Monitoring of Systemic Light-Chain Amyloidosis Patients Using Mass Spectrometry (M-inSight) in Serum

Abstract

Introduction: Systemic light-chain amyloidosis (AL) is a rare disorder characterized by extracellular deposition of monoclonal light-chain immunoglobulins forming insoluble aggregates in different organs. Kidney, heart and liver are principal organs affected by the disease inducing dysfunctions and failures. In this disease, even a low level of circulating light chain can cause significant damages, making challenging the measure of disease burden via peripheral blood. Although a bone marrow aspiration is required to confirm the diagnosis, the evolution of the disease is usually monitored using free light chains dosage, serum and urine electrophoresis, as well as immunofixation. However, these techniques have demonstrated some limitations in terms of sensitivity. Sebia had recently introduced M-inSight TM, a novel, ultra-sensitive and non-invasive assay for monitoring low level disease on blood using targeted mass spectrometry. In this pilot study, M-inSight TM technology is employed to assess the use of mass spectrometry to monitor AL patients. Methods: 14 AL patients were selected to assess the use of M-InSight to detect and quantify AL light chain. 8/14 patients were selected from the AMYDARA study (NCT02816476), consisting in refractory patients treated with Daratumumab. Serum free light-chain assay (Freelite TM), immunofixation on serum/urine as well as bone marrow collection for DNA (ClonoSeq) and RNA (RACE-RepSeq) immunoglobulin next generation sequencing were used to monitor the disease at baseline and end of treatment (EOT) and compared to M-inSight TM. Additionally, 6/14 multicenter patients with normal free light chain level at diagnosis were selected to evaluate the sensitivity of M-InSight. Total mRNA was extracted from BM samples, retrotranscribed to cDNA and then used for immunoglobulin repertoire high-throughput sequencing assay (RACE-RepSeq). cDNA assembly pipeline using IMGT/HighV-QUEST and Vidjil software were used to construct clonotypes and to identify clonal molecular fingerprints and finally their clonotypic peptides for M-InSight analysis. This technology consists of sequence the AL light chain from serum sample and choose clonotypic peptides from the hypervariable regions which then tracked down in the follow-up sample. Results Clonotypic peptides were identified in all eight patients from the baseline sample, which were then confirmed by mass spectrometry. Out of the 8 AMYDARA patients, 5/8 patients achieved very good partial response (VGPR), 2/8 achieved partial response (PR) and 1/8 resulted in complete response (CR). 6 patients had a decrease of the dFLC ratio measure with Freelite TM of 80% or more. 6 out of these 8 patients had MS data highly concordant with that of the serum free light-chain assay. 2 patients had a strong decrease in dFLC while MS measurements were stable or increased. However, detection of the clonotype using RACE-RepSeqand Clonoseq remained elevated, which was more in line with MS than Freelite TM. All patients with normalization of their dFLC were still detected using M-inSight, even those reaching MRD negativity (1 patient) or low number of cells (~10-5, 2 patients) by ClonoSeq. These results were consistent with the RACE-RepSeq which detected the light chain clonotypes in all patients at EOT. Clonotypic peptides using M-InSight were also detected in all 7 patients with normal free light chain at diagnosis. However, in one bi-clonal patient, peptides from only one of both chains were detected, suggesting that only one clone was secreting light chain. Conclusion The goal of this study was to evaluate the M-InSight technique to monitor the evolution of systemic AL amyloidosis in blood samples. The results showed a high concordance with bone marrow MRD measurement and a higher sensitivity than dFLC to monitor the disease. This study shows that MRD monitoring on blood is feasible, using an ultra-sensitive MS technique, avoiding invasive bone-marrow aspirate. M-inSight can track low level AL disease.