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

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