP and sFLC results were identified for two 12-month periods: Group A (7/1/2020- 6/30/2021) and Group B (7/1/2021-6/30/2022). Within each time frame, we also identified patients without an M-protein on SPEP but a positive sFLC which was defined as an elevated kappa or lambda free light chain and a ratio either &amp;gt;1.65 or &amp;lt;0.26 (Groups C and D, correlating with earlier and later time frames, respectively). Groups E (pre-order change) and F (post-order change) included the subsets of sFLC positive only pts from each time frame with an sFLC ratio &amp;gt;3.0 or &amp;lt;0.26 . These more restrictive groups were chosen to minimize those with polyclonal light chain findings which may be seen in renal disease. Results: Group A: 4192 SPEPs, 1876 sFLCs (45% of SPEPs); Group B: 3966 SPEPs, 3966 sFLCs (100% of SPEPs) Group C: those from A with a negative SPEP and a positive sFLC defined by an elevated kappa or lambda free light chain and a ratio either &amp;gt;1.65 or &amp;lt;0.26: 30 (1.6%) were identified. Group D: those from B with a negative SPEP but a positive sFLC defined by an elevated kappa or lambda free light chain and a ratio either &amp;gt;1.65 or &amp;lt;0.26: 102 (2.6%) were identified. Group E: those from A without an M-protein on SPEP but a positive sFLC defined by an elevated kappa or lambda free light chain and a ratio either &amp;gt;3 or &amp;lt;0.26: 17 (0.9%) were identified. Group F: those from B without an M-protein on SPEP but a positive sFLC defined by an elevated kappa or lambda free light chain and a ratio either &amp;gt;3 or &amp;lt;0.26: 28 (0.7%) were identified. For the total sFLC positive only groups (C and D): Average age= 74 (49-93); Race: 51% white, 42% AA, 7 % UK; sex: 93% male; light chain type: 91% kappa, 9% lambda; K:L ratio: med 4.5 (range 0.01-165). For Group E (pre-order change, ratio &amp;gt;3 or &amp;lt;0.26): of the 17 cases, most were diagnosed with MGUS (10), 1 SMM and 6 with MM (all of whom were symptomatic at time of testing). For Group F (post-order change, ratio &amp;gt;3 or &amp;lt;0.26): of the 28 cases, most were diagnosed with MGUS (23), 2 CLL, 2 SMM, 1 B-cell lymphoma and 1 with MM (who was asymptomatic at time of testing). Overall, in the 12 months post-order change there were 2090 more sFLCs performed which identified 72 additional sFLC positive only cases (an increase of 340%). Conclusions: Combining the SPEP and sFLC into one order test from two separate tests improved adherence to IMWG guidelines for getting both tests ordered (from 45% to 100%). This increased the number of sFLC tests being performed but also increased the number of PCDs diagnosed. Although most of these were MGUS, early detection of this diagnosis has been suggested in other studies to result in closer follow-up and better outcomes. In addition, in our series there was a case of asymptomatic MM which would have been missed and diagnosis delayed if just an SPEP had been ordered. M-protein analysis should be considered as a combined screening test order at other institutions.

Prognostic Significance of Monoclonal Paraproteinemia in Marginal Zone Lymphoma

Introduction and aim:Marginal zone lymphoma (MZL) is an indolent lymphoma which can progress with frequent relapses and transform to aggressive lymphoma. Therefore, the factors that affect prognosis are important fields of interest. This study was aimed to investigate the effect of immunoglobulin (Ig) paraproteinemia on MZL prognosis and survival. Material and method: Between January 2000 and June 2022, 73 patients who applied to Dokuz Eylül University Faculty of Medicine Hospital Department of Hematology, diagnosed with MZL, and continued their follow-up in our center were assessed. Patients whose paraproteinemia was investigated by immunofixation (SIFE), serum protein electrophoresis (SPEP), or free kappa and lambda free light chain (FLC) assays were included in the study. Demographic characteristics and laboratory data were retrospectively analyzed. Results: Ig paraproteinemia was ascertained in 20 patients which 11 of 20 was IgM type. The median follow-up was 58 (3-200) months. Median overall survival (OS) and progression free survival (PFS) were not reached. Mean OS and PFS were 169±8.5 and 146±10.6 months, respectively. Serum Ig paraproteinemia was detected in 20 of 73 patients (27.3%). Most were IgM- κ (9, 45%), followed by IgG-κ (7, %35), IgM-λ (2, %10), IgA-λ (1, %5), and IgA-κ (1, %5). The presence of Ig paraproteinaemia was significantly associated with advanced age (p&amp;lt;0.001), advanced disease stage (III-IV) (p=0.023), elevated ß2 microglobulin (ß2MG) level (p=0.001), decreased hemoglobin level (p=0.002), decreased albumin level (p&amp;lt;0.001), and bone marrow involvement (p=.026). Gender, elevated lactate dehydrogenase (LDH) level, and elevated Ki-67 were not significantly associated with the presence of Ig paraproteinaemia. In our cohort Ig paraproteinemia was shown in 35%, 10% and 55% of extranodal MZL, splenic MZL and nodal MZL respectively (p=0,009) which favors nodal MZL dominance. The prognostic value of Ig paraproteinaemia and other clinical outcomes are summarized in Table 1. The OS rate at 5 and 10 years was %87.2 and %81.9, respectively. The PFS rate at 5 and 10 years was %71.5 and %68.3 respectively. Patients with Ig paraproteinaemia has shorter OS compared to patients without any paraproteinemia (73,8 mo. vs 178,8 mo. p&amp;lt;0,05). This survival disadvantage was more pronounced in patients with IgM paraproteinemias. Kaplan-Meier survival curve analysis of PFS according to the occurrence of Ig paraproteinaemia are shown in Figure 1. Conclusion: Although MZL is an indolent lymphoma type and there are many prognostic factors affecting its prognosis, it may show relapsed or refractory disease course or histological transformation. 55% of our nodal MZL cohort had Ig paraproteinemia which is higher than all reported cohorts in the literature so far. Several studies have previously shown that Ig paraproteinemia may be a potential prognostic factor for NHL and MALT lymphoma 1-3. Ren et al. showed that Ig paraproteinemia was an independent prognostic predictor for MALT lymphoma 4. Epperla et al. showed that patients diagnosed with MZL with Ig paraproteinemia had inferior survival in comparation with no-M protein group 5. In conclusion, this study provides evidence that Ig paraproteinaemia and especially of IgM type shortens PFS and OS in patients with MZL. Thus, our study is the first in literature to show survival disadvantage for MZL patients specifically with IgM paraproteinemia and deserves confirmation with further studies.

Genetic Basis of Relapse after GPRC5D-Targeted CAR T Cells

GPRC5D-targeted CAR T cell therapy was recently shown to be safe and active in patients with relapsed or refractory multiple myeloma with limited or no other therapeutic options, such as those with triple-refractory disease (to a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody) and disease progression on a BCMA antibody-drug conjugate, bi-specific T-cell engager, and/or CAR T cell therapy (Mailankody S. et al. NEJM 2022). Among the 17 patients treated in our phase 1 study of the GPRC5D-targeted CAR T cell therapy MCARH109, 12 patients had an objective response and 6 patients relapsed after an initial response of 3-9 months. While there are currently no validated clinical assays to assess GPRC5D expression, we reported decreased or loss of protein expression using immunohistochemistry in all 6 patients who relapsed. This finding suggests that genetic alterations may be an important mechanism of antigen escape after GPRC5D-directed CAR T cell therapy. With BCMA-directed CAR T cell therapies recently approved for multiple myeloma, antigen loss due to genetic alterations is a rare mechanism of resistance. We first investigated the genetic status of GPRC5D in a patient (Patient 15) who underwent biopsies of a left posterior chest wall plasmacytoma at baseline and a recurrent plasmacytoma at the same site at relapse with nearly 100% tumor. This patient received the maximum tolerated dose of 150×10 6 GPRC5D-directed CAR T cells and achieved a best response of stringent complete response without minimal residual disease in the bone marrow. Unfortunately, 6 months after infusion, he relapsed with recurrent FDG-avid lesions in similar locations on PET, extensive bone marrow involvement (90-95%), rapidly rising lambda free light chain in the serum, and significantly decreased but still detectable circulating CAR T cells (CAR vector copies per mL: 666) ( Figure A). We performed droplet digital PCR (ddPCR) on extracted DNA from the plasmacytoma biopsies and estimated GPRC5D copy number to be two at baseline and 0.1 at relapse, consistent with bi-allelic loss in a significant majority of plasma cells. We then performed whole exome sequencing which revealed two balanced copies of GPRC5D at baseline and bi-allelic loss at relapse consisting of a broader deletion (~8756 kb) in chromosome 12p in one allele and a more focal deletion (~145 kb) encompassing the GPRC5D gene locus in the second allele ( Figure B). Beyond this patient, we identified that relapses after GPRC5D-directed CAR T cell therapy are associated with decreased or loss of GPRC5D mRNA expression. We measured GPRC5D mRNA expression by ddPCR on patient bone marrow or plasmacytoma samples at baseline prior to CAR T cell infusion and at disease progression. The results corresponded with those from immunohistochemistry, and all patients who relapsed showed reduced or absent GPRC5D mRNA and protein expression at disease progression. Given that two patients who responded and relapsed had low or no GPRC5D expression at baseline (Patients 10 and 13), further studies are warranted to characterize the heterogeneity of GPRC5D expression and how well immunohistochemistry and ddPCR capture its expression. Here we report that antigen escape after GPRC5D-directed CAR T cell therapy can be mediated by structural chromosome alterations resulting in bi-allelic loss of the GPRC5D gene locus in a patient with initial excellent response. We also note transcriptional downregulation of GPRC5D in other patients at relapse, but it remains to be determined whether this is transient or durable and whether it can be explained by similar on-target genetic alterations or non-genetic mechanisms. Our patients with heavily pretreated multiple myeloma may harbor greater tumor heterogeneity and genomic instability than previously described, which can facilitate clonal outgrowth of antigen-negative tumor cells under continuous selective pressure of GPRCRD-targeted CAR T cells. Potential strategies to mitigate antigen escape-mediated relapse in myeloma patients receiving T-cell engaging therapies including earlier use of these therapies, multi-antigen targeting, or combination approaches are being evaluated in ongoing trials.

Lambda-free light chain: A serum marker of dengue disease via NS3 protease-mediated antibody cleavage

ABSTRACT Dengue poses a significant global public health threat, with diverse clinical manifestations due to complex interactions between the host and the pathogen. Recent reports have highlighted elevated serum-free light chain (FLC) levels in viral infectious diseases. Hence, our study aimed to investigate serum FLC levels in dengue patients. The findings revealed elevated serum λ FLCs, which were associated with the severity of dengue. Receiver operating characteristic curve (ROC) analysis demonstrated that λ FLCs may serve as a serum marker for identifying dengue disease (AUC: 0.7825, sensitivity: 80, specificity: 71.43) and classifying severe dengue (AUC: 0.8102, sensitivity: 75, specificity: 79.52). The viral protease, Dengue virus (DENV) nonstructural protein 3 (NS3), acts as a protease that cleaves viral polyproteins as well as host substrates. Therefore, we proposed that antibodies might be potential targets of NS3 protease, leading to an increase in FLCs. LC/MS-MS analysis confirmed that λ FLCs were the predominant products after antibody degradation by NS3 protease. Additionally, purified NS3 protease cleaved both human IgG and DENV2-neutralizing antibodies, resulting in the presence of λ FLCs. Moreover, NS3 protease administration in vitro led to a reduction in the neutralizing efficacy of DENV2-neutralizing antibodies. In summary, the elevated serum λ FLC levels effectively differentiate dengue patients from healthy individuals and identify severe dengue. Furthermore, the elevation of serum λ FLCs is, at least in part, mediated through NS3 protease-mediated antibody cleavage. These findings provide new insights for developing diagnostic tools and understanding the pathogenesis of DENV infection.

Revised Definition of Free Light Chains in Serum and Light Chain Monoclonal Gammopathy of Undetermined Significance: Results of the Istopmm Study

Background: Serum free light chain (FLC) measurement, consisting of serum free kappa, serum free lambda, and a calculated FLC ratio (kappa/lambda), plays a pivotal role in the diagnosis, risk stratification and management of plasma cell disorders. Light chain monoclonal gammopathy of undetermined significance (LC-MGUS), is defined as abnormal FLC ratio with elevation of the involved FLC without evidence of heavy chain M protein or end-organ damage attributed to the plasma cell disorder. Several years ago, reference intervals for serum kappa (3.3-19.4 mg/L) and lambda (5.7-26.3 mg/L) FLC and FLC ratio (0.26-1.65) were defined in a small retrospective cohort (N = 282) of healthy individuals. Limitation of these reference intervals include inaccurate distributions among individuals with impaired kidney function. Recently, we addressed this matter in a large population-based study focusing on individuals with estimated glomerular filtration rate (eGFR) &amp;lt; 60 mL/min/1.73m 2, the results of which led to redefined reference intervals for patients with chronic kidney disease (Long et al, Blood Cancer J. 2022). Here, using a large cohort (N=41,882) of screened individuals, we aimed to determine FLC reference intervals in individuals with normal kidney function (eGFR ≥ 60 mL/min/1.73m 2). Methods: Data were collected from the ongoing Iceland Screens, Treats, or Prevents Multiple Myeloma (iStopMM) study, which focuses on population-based screening for MGUS. A total of 75,422 individuals aged ≥ 40 years (representing 51% of this age group in the Icelandic population) were screened for MGUS by serum protein electrophoresis, immunofixation, and serum FLC assay (Freelite®), and two-thirds of MGUS cases were randomized to active follow-up. Participants' eGFR was calculated using serum creatinine (CKD-EPI) closest to the time of screening. Participants with heavy chain M protein, known lymphoproliferative disorder, unknown eGFR or eGFR &amp;lt; 60 mL/min/1.73m 2, were excluded. The 0.5 and 99.5 percentiles of kappa FLC, lambda FLC, and the FLC ratio distributions were assessed for the whole group, and subgroups of age, sex, and different levels of eGFR. A nonparametric bootstrapping method was used to calculate the 95% confidence intervals. Partitioning was determined based on the proportion of subgroups (age, sex and eGFR) outside the whole group reference interval. Results: After application of the exclusion criteria, 41,882 participants were included for further analysis. The median (interquartile range, IQR) serum free kappa was 14.3 (11.6-17.8) mg/L, serum free lambda 14.2 (11.6-17.5) mg/L, and the FLC ratio was 1.02 (0.85-1.21). The median (IQR) age was 60 (52-68) years, eGFR 84 (74-94) mL/min/1.73 m 2, and 43% were male. A strong correlation was found between age and serum kappa FLC (ρ = 0.27, p &amp;lt; 0.001), lambda FLC (ρ = 0.14, p &amp;lt; 0.001), and the FLC ratio (ρ = 0.16, p &amp;lt; 0.001). Use of standard reference intervals yielded abnormal results for 17.5%, 3.9%, and 4.5% of serum kappa, lambda, and FLC ratio determinations, respectively, and a prevalence of LC-MGUS of 2.0% (96% kappa and 4% lambda). Based on these findings, we established new reference intervals for serum kappa and lambda FLC and FLC ratio, partitioned by age &amp;lt; 70 years and ≥ 70 years (Table). Utilizing the new reference intervals the crude prevalence of LC-MGUS was 0.3% (54% kappa and 46% lambda), yielding a relative decrease of 83%. Among the group of individuals diagnosed with LC-MGUS based on standard reference intervals who did not meet the diagnostic criteria using our revised reference intervals - none had progressed to a lymphoproliferative disorder after a median follow-up time of 42 months. Conclusion: Based on prospective screening of more than 40,000 individuals and after 3.5 years of follow-up, we show that standard reference intervals for serum FLC and FLC ratio appear inaccurate for persons with preserved kidney function. We propose a revision of the reference intervals for serum FLC and FLC ratio, and a new definition of LC MGUS (Figure). Implementation of new reference intervals will decrease the rate of false positive diagnosis of LC-MGUS in individuals with preserved kidney function by more than 80%. This, in turn, will reduce the unnecessary psychological and financial burden driven by clinical evaluation and lifelong monitoring of these individuals.