P-081: Sensitive detection of M-proteins and FLC in blood for diagnosis and monitoring of multiple myeloma

Introduction: Multiple myeloma (MM) is a cancer of plasma cells (myeloma cells) that accumulate in the bone marrow and produce abnormal monoclonal immunoglobulins (mAb, M-proteins). Despite the development of new therapies, MM is still associated with a high likelihood of persistent minimal residual disease (MRD) and thus earlier relapse. Relapse in MM is caused by clonal heterogeneity and evolution, which can lead to selection of drug-resistant malignant plasma cell (PC) clone(s), contributing to treatment failure. Detection of persistent clones, after treatment, is currently limited due to the invasiveness of bone marrow biopsy and the very low frequency of sampling. Therefore, the main goal of our work was to develop a method for sensitive detection of low-abundant PC clones based on the detection of M-proteins and free light chains (FLC) in human serum.

Methods: The newly developed method combines selective affinity isolation of M-proteins and FLC from a blood sample and their sensitive analysis by high-performance liquid chromatography with UV detection (af-HPLC/UV). Af-HPLC/UV also served as the first step for targeted detection of the variable region (CDR3) of M-proteins by high-resolution mass spectrometry after prior sequencing of the corresponding DNA regions. The reliability and sensitivity of the method were tested on three types of mAb standards added to human sera. Patient samples before and after treatment were also included in this study.

Results: The af-HPLC/UV workflow was able to detect mAb in serum with high sensitivity (≤100 mg.Le-1). Selective isolation of M-protein and FLC allowed quantitative separation of mAbs by light chain types, which has great potential for the diagnosis of oligoclonality. HPLC with UV detection is then suitable for accurate LC and partial HC classification, as well as for the determination of therapeutic mAbs, which usually complicate analysis by immunofixation electrophoresis. In addition, the sensitivity of the entire workflow can be greatly enhanced when combined with LC-MS/MS of the CDR3 region, which is unique to each antibody clone. These regions can then be monitored in a targeted manner by mass spectrometry based on initial analysis of myeloma cell DNA. The combination of very high sensitivity and the ability to detect multiple clones thus has the potential to sensitively detect resistant and emerging myeloma cell clones in MRD based on M-proteins and FLCs detection directly in the blood.

Conclusions: With these new MM detection methods, it may be possible in the future to carefully monitor patients at short time intervals and detect not only clones from the time of diagnosis, but also newly formed clones with high sensitivity from blood. The method then has the potential to replace invasive and often problematic bone marrow aspiration.