Role of Serum Free Light Chain Assay in Relapsed/Refractory Multiple Myeloma. A Real-Life Unicentric Retrospective Study

Background: In the era of novel drugs a growing number of multiple myeloma (MM) patients are treated until disease progression. Serum free light chain (sFLC) assay is recommended for disease monitoring in oligo-secretory and micromolecular MM. Methods: In this real-life survey, a total of 130 relapsed/refractory MM patients treated at our center with at least three lines were investigated as a retrospective cohort. Results: The median age at diagnosis was 64 years and more than half of patients were male. A total of 24 patients (18%) had oligo-secretory/micromolecular disease at diagnosis. More than 20% of 106 normo-secretory patients had oligo-secretory/micromolecular escape. In order to evaluate potential role of sFLC assay before (“pre”) and after (“post”) every treatment line, involved serum free light chain values (iFLC) less than 138 mg/mL and serum free light chain ratios (FLCr) <25 were identified by using ROC curve analysis. The analysis of the entire cohort throughout four treatment lines demonstrated a statistically significant negative impact on progression-free survival (PFS) for both involved pre-sFLC and its ratio (respectively p = 0.0086 and p = 0.0065). Furthermore, both post-iFLC and post-FLCr greater than the pre-established values had a negative impact on PFS of the study cohort; respectively, p = 0.014 and p = 0.0079. Odds ratio analysis evidenced that patients with both involved post-sFLC greater than 138 mg/mL and post-FLCr above 25 at disease relapse had a higher probability of having clinical relapse (respectively p = 0.026 and p = 0.006). Conclusions: Alterations of sFLC values, namely iFLC and FLCr, both prior to treatment initiation and in the course of therapy at every treatment line, could be of aid in relapse evaluation and treatment outcome. We therefore suggest close periodical monitoring of sFLC assay, independently from secretory status.

Impact of Light Chain Isotype on Clinical Features and Outcomes in Systemic AL Amyloidosis

Purpose: In systemic AL amyloidosis, light chain (LC) isotype has been shown to predict outcome in patients undergoing upfront autologous transplantation. However, less than one-third of newly diagnosed AL patients are transplant-eligible. The objective of our study was to characterize the relationship between LC isotype, organ involvement, response, and survival in patients with newly diagnosed AL amyloidosis irrespective of transplant-eligibility. Methods: We performed a retrospective cohort study on 112 patients with AL amyloidosis who had treatment records at Columbia University Irving Medical Center (CUIMC) between 2015-2019. Two-sided Fisher's exact test was used for categorical variables and two-sided Wilcoxon rank sum test was used for continuous variables. Survival analysis was done using Kaplan Meier method. Overall survival (OS) was the time from randomization to death due to any cause. Event-free survival (EFS) was the time to initiation of a subsequent line of therapy or death due to any cause, whichever was earlier. Two-sided log-rank test was used to test differences between survival curves. Results: Kappa LC isotype was seen in 26% of patients. The proportion of patients with Mayo 2004 stage IIIA or IIIB disease in kappa and lambda isotype was 33% and 35% respectively (p=0.91). Bortezomib-based and daratumumab-based initial therapy was administered in 70% and 8% of patients respectively, with 36% having auto-transplant as part of first-line therapy. Patients with kappa LC isotype had a significantly higher baseline difference in free light chain (dFLC) and involved/uninvolved serum free light chain ratio (sFLCr) compared to lambda (median dFLC, 61.5 vs 21.6 mg/dl respectively, p=0.02; and median sFLCr, 63.5 vs 10.6 respectively; p&lt;0.01). Kappa LC isotype was also associated with a near-significant higher bone marrow plasma cell (BMPC) burden at baseline (median BMPCs, 20% vs 10% respectively; p=0.06). Patients with lambda LC isotype had a significantly higher incidence of kidney involvement compared to kappa (64% vs 38% respectively, p=0.02). There was no significant difference in the incidence of t(11;14) between the two groups. The incidence of hematologic very good partial response or better (≥VGPR), involved free light chain less than 20 mg/dl (iFLC&lt;20), and dFLC less than 10 mg/dl (dFLC&lt;10) at the end of first-line therapy was similar in the two groups (Table in Figure 1). There was no difference in cardiac or renal response rates between the two groups. The proportion of patients requiring a subsequent line of therapy in kappa and lambda isotype was 41.4% and 39.8% respectively (p=0.88). At a median follow-up of 43 months for surviving patients, there was no significant difference in event-free survival (hazard ratio (HR) [kappa/lambda]: 0.76 [0.43-1.38]; p=0.37). However, there was a non-significant trend toward superior OS (HR [kappa/lambda]: 0.47 [0.18-1.23], p=0.12; 4-year OS: 86% vs 67% respectively) in patients with kappa LC isotype. Achievement of hematologic ≥VGPR, iFLC&lt;20 mg/dl, and dFLC&lt;10 mg/dl at end of first-line therapy was associated with a superior OS in our cohort irrespective of light chain isotype. Conclusion: Lambda AL amyloidosis is associated with significantly higher renal involvement compared to kappa. Despite a higher baseline disease burden (dFLC, sFLCr, and percent BMPC) in kappa compared to lambda, there was a trend toward superior OS in the former, which may imply decreased tissue toxicity of kappa FLCs. Further studies should investigate proteomic signatures and immunoglobulin gene usage according to light chain isotype and correlation with clinical phenotype. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Lambda monoclonal free light chain abnormalities detected by a serum immunofixation electrophoresis assay are underrepresented by quantitative serum free light chain results

Protein and immunofixation (IFIX) electrophoresis are used to diagnose and monitor monoclonal gammopathies. While IFIX detects clonal production of intact immunoglobulins and free light chains (FLC), the latter can also be quantified using a serum free light chain (SFLC) assay, in which polyclonal antisera detects epitopes specific for free kappa (KFLC) or lambda light chains (LFLC). An abnormal KFLC: LFLC ratio (KLR) serves as a surrogate for clonality. While the SFLC assay is highly sensitive, normal LFLC (&lt;2.63mg/dL) and KLR results (&gt;0.26 & &lt;1.65) were found in samples with distinct lambda monoclonal free light chains visualized by IFIX (X-LMFLC). To investigate this discordance, contemporaneous SFLC or KLR values were evaluated for their ability to accurately classify monoclonal FLCs identified by IFIX. We performed a retrospective analysis of serum and urine IFIX (Sebia Hydrasys) and SFLC (Freelite®, Binding Site) results from our institution between July 2010 through December 2020, using R 4.0.2 and Tidyverse packages. From among 9,594 encounters in which a single monoclonal component was initially identified by IFIX, 157 X-LMFLC and 131 X-KMFLC samples were analyzed. Elevated LFLC with normal KFLC was identified in 105/157 X-LMFLC samples (67%), while both LFLC and KFLC were elevated in 42/157 samples (27%). Concordance between X-KMFLC and KFLC was markedly higher, where 122/131 samples (93%) displayed elevated kappa FLC (&gt;1.94mg/dL) with normal LFLC, and only 7/131 X-KMFLC samples (5%) possessed both elevated KFLC and LFLC. The use of KLR to identify pathogenic monoclonal free light chains improved lambda concordance to 85%; however, 19/157 (12%) of X-LMFLC samples still exhibited normal KLR. High concordance of 98% was again observed for X-KMFLC with abnormal KLR. When samples were segregated according to normal or impaired renal function (eGFR &gt; or ≤60mL/min/1.73m², respectively), this disparate identification of X-LMFLC and X-KMFLC by the SFLC assay persisted, suggesting that renal dysfunction (as measured by eGFR) does not underlie this phenomenon. Lastly, we corroborated the above findings in a larger sample population by examining patients with urine Bence Jones FLC identified by IFIX who had free or intact monoclonal components in serum (N=724), grouped by lambda or kappa light chain involvement. The cause(s) of the discrepant performance by the Freelite® SFLC assay, relative to the Sebia Hydrasys IFIX assay, for identifying lambda FLC components is currently unclear. Possible contributory factors include assay reference range cutoffs, other patient disease parameters, and differences in assay-specific polyclonal antisera. Future analyses of these factors will help to further characterize SFLC assay performance and elucidate how interpretation of composite serum FLC test results can be improved to better guide patient management.

Evaluation of the siemens N Latex free light chain assay and comparison to Binding Site Freelite™ assay

Introduction/Objective The International Myeloma Working Group (IMWG) guidelines include serum free light chain (sFLC) level and κ/λ ratio as excellent indicators of clonality. The Binding Site Freelite ™ was the first FDA approved assay for quantitative measurement of sFLC, the assay was based on a mixture of polyclonal antibodies directed against a variety of FLC epitopes. The Siemens N-Latex assay employs a probe mixture of mouse monoclonal antibodies. Both assays can be run on nephelometers. We assessed the analytical performance of the N-Latex assays and compared it with the Freelite™ assays. Methods/Case Report Analytical accuracy, precision, reproducibility and linearity were evaluated according to the regulatory standards. Method comparison was performed with 220 clinical samples for statistic correlation and clinical concordance analysis. Results (if a Case Study enter NA) The N-Latex FLC κ and λ assays had coefficient variation of 1-5% with-in run and 3-8% between run precision. Accuracy was verified using assayed controls in the duration of 21 days. Within analytical measuring range, almost perfect linearity was achieved for both κ and λ FLC assays. In comparison study to Freelite™ with 220 clinical samples, good agreement in classification was observed for κ, λ and κ/λ ratio (Cohen’s κ 0.73, 0.82 and 0.87). Pearson correlation analysis showed correlation coefficient value r &gt;0.90 for all the analytes. Conclusion The N-Latex FLC assay has good analytical performance, did not exhibit gross antigen excess and can be used in clinical practice. However, it showed markedly lower absolute values for κ/λ ratio compared with Freelite™. Our data demonstrated that although good clinical concordance between N-Latex and Freelite™ FLC assays was achieved, the absolute values from the two assay are not interchangeable. Further studies in modification of the assay-specific diagnostic (involved FLC/non-involved FLC) thresholds for smoldering multiple myeloma (SMM) and multiple myeloma (MM) are needed.