Free Light Chain Measurements Research Articles

THU0047 Baff and B2m, but not Free Light Chains, Correlate with Disease Activity in Primary SjÖGren's Syndrome (PSS)

BackgroundBoth kappa and lambda free light chain (FLC) levels appear to be elevated the serum of patients with pSS, in comparison to healthy controls [1,2]. Consequently, FLCs may represent potential...

Serum free immunoglobulin light chain evaluation as a marker of impact from intraclonal heterogeneity on myeloma outcome

AbstractIntraclonal heterogeneity was recently described in multiple myeloma (MM), but its full impact on disease progression and relapse has not been entirely explored. The immunoglobulin type produced by myeloma cells provides an excellent marker to follow changes in clonal substructure over time. We have prospectively evaluated serial paraprotein and serum free light chain (FLC) measurements and found that 258 of 520 and 54 of 520 patients who presented with a whole paraprotein relapsed with paraprotein only (PO) and “FLC escape,” respectively. The median overall survival of PO patients was longer, when compared with patients whose relapse manifested as an increase in FLC both alone and with a whole paraprotein, as a result of a significantly shorter survival from relapse of the latter groups. These observations fit a model in which 1 clone is able to produce a complete antibody, whereas the other secretes only FLC; the type of relapse represents the outgrowth of different clones, some of which are more resistant to therapy. To our knowledge, this is the largest series describing patients who have relapsed with FLC escape and highlights the importance of monitoring FLC when there is a suspicion of clinical relapse. This study was registered at www.isrctn.org as ISRCTN68454111.

Cerebrospinal Fluid Immunoglobulin Kappa Light Chain in Clinically Isolated Syndrome and Multiple Sclerosis

BackgroundOligoclonal bands (OCB) are the most widely used CSF test to support the diagnosis of MS and to predict conversion of clinically isolated syndrome (CIS) to multiple sclerosis (MS). Since OCB tests are based on non-quantitative and difficult to standardise techniques, measurement of immunoglobulin kappa free light chains (KFLC) may represent an easier to use quantitative test.MethodsKFLC were measured in CSF and serum of 211 patients using ELISA. These include patients without any inflammatory central nervous system reaction (NIND, n = 77), MS (n = 20), viral CNS infections (V-CNS-I, n = 10), neuroborreliosis (NB, n = 17) and other bacterial CNS infections (B-CNS-I, n = 10). Furthermore a cohort of 77 patients with CIS, including 39 patients that remained CIS over follow-up of two years (CIS-CIS) and 38 patients that developed MS over the same follow-up time (CIS-MS).ResultsCSF-serum ratio of KFLC (Q KFLC) was elevated in all patients with MS, 86.8% of patients with CIS-MS and 61.5% of patients with CIS-CIS. It was significantly elevated in CIS with presence of OCB (p<0.001). Q KFLC significantly correlated with other CSF variables such as CSF leukocyte count (p<0.001, R = 0.46), CSF CXCL13 levels (p<0.001, R = 0.64) and also intrathecal IgG synthesis (p<0.001, R = 0.74) as determined by nephelometry and quotient diagram. OCB were detected in 66.7% of CIS-CIS and in 92.1% of CIS-MS.ConclusionsAlthough the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS.

Serum free light chains and the adaptive immune response in usual and alpha-1-antitrypsin deficiency-related chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is a chronic inflammatory condition with a complex pathophysiology. In COPD, the immune response involves the adaptive immune system, although the antigenic stimulus driving this response is unknown. During the process of antibody synthesis an excess of free light chains (FLCs) are produced and secreted into the circulation. High polyclonal FLC concentrations have been found in several autoimmune and inflammatory conditions such as systemic lupus erythematosus and rheumatoid arthritis. Thus, FLCs are being investigated as a biomarker of immune activation, disease activity, and severity in these conditions. Our aim was to establish whether serum FLCs could be a marker of phenotypic variation or predict poor outcome in patients with usual and alpha-1-antitrypsin deficiency (A1ATD)-related COPD. MethodsWe measured FLC concentrations in 294 COPD patients with A1ATD-related and 85 patients with usual COPD using the Freelite serum FLC assay (The Binding Site Ltd, Birmingham, UK). We then compared combined (κ and λ) FLC (cFLC) concentrations in different subgroups defined by the presence of chronic bronchitis, colonisation of the lower respiratory tract, and subsequent mortality. In addition, we measured the estimated glomerular filtration rate (eGFR) of patients included in the study. FindingsOur preliminary data showed that in A1ATD-related COPD and usual COPD, cFLC concentrations were significantly higher in patients with chronic bronchitis than in those without bronchitis (p=0·008 vs p=0·047). Patients with A1ATD-related COPD who were chronically colonised with pathogenic organisms in their stable state also had significantly higher cFLC concentrations than did those who were not colonised (p=0·036). A multiple regression analysis showed that eGFR and the presence of chronic bronchitis significantly predicted cFLC concentrations in patients with A1ATD: renal function was the strongest predictor (β=−0·326, p<0·0001) followed by the presence of chronic bronchitis (β=0·147, p=0·01). A similar trend was seen in the usual COPD group although this did not meet statistical significance (eGFR β=−0·220, p=0·055; chronic bronchitis β=0·224, p=0·061). The 33 A1ATD patients who subsequently died also had significantly higher cFLC concentrations (p=0·005) than did those still alive. InterpretationThe higher cFLC concentrations in patients with A1ATD-related COPD who have chronic bronchitis and colonised lower respiratory tracts could mean that there is a greater adaptive immune response in these individuals. The drive behind this response might come directly from microorganisms or through self-perpetuating autoimmune events. Within the specialty of COPD clinical management, there is a move towards a more individualised approach to treating different clinical phenotypes. Measurement of serum FLCs might have a potential role as a biomarker to help stratify patients. The higher cFLC concentrations in patients with renal impairment is unsurprising in view of their renal clearance, but raised polyclonal FLCs have also been shown to be a predictor of mortality in the general population. Our mortality results suggest that FLCs could have a role in risk stratification for patients with COPD, but further investigation is needed. FundingGrifols Therapeutics.

Free light chain levels in pediatric patients with inflammatory conditions.

Because free light chain assays measure polyclonal as well as monoclonal free light chain components, some previous studies focused on the potential utility of the free light chain assay for detecting chronic immune stimulation, which occurs in autoimmune diseases and allergies. However, most of these trials have been limited to adult patients. In this study, we evaluated the paired serum and urinary free light chain levels of pediatric patients with various inflammatory conditions to investigate the clinical significance of free light chain measurement in pediatrics. The study included 227 paired serum and urine specimens from 134 pediatric patients at our hospital between January and February of 2012. Serum and urinary FLC levels were measured using a Freelight Kit (The Binding Site, Ltd., Birmingham, UK). The serum lambda and urine kappa and lambda components were significantly increased only in the renal impairment group, not in the mild inflammatory group. FLC ratios were not significantly different among these groups. In serum, only the L components were significantly increased. This result may indicate the presence of a dimeric L structure, in contrast with monomeric K. FLC levels might also be influenced by renal conditions other than mild inflammation. Therefore, as shown in previous studies of adult patients, renal reference ranges might be needed to interpret FLC results, especially for dimeric L components.

Serum Free Light Chains Identify Patients With Monoclonal Gammopathy At Increased Risk Of Developing Renal Disease

BackgroundMonoclonal gammopathy (MG) is frequently associated with kidney disease (monoclonal gammopathy of renal significance; MGRS). International guidelines recommend the use of the serum free light chain (FLC) assay when screening for monoclonal gammopathies; an abnormal FLC ratio and an elevation of at least one FLC isotype indicates an Ig LC clone. However it is not known if, in patients with normal kidney function, the presence of a FLC clone predisposes to the future development of kidney disease. Patients and MethodsA 22485 patient database with multiple laboratory tests and up to 15-year follow up (Regional Medical Laboratory, Tulsa, OK, USA) was analysed. There were 425 confirmed MG patients with no progression to multiple myeloma. Median age was 72 years (22-97 years) and M/F ratio was 177/248. FLC kappa and lambda were measured nephelometrically with Freelite® (The Binding Site Ltd, Birmingham, UK). Baseline was defined on the first available FLC measurement; this was available at inception in 258 (61%) patients and during follow-up in 167 (39%) patients. 128 patients with MG and an eGFR <60ml/min/1.75m2 and/or a clinical diagnosis of renal disease (ICD9 code) prior to and up to 100 days after baseline were excluded from the analysis. Statistical analyses, including Receiver Operating Characteristics (ROC) to identify monoclonal FLC (iFLC) cut-off were carried out using SPSS v21. Results297 (of 425) patients had a MG with normal renal function at baseline; 118 (40%) patients had an abnormal FLC ratio (88 kappa, median: 35.5 (7.7-2675) mg/L; 30 lambda, median: 83.6 (0.9-3552) mg/L). 21/297 (7%) patients developed renal impairment during the course of follow up (median: 852 (114-2388) days); 15/21 (71%) had monoclonal FLC production. In these patients there was no association between iFLC (median: 47.8 (0.9-3552) mg/L) and creatinine (median: 0.9 (0.5-1.5) mg/dL) levels (p=0.3966). Time to renal impairment (TTRI) was significantly shorter for patients with an abnormal v normal FLC ratio (75% TTRI: 1261 days v not reached (NR), hazard ratio (HR): 5.31; p<0.001). Age between groups was similar (median: 68 (22-97) v 72 (39-92) years; p=0.0544). In 118 patients withan abnormal FLC ratio, Cox regression analysis identified iFLC concentrations as being associated with the development of impaired renal function (p=0001). Furthermore, patients with iFLC>100mg/L (n=25) were at a significantly increased risk of developing renal impairment compared with patients with iFLC<100mg/L (n=93) (75% TTRI: 874 days v NR, respectively; HR: 6.10; p<0.001). ConclusionsThese results indicate that the presence of an Ig LC clone is associated with an increased risk of people with MG and normal kidney function progressing to renal disease. Disclosures:No relevant conflicts of interest to declare.

Serum Free Light Chain Levels Post Allogeneic Stem Cell Transplantation Are Associated With Improved Overall Survival In Patients With Acute Myeloid Leukemia

Introduction Development of chronic graft vs host disease (cGvHD) and maintained complete remission at day 100 post-transplant (CR day 100) are associated with improved survival post-allogeneic transplant in acute myeloid leukemia (AML) patients. However, earlier prognostic markers could be beneficial in guiding clinical management post-transplantation. As survival is associated with successful engraftment and B cells play a positive role in cGvHD, we investigated serum free light chain (FLC) measurements, as markers of B cell activation, to determine if they provide prognostic information before day 100 post-transplant. Materials and Methods Serum FLCκ and FLCλ (Freelite®, The Binding Site Group Ltd, Birmingham UK) concentrations were measured retrospectively in stored sera from 42 AML patients at 5 weeks post-transplantation (wk5). FLCκ and FLCλ results were summated to give a combined FLC (cFLC) value, which was assessed with respect to cGvHD and 2 year survival. All statistics were performed using SPSS v21.0. At wk5 median cFLC levels were 17.9mg/L (range: 1.7-53.3).Age at transplant ranged from 19-71 years (median: 53); male/female ratio was 23/19. 27/42 (64%) were transplanted at CR1, 12/42 (29%) at CR2 and 3/42 (7%) at CR3. A myeloablative (MA) conditioning regimen was used in 13/42 (31%) patients; 29/42 (69%) received reduced intensity conditioning (RIC). 21/42 (50%) developed acute GvHD (aGvHD), 18/42 (43%) developed cGvHD and the 2 year mortality rate (MR) was 43% (18/42). Results Development of cGvHD (HR 3.2, p=0.04) and maintained CR day 100 (HR 56.8, p=0.001) were associated with improved 2 year survival. By comparison, other routinely evaluated markers were not prognostic, including: 1) type of donor (sibling or match unrelated; HR 0.6, p=0.81); 2) CR1 v CR2/3 prior to transplant (HR 1.2, p=0.72); 3) RIC v MA conditioning (HR 1.0, p=0.97); 4) T-cell depletion/Campath therapy (HR 1.0, p=0.89); 5) CMV reactivation (HR 0.8, p=0.66); 6) donor lymphocyte infusion (HR 21.7, p=0.62); 7) donor/recipient gender mismatch (HR 0.6, p=0.47); and 8) aGvHD development (HR 0.2, p=0.57). At wk5, patients with elevated cFLC levels (&gt;22.6mg/L; top tertile) had a better 2 year survival compared to the lower two tertiles (Cox regression analysis: HR 5.6, p=0.02; Kaplan Meier analysis: &gt;22.6mg/L: n=14 (2 deaths, 14% MR), median 2 year survival not reached (NR);&lt;22.6mg/L: n=28 (16 deaths, 57% MR), median 2 year survival 408 days, p=0.01). However, cFLC measurements at wk5 were not associated with development of cGvHD (HR 1.15, p=0.78). Finally, wk5 cFLC levels stratified patients with a maintained CR at day 100 (cFLC&lt;22.6mgL: n=25 (13 deaths, 52% MR), median 2 year survival: 517 days; cFLC&gt;22.6mg/L: n=13 (1 death, 8% MR), median 2 year survival NR, p=0.009) and tended towards significance in patients who developed cGvHD (cFLC&lt;22.6mgL: n=15 (6 deaths, 40% MR), median 2 year survival: 394 days; cFLC&gt;22.6mg/L: n=6 (0 deaths), median 2 year survival NR, p=0.09). Conclusion Our data suggests assessment of cFLC levels at wk5 post-transplantation provides prognostic information in patients with AML, irrespective of maintained response post-transplantation and cGvHD development. Further studies are needed to confirm the utility of this observation Disclosures: Young: The Binding Site Group Ltd: Employment. Adie:The Binding Site Group Ltd: Employment. Harding:The Binding Site Group Ltd: Employment.

Isotype Matched Paired Immunoglobulin Suppression Identifies High Risk Multiple Myeloma Patients and Is Independent Of Bone Marrow Infiltration Suggesting Significant Variability In The HLC Matched Pair Suppressive Activity Of Individual Myeloma Clones

IntroductionMultiple myeloma (MM) is characterised by the production of monoclonal immunoglobulins (MIg), both intact and a free light chain (FLC) component. The production of MIg can be associated with suppression of the non-involved isotypes which can easily be measured by conventional techniques while identification of a possible suppression of the HLC matched pair isotype was not possible before the introduction of the HLC assay. Here we assess the frequency and biologic relevance of the suppression of HLC pair matched isotypes and non-involved isotypes and correlate these findings and those of MIg measurements with bone marrow plasma cell (BMPC) infiltration. Patients and Methods113 previously untreated MM patients (72 IgG (46κ, 26λ) and 41 IgA (23κ, 18λ)) with a median age of 65 years (range 32-86 years) were enrolled and followed for up to 13 years (median: 4 range 0.25-13 years). Serum FLC (FLCκ/FLCλ) and HLC concentrations (IgGκ/IgGλ and IgAκ/IgAλ) measurements were made using polyclonal antisera assays (FreeliteTM, Binding Site, Birmingham, UK. and, HevyliteTM, Binding Site, Birmingham, UK) and results compared to standard serum assessments (SPE, IFE, β2M, Albumin, LDH and CRP) and bone marrow plasma cell (BMPC) infiltration. Severe non-involved isotype or HLC pair suppression was defined as >50% lower of than the published lower normal ranges for the relevant isotypes. Correlation and survival analysis was performed using the SPSS v 21 programme. ResultsMedian overall survival was 46 months (range 3-158). IgA MIg production correlated weakly to BMPC infiltration at presentation (Pearsons 0.327; p=0.026) but to no other marker. Uninvolved IgA HLC did not correlate to BMPC infiltration but had a weak correlation to LDH (Pearsons 0.436; p=0.002). By contrast, IgG MIg production showed significant correlation to ß2M (Pearsons 0.53; p<0.001), with uninvolved IgG HLC having no significant correlations. There was a greater degree of monoclonal production in IgG patients than in IgA patients (p=0.049). Severe IgG and IgM suppression was noted in 13/41 (31%) of IgA patients with 78% of patients having either isotype suppressed. In IgA MM patients IgM suppression was more commonly identified than IgG (32/41 v 13/41 patients respectively) and IgG suppression was always concomitant with IgM suppression. Severe IgA and IgM suppression was noted in 36/72 (50%) of IgG patients with 69% of patients having either isotype suppressed. In contrast to IgA patients there was no difference between the incidence of IgA or IgM suppression. Severe isotype matched pair suppression was more frequently noted in IgG compared to IgA patients (66% vs. 60%, p<0.01). Severe HLC pair suppression correlated with a decreased OS (46.6 vs. 84.4 months, p<0.044). By contrast neither non-involved isotype nor monoclonal Ig production was correlated with outcome. Discussion and ConclusionThe correlation between IgG MIg and BMPC infiltration indicates a greater uniformity in the inter-individual MIg production rate in patients with IgG compared to those with IgA MIg, which were only weakly correlated with BMPC infiltration. Part of this finding may also be due to greater variations in the catabolic rate of individual IgG MIgs. The absence of a correlation between the degree of HLC pair suppression and BMPC infiltration suggests significant variation in the ability of individual clones in suppressing the isotope matched pair. This uncoupling between the proportion of myeloma cells and isotype suppression was previously unknown. Myeloma clones with greater HLC matched isotype pair suppression reflect a more aggressive phenotype which correlates with short survival. Suppression of the non-involved isotype, in contradistinction, showed no prognostic relevance. Disclosures:Young:The Binding Site Group Ltd: Employment. Harding:The Binding Site Group Ltd: Employment.

In Patients With Chronic Lymphocytic Leukemia (CLL) Ibrutinib Effectively Reduces Clonal IgM Paraproteins and Serum Free Light Chains While Increasing Normal IgM, IgA Serum Levels, Suggesting a Nascent Recovery Of Humoral Immunity

IntroductionThe Bruton's tyrosine kinase (BTK) inhibitor ibrutinib induces objective clinical responses in the majority of CLL patients (Byrd et al., NEJM 2013). Ibrutinib covalently binds to BTK and with once daily dosing (420 mg, PO) results in > 90% inhibition of kinase activity. Germline inactivating mutations in BTK lead to an immunodeficiency syndrome first described by the pediatrician Dr. Bruton in boys suffering from recurrent bacterial infections. These kids, diagnosed with what is now known as Bruton's agammaglobulinemia, have a severe defect in B cell maturation resulting in the virtual absence of immunoglobulins. Hypogammaglobulinemia is a common complication of CLL and likely is a significant contributor to the increased rate of infections that are a leading cause of death in CLL. Thus, to what degree ibrutinib affects normal B cell function and immunoglobulin levels may in part determine the safety profile of continuous treatment with this agent. Patients and MethodsHere we present data from a phase II trial (NCT01500733) of ibrutinib 420 mg daily on 28 day cycles for relapsed/refractory (RR) and treatment naïve (TN) CLL/SLL patients (pts). Serum immune globulins (IgG, IgM, IgA), serum free light chains, and immunofixation electrophoresis were obtained at baseline, and every 6 months thereafter. For statistical analysis of pre-treatment to on-treatment measurements the paired Student t-test was used. ResultsHere we report on 25 patients (10 TN, 15 RR) who completed >12 months on ibrutinib and never received immunoglobulin replacement therapy. By 6 and 12 months, there was a non-statistically significant trend toward decreased IgG levels (ref. range 642-1730) from a pre-treatment median of 601 to 587 mg/dL (at 6 months) and 495 mg/dL (at 12 months; P = 0.14). In contrast, median serum IgA (ref. range 91-499) rose from 42 (baseline) to 58 (at 6 mo) to 61 mg/dL by 12 months (P< 0.005). Three patients had a clonal IgM on electrophoresis, which decreased with treatment. In the remaining 22 patients IgM (ref. range 34-342) rose from 16 (baseline) to 25 (6 months) to 23 mg/dL by 12 months (P<0.01). TN patients had higher IgA and IgM levels at baseline and achieved the higher absolute increase by 12 months. However, the relative rate of increase from baseline was similar for both groups, suggesting that ibrutinib enables a recovery of IgA and IgM levels equally in both TN and RR patients. In 20 patients serum free light chain measurements were available, with an abnormal pre-treatment kappa/lambda ratio in 17. In 11 patients the CLL cells were kappa clonal by flow cytometry and in 9 they were lambda clonal. Eight of 11 pts with a kappa CLL clone had kappa serum free light chain (KSFLC, ref. range 0.57 – 2.22 mg/dL) levels > upper limit of normal (median 5.7 mg/dl). At 6 and 12 months there was a 76% and 72% reduction of the KSFLC (P< 0.01), and in 7 pts the level normalized by 6 months. In contrast, prior to therapy the lambda serum free light chains (LSFLC, ref. range 0.66-2.32 mg/dL) were low (median 0.62 mg/dL) in these patients and increased by 68% (P<0.005) to normal levels by 6 months in all of them. Conversely, 8 of 9 patients with lambda clonal CLL by flow cytometry had LSFLC > upper limit of normal (median 8.4 mg/dL), which decreased on ibrutinib by > 80% (P< 0.03) and normalized in 88% of pts by 12 months. The KSFLC in most of these patients was in the low normal range and only increased by 19% from baseline by 12 months. Thus, ibrutinib effectively reduces the clonal light chain, a correlate of tumor control, while the non-clonal light chains, presumably in part reflecting normal B-cells, are low pre-treatment and increase during treatment. ConclusionConsistent with other reports we see little change in IgG levels in the first 12 months. Importantly, ibrutinib leads to a significant increase in both IgA and IgM serum levels, suggesting a beginning recovery of humoral immunity. The reduction of clonal light chains, a tumor marker, correlates with clinical response. In contrast, the increasing levels of the non-clonal light chain may herald a recovery of the normal B-cell (and possibly plasma cell compartment) raising the possibility that ibrutinib may selectively target CLL cells while allowing the re-growth of normal B-cells. We are currently investigating this further.Supported by the Intramural Research Program of NHLBI. We thank our patients for participating and acknowledge Pharmacyclics for providing study drug. Disclosures:Off Label Use: Ibrutinib not FDA approved for CLL.

Verification of serum reference intervals for free light chains in a local South African population

Monoclonal serum free light chain measurements are used to follow up and manage patients with monoclonal gammopathies, and abnormal serum free light chain ratios are associated with risk of progression...

The significance of free light chain measurements in the diagnosis of myelomatous pleural effusion

The significance of free light chain measurements in the diagnosis of myelomatous pleural effusion

Development of a highly-sensitive multi-plex assay using monoclonal antibodies for the simultaneous measurement of kappa and lambda immunoglobulin free light chains in serum and urine

Monoclonal κ and λ immunoglobulin free light chain (FLC) paraproteins in serum and urine are important markers in the diagnosis and monitoring of B cell dyscrasias. Current nephelometric and turbidimetric methods that use sheep polyclonal antisera to quantify serum FLC have a number of well-observed limitations. In this report, we describe an improved method using specific mouse anti-human FLC monoclonal antibodies (mAbs). Anti-κ and anti-λ FLC mAbs were, separately, covalently coupled to polystyrene Xmap® beads and assayed, simultaneously, in a multi-plex format by Luminex® (mAb assay). The mAbs displayed no cross-reactivity to bound LC, the alternate LC type, or other human proteins and had improved sensitivity and specificity over immunofixation electrophoresis (IFE) and Freelite™. The assay gives good linearity and sensitivity (<1mg/L), and the competitive inhibition format gave a broad calibration curve up to 437.5mg/L and prevented anomalous results for samples in antigen excess i.e. high FLC levels. The mAbs displayed good concordance with Freelite™ for the quantitation of normal polyclonal FLC in plasma from healthy donors (n=249). The mAb assay identified all monoclonal FLC in serum from consecutive patient samples (n=1000; 50.1% with monoclonal paraprotein by serum IFE), and all FLC in a large cohort of urine samples tested for Bence Jones proteins (n=13090; 22.8% with monoclonal κ, 9.0% with monoclonal λ, and 0.8% with poly LC detected by urine IFE). Importantly this shows that the mAbs are at least close to the ideal of detecting FLC from all patients and neoplastic plasma cell clones. Given the overall effectiveness of the anti-FLC mAbs, further clinical validation is now warranted on serial samples from a range of patients with B cell disorders. Use of these mAbs on other assay platforms should also be investigated.

Serum free light chain reference values: A critical approach

ObjectivesThe clinical usefulness of serum free light chain (FLC) measurement in the management of patients with plasma cell proliferative disorders has been reported in several papers, and most clinical studies use the reference ranges declared by the manufacturer. The aim of the present study was to evaluate the reproducibility of FLCs immunoassay and to validate the reference range, before introducing it in routine setting. Design and methodsInternal quality control materials and a pool of fresh serum samples were used to evaluate imprecision; 162 fresh sera from healthy blood donors were analyzed to evaluate the reference range for FLCs. In order to verify the κ/λ FLC ratio, 43 sera from patients with polyclonal hypergammaglobulinemia were tested. The FLC immunoassay was performed using a nephelometer with the Freelite reagents. ResultsThe imprecision studies performed using a serum pool tested with two different lots of reagents showed a mean CV of 16.09% for κFLC and of 16.72% for λFLC. Lower CV%s and different mean values were found by calculating the results from each specific lot separately, while different results were obtained using the control materials provided by the manufacturer. In reference subjects, the 2.5–97.5th percentiles were found to be 4.52–22.33 and 4.84–21.88mg/L for κFLC and λFLC, respectively. The range for κ/λ ratio (0.65–2.36) was validated with the values obtained from subjects with polyclonal hypergammaglobulinemia. In retesting 15 samples from blood donor subjects with a different lot of reagents, mean bias percentages of 17.60 for κFLC and 15.26 for λFLC were obtained. ConclusionsThese findings confirm the lot-to-lot variability of the FLC assays also in the measurement of polyclonal light chains, as well as the need to carefully validate the reference values.

Polyclonal free light chains: a biomarker of inflammatory disease or treatment target?

Free light chains are proteins produced by B lymphocytes during the process of antibody synthesis. Their production, as a reflection of B cell activation, can give insight into the activity of the adaptive immune system. In recent years, an automated immunoassay that provides quantitative measurement of free light chains in the serum has been developed. This assay has not only revolutionised the investigation of monoclonal light chain overproduction in plasma cell diseases, but has also allowed for the quantification of polyclonal free light chains in serum. The discovery of high levels of polyclonal free light chains in a number of inflammatory and auto-immune conditions has led to the examination of their value as a biomarker of disease activity. Research into their bio-activity has also highlighted their potential role in the pathogenesis of inflammatory disease, making them an attractive target for novel therapies.

Defining the impact of individual sample variability on routine immunoassay of serum free light chains (sFLC) in multiple myeloma

Serum free light chain (sFLC) measurement has gained widespread acceptance and is incorporated into various diagnostic and response criteria. Non-linearity and antigen excess are the main causes of 'variability' in the measurement of sFLC using immunoassay, but the impact of these on measurement has been unclear. We performed a retrospective evaluation using a dilutional strategy to detect these phenomena. A total of 464 samples in 2009 and 373 samples in 2010 were analysed for sFLC. Non-linearity was detected in both high and apparently normal sFLC. Major non-linearity of more than twofold is common in high kappa (20·2%) and lambda (14·1%). It is less common in samples with apparently normal levels - kappa (6·4%) and lambda (9·5%). 9·4% of kappa and 15·5% of lambda showed antigen excess at screening dilutions. 34·4% of the samples had either non-linearity or antigen excess. We conclude that significant measurement variability is common in the measurement of sFLC. There is currently no reliable technique to detect non-linearity phenomena unless a serial dilution strategy is applied to every analysis. We recommend that laboratories routinely reporting sFLC results for clinical services need appropriate strategies for addressing these issues. Clinicians should be aware of these limitations in interpretation of sFLC assay for individual patients. Future guidelines should adopt action thresholds which are grounded firmly in test performance parameters.

Computational Algorithm Based On Serum Immunoassays for the Identification of Haematological Disease May Identify Patients with Other Acute Disorders

AbstractAbstract 4001 IntroductionSerum free light chain (FLC) measurements are used routinely to identify monoclonal immunoglobulin (M-Ig) production in patients. In addition, summated kappa + lambda concentrations have recently been shown to have prognostic value in chronic lymphocytic leukaemia (CLL), Hodgkins lymphoma and HIV patients; presumably reflecting immune stimulation. A recently developed companion assay to FLC testing measures intact immunoglobulin heavy chain/light chain (HLC) pairs in serum calculating Ig’κ/Ig’λ ratios as a reflection of clonality. Here we report on a computational approach combining FLC and HLC measurements to identify clonal disease, hyper- and hypo-gammaglobulinemia, and as markers of immune dysfunction. Furthermore, we comment on the potential of such an algorithm to risk stratify haematological and non-haematological malignancies. Patients, Materials and Methods1468 patients referred to the Royal Wolverhampton Hospital for haematological evaluation were enrolled on to the study. Median patient age was 64 years, with a slight female preponderance (60%). Patient sera were analysed at presentation using routine electrophoresis tests (serum protein electrophoresis, SPEP and immunofixation, IFE). FLC and HLC (IgG, IgA and IgM) were measured by nephelometric immunoassay. Each immunoassay result was assessed with respect to individual normal ranges (FLCκ = 3.3–19.4mg/L, FLCλ = 5.71–26.30mg/L, IgGκ = 4.03–9.78g/L, IgGλ = 1.97–5.71g/L, IgAκ = 0.48–2.82g/L, IgAλ = 0.36–1.98, IgMκ = 0.29–1.82g/L, IgMλ = 0.17–0.94g/L) and ratios (FLCκ/FLCλ = 0.26–1.65, Gκ/Gλ = 0.98–2.75, Aκ/Aλ = 0.8–2.04, Mκ/Mλ = 0.96–2.3). The results were used to create an algorithm which assessed the degree of abnormality individually and with respect to the other results generated. Diagnosis was recorded ∼3 months after the analysis and patients were followed for up to 3 years. Results293/1468 (19%) samples had an abnormal SPEP of which 95/293 were confirmed by IFE including: 10 intact immunoglobulin Multiple Myeloma (MM, 6 IgGκ, 1 IgGλ, 2 IgAκ, 1 IgAλ), 6 light chain MM (LCMM, 4 FLCκ, 2 FLCλ), 2 Waldenstrom macroglobulinemia (2 IgMκ), 1 IgMκ cryoglobulinemia, 3 CLL, 1 mantle cell lymphoma, 3 other lymphoma, 1 IgGκ plasmacytoma, and 68 MGUS patients, 26/68 were confirmed MGUS (1 FLCκ, 13 IgGκ, 4 IgGλ, 4 IgAκ, 2 IgMκ, 2 IgMλ), 42 were laboratory findings requiring follow up (1FLCκ, 3 FLCλ, 19 IgGκ, 5 IgGλ, 4 IgAκ, 1 IgAλ, 8 IgMκ, 1 IgMλ).FLC/HLC algorithm identified 85/95 IFE positive patients; of the 10 patients reported not identified by the algorithm, 3 had oligoclonal banding indicative of infection and 7 had monoclonal bands secondary to other diagnoses (including 2 colon cancer patients and 2 rheumatoid arthritis patients). In addition the algorithm identified 15 IFE negative patients with M-Ig production including: 2 AL amyloid patients, 1 asymptomatic LCMM, 1 patient with ∼1g/L FLCκ (lost to follow up), 1 follicular lymphoma patient and 1 CLL; diagnosis was not available for 9/15 patients. Furthermore, the algorithm identified 205/1468 patients as having elevated polyclonal FLC (cFLC) >50mg/L without evidence of clonal production. In a subset analysis comparing matched numbers of patients with elevated or normal cFLC concentrations, cFLC >50mg/L predicted all cause mortality (logistic regression odds ratio 9.96, 95% CI 4.72–21.0, p<0.001), and was associated with poorer overall survival (Kaplan Meier p<0.001, Cox regression hazard ratio 8.73, 95% CI 4.47–17.02). DiscussionMM presents with disparate and vague symptoms pervasive in an elderly population. Current guidelines recommend SPEP and FLC analysis reflexing to IFE for confirmation in the event of abnormality. However, SPEP and IFE require interpretation making the assessment variable. Standardised immunoassays (FLC+HLC) may obviate the need for interpretation and in this study identified additional haematological malignancies. Furthermore, standard assessments can be used to identify M-Ig, but in the absence of this finding may offer little information pertaining to the symptoms that prompted referral. By contrast this algorithm identified patients with elevated cFLC which in agreement with other reports was associated with increased risk of mortality. Further work is required to assess the algorithm, and determine if cFLC measurements should be used as a prompt for additional diagnostic investigations. Disclosures:Faint:The Binding Site Group, Ltd.: Employment. Harding:The Binding Site Group Ltd.: Membership on an entity's Board of Directors or advisory committees. Mirbahai:The Binding Site Group, Ltd: Employment.

Long-Term Outcomes of Patients with Systemic Light Chain Amyloidosis (AL) Treated At Diagnosis with Risk-Adapted Stem Cell Transplant and Consolidation with Novel Agents.

AbstractAbstract 3150▪▪This icon denotes a clinically relevant abstract Background:AL is a plasma cell dyscrasia characterized by the pathologic production of monoclonal light chains which misfold, deposit in various organs, including the heart, and can cause early death. High dose melphalan with stem cell transplant (SCT) results in high hematologic response rates and is a standard treatment for eligible patients. Achieving a complete hematologic response (CR) to SCT results in extended event-free and overall survival (OS), up to 8 and 13 years respectively in one large series. (Blood 2011; 118:4346) We have studied the addition of novel agents as consolidation following risk-adapted SCT (RA-SCT) in order to improve hematologic response (HR) rates and therefore outcomes. (Br J Haem 2007;139:224; Amyloid 2010;17:80a) In this report we examine the long-term outcomes of patients who received initial therapy with RA-SCT followed by consolidation for hematologic response less than CR (HR < CR). Methods:We performed a retrospective study to assess the HR rates, incidence of hematologic progression and overall survival (OS) of AL patients enrolled at diagnosis on two consecutive phase II trials using RA-SCT with consolidation for HR < CR (NCT01527032 and NCT00458822). OS was calculated from date of transplant to date of death or last follow up. Median event free survival (EFS) and OS were estimated by the method of Kaplan Meier. Cumulative incidence function was used to estimate the incidence of progression and death. Results:Between 2002 and 2011, 83 patients were enrolled and underwent RA-SCT on these trials and, following RA-SCT, those with HR < CR received consolidation with thalidomide and dexamethasone (TD) in the first and bortezomib and dexamethasone (BD) in the second trial. Thirty-six patients had cardiac involvement (43%) and all patients had free light chain measurements employed to score hematologic response and progression using consensus criteria (Am J Hematol 2005;79:319; Blood 2010;116:1364a). The frequency of CR following SCT was 24% and increased to 48% with post-SCT consolidation. The CR rates increased at 1 year compared to 3 months post-SCT from 21% to 36% with TD and from 28% to 62% with BD. With a median follow up of 5.1 years, the EFS is 4.5 years (95% CI: 2.6 to not reached) and the OS of all patients has not been reached (Figure 1). Sixteen patients died prior to hematologic progression and 26 patients have progressed with a cumulative incidence of hematologic progression of 8%, 18%, and 29% at 1, 2 and 3 years, respectively (Figure 2). Thirty-one percent (8/26) of relapsed patients have not required second-line therapy while among those who have, 78% (14/18) have responded including 44% (8/18) with CR. The median OS following hematologic progression was 5 years (95% CI: 2.6–5.8). Conclusions:Half of the AL patients on initial therapy trials employing RA-SCT and consolidation for HR < CR achieved CR with 36% of pts on the TD and 62% on the BD consolidation trial in CR at 1 year post-SCT respectively. At 3 years post-SCT the cumulative incidence of relapse was 29% and a third of relapsed patients did not require therapy, likely due to the very sensitive serum free light chain assay that detects low level hematologic progression in the absence of organ progression. Almost 80% of patients requiring second-line therapy responded, over half with CR, and median OS after relapse was 5 years. These results indicate that initial therapy with RA-SCT and consolidation is an effective initial treatment strategy for patients with AL in the era of novel agents. With over 5 years of follow up the median OS has not been reached. [Display omitted] [Display omitted] Disclosures:Comenzo:Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Use of the investigational agent MLN9708, an oral proteasome inhibitor, in the treatment of relapsed or refractory light-chain amyloidosis. Hassoun:Millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding. Giralt:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding. Landau:Millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Research Funding.

Lack of a Response by Serum Free Light Chains Performed Early Post Stem Cell Transplantation in Patients with Multiple Myeloma Predicts Inferior Progression Free and Overall Survival.

AbstractAbstract 3127▪▪This icon denotes a clinically relevant abstract BackgroundMultiple myeloma (MM) is an incurable cancer commonly treated with autologous stem cell transplantation (SCT). Response evaluation has traditionally been evaluated at 100 days after SCT, both to allow for hematopoietic reconstitution and due to the long half-life of immunoglobulins. Free light chains (FLC) are useful in the diagnosis, risk stratification, and treatment response assessment of plasma cell disorders. FLC have significantly shorter half-lives, and may provide evidence of response or treatment failure earlier after SCT; however their role in this setting has not been validated. MethodsPatients were included in the study if they received intermediate or high dose chemotherapy (melphalan ≥ 100 mg/m2) with autologous stem cell support and had FLC measurements early after SCT, defined as 30 or 60 days after SCT.Using the difference between involved and uninvolved FLC (dFLC), we established two criteria to identify patients at risk of early relapse:1.dFLC rise: 10% increase in dFLC with an absolute increase of 25 mg/L2.dFLC stable: in patients with pre-SCT dFLC greater than 100 mg/L, failure to reduce dFLC by 50%.These criteria are less stringent than standard relapse criteria, as they were chosen to identify patients at risk of relapse before they meet full criteria for relapse. While there is an intra-patient variability in FLC that may argue for more stringent criteria, in the setting of high dose melphalan the natural course is rapid and deep reductions in FLC, which could allow smaller rises in FLC to have prognostic value. ResultsEight-one patients met the inclusion criteria. Thirty-nine underwent SCT as part of the first line of therapy and the remaining 42 as a second line or higher therapy (median 3rd line). Patients with dFLC rise had shorter progression free (PFS; 98 vs. 393 days, p=0.04; Figure 1) and overall survival (OS; 10 versus 38 months, p=0.021; Figure 2). Patients meeting a composite of either high-risk criterion had shorter PFS, though not statistically significant (161 versus 430 days, p=0.192), whereas the OS was shorter (median not reached, p=0.009).The majority of patients meeting the high-risk criteria were undergoing SCT as part of a 2nd or greater line of therapy. When analyzing this subset of patients, PFS was 96 days for those with dFLC rise compared to 229 days for standard risk patients (p<0.001), and OS was 310 days compared to 954 days (p=0.103). For patients meeting either high-risk criterion, PFS was 96 days compared to 244 days (p=0.013). Similarly, OS for these patients was 314 days compared to 954 days for normal risk patients (p=0.022).To adjust for potentially confounding factors with known prognostic importance, we performed a multivariate cox regression with the covariates International Staging System stage, number of prior lines of therapy (0, 1–2, ≥3), and high-risk cytogenetic or FISH abnormalities. After adjusting for these covariates, early FLC assessment remained a significant prognostic factor for PFS and OS. ConclusionsAs newer treatments for myeloma provide superior responses, more patients are entering SCT with low or unmeasurable M-spikes. Due to their very short half-lives FLC are a potentially important prognostic factor after SCT. In our study, FLC assessment either one or two months post-SCT, particularly in those patients receiving SCT as second line or later regimen, was able to identify a subset of patients at high risk of early relapse. Our results offer the potential of early identification of patients who may benefit from early non-myelosuppressive or novel therapeutic interventions. [Display omitted] [Display omitted] Disclosures:Jagannath:Onyx Pharmaceuticals, Inc.: Consultancy; Celgene Corporation: Scientific Advisory Board, Industry sponsored lecture, Scientific Advisory Board, Industry sponsored lecture Other; Millennium Pharmaceuticals, Inc. (Takeda Pharmaceutical Company Limited ): Scientific Advisory Board, Scientific Advisory Board Other; Merck & Co., Inc.: Industry Sponsored Lecture, Industry Sponsored Lecture Other; PharmaMar (Zeltia Group): Data Safety Monitoring Board Other. Chari:Array BioPharma: Scientific Advisory Board Other; Celgene Corporation: Scientific Advisory Board, Industry sponsored lectures, Scientific Advisory Board, Industry sponsored lectures Other; Onyx Pharmaceuticals, Inc: Scientific Advisory Board, Scientific Advisory Board Other.

New Criteria for Response to Treatment in Immunoglobulin Light Chain Amyloidosis Based on Free Light Chain Measurement and Cardiac Biomarkers: Impact on Survival Outcomes

To identify the criteria for hematologic and cardiac response to treatment in immunoglobulin light chain (AL) amyloidosis based on survival analysis of a large patient population. We gathered for analysis 816 patients with AL amyloidosis from seven referral centers in the European Union and the United States. A different cohort of 374 patients prospectively evaluated at the Pavia Amyloidosis Research and Treatment Center was used for validation. Data was available for all patients before and 3 and/or 6 months after initiation of first-line therapy. The prognostic relevance of different criteria for hematologic and cardiac response was assessed. There was a strong correlation between the extent of reduction of amyloidogenic free light chains (FLCs) and improvement in survival. This allowed the identification of four levels of response: amyloid complete response (normal FLC ratio and negative serum and urine immunofixation), very good partial response (difference between involved and uninvolved FLCs [dFLC] < 40 mg/L), partial response (dFLC decrease > 50%), and no response. Cardiac involvement is the major determinant of survival, and changes in cardiac function after therapy can be reliably assessed using the cardiac biomarker N-terminal natriuretic peptide type B (NT-proBNP). Changes in FLC and NT-proBNP predicted survival as early as 3 months after treatment initiation. This study identifies and validates new criteria for response to first-line treatment in AL amyloidosis, based on their association with survival in large patient populations, and offers surrogate end points for clinical trials.

Clinical and preclinical validation of the serum free light chain assay: identification of the critical difference for optimized clinical use

The use of the assay for the measurements of free light chains in serum (sFLCs) is increasing. However, there are technical limitations that potentially affect the use in serial measurements. We need further knowledge on the standards of analytical precision, the utility of conventional population-based reference values and the critical difference (CD) between serial results required for significance. To answer these questions, the biological variation must be known. We determined the biological variation in healthy individuals and patients with plasma cell dyscrasia (PCD). We assessed the imprecision of the analysis in use from FreeLite™. We determined the reference interval (RI) in 170 healthy individuals. The biological variation is identical for healthy individuals and patients with PCD. The imprecision of the sFLC analysis cannot fulfil the desirable performance standards for a laboratory test, but are within the manufacturer's ±20% variation for quality control samples. RI showed a significant increase for κ FLC and κ/λ ratio with age, but not for λ. Critical difference was calculated to be 24% and 23% for κ and λ, respectively. We suggest the use of an age-dependent RI. When monitoring patients with PCD, their own former results are the best reference, and knowledge on CD is a valuable tool, which we describe for the first time. Also, it challenges the recently proposed International Myeloma Working Group 'paraprotein relapse criteria', recommending an increase of more than 25% in the involved FLC to indicate the need for initiation of retreatment. We recommend revision of this criterion.