Circulating Tumor Cells By Next Generation Flow Cytometry May be a New Prognostic Biomarkers Among Patients with Asymptomatic Monoclonal Gammopathies

Monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic (smoldering) multiple myeloma (sMM) precede symptomatic myeloma (MM); however, these early lesions are characterized by significant heterogeneity in terms of their clinical course. Currently no single molecular or clinical feature can identify patients (pts) who should be considered for immediate therapy; watchful wait remains the standard approach and clinical biomarkers (serum FLCs, bone marrow biopsy infiltration and M-protein amount) are used to stratify progression risk. Circulating tumor cells (CTCs - clonal plasma cells) are detected in most pts with symptomatic MM and have independent prognostic significance. The use of next generation flow cytometry (NGF) can assess CTCs accurately and provides a non-invasive tool to assess tumor cells. Few studies have addressed the prognostic significance of CTCs as a potential prognostic biomarker in SMM and MGUS. We report results from the prospective evaluation of CTCs, detected with NGF, in pts with SMM or MGUS to determine their prognostic significance.

We analyzed the data and outcomes of 254 consecutive pts with MGUS or SMM by IMWG2014 criteria, diagnosed and followed in the Department of Clinical Therapeutics, Athens. Pts were evaluated prospectively for the detection of CTCs at the time of initial diagnosis. CTCs were assessed in peripheral blood (PB) according to the EuroFlow guidelines, using two independent 8-color panels, both containing CD19-PC7, CD27-BV510, CD38-FITC, CD45-PerCPCy5.5, CD56-PE, CD138-BV421, and additionally CD117-APC and CD81-APCC750 only in the surface tube or CyIgκ-APC and CyIgλ-APCC750 only in the intracytoplasmic tube. A median number of 5 million events (range 3.9x106-6.1x106) were acquired for each tube and median LOD was 2.3x10-6 (range 2x10-6- 3.1x10-6).

The median age of the cohort was 65 years (range 28-87), 60% were females, 118 (46.5%) had non-IgM MGUS and 136 (53.5%) SMM. The isotype was IgG in 83% and 74%, IgA in 16% and 22% and light chain only in 1% and 3% of MGUS and SMM pts respectively. Among MGUS pts, 46% had abnormal FLC ratio and the median M-spike was 0.59 gr/dl (range: 0-1.87). For SMM pts, 17% had FLC ratio >20, 13% had M-spike >2 gr/dl and 29% had BM plasma cell infiltration >20%. Per IMWGs 20/2/20, 42% were low risk (no risk factors), 32% intermediate (one risk factor) and 11% high risk (2-3 risk factors). Cytogenetics were available in 60 pts (19 MGUS and 41 SMM): t(4;14) was present in 10% of SMM, t(11;14) in 22% of SMM and MGUS, t(14;16) in 8% if SMM, +1q21 in 31% of SMM and 6% of MGUS, del17p in 2% of SMM and 5% of MGUS (one patient each) and del13q in 29% of SMM and 17% of MGUS.

Clonal plasma cells (CTCs) were detectable in 39% of cohort’s pts: in 24% of MGUS and in 52% of SMM pts (p<0.001). Among those with detectable CTCs, median level was 0.0019% (range 0.0002% to 0.22%) and did not differ among MGUs and SMM pts. The presence of detectable CTCs was associated with higher BM infiltration (p<0.001) but did not correlate with SMM risk group: among low-risk SMM pts, 50% had detectable CTCs (median 0.0033%) vs 52% of pts with intermediate (median 0.0031%) and 62% (median 0.0004%) with high risk SMM (p=0.864). The median follow-up of the whole cohort is 24 months; 23 pts have progressed and 2-year progression rate was 9% for the whole cohort, 2% for MGUS and 12% for SMM pts. Among SMM pts, 2-year progression rate was 0%, 7% and 15% for low, intermediate and high risk SMM (p=0.1). The presence of CTCs was associated with a higher risk of progression to symptomatic MM among all pts in the cohort (HR: 2.99; 95% CI 1.15-7.7, p=0.024). Among SMM pts the presence of detectable CTCs was also associated with a higher risk of progression (HR: 2.99; 95% CI 1.02-9.5, p=0.045). For MGUS pts the number of events was low to draw statistical conclusions. We also evaluated 0.015% as a CTCs cutoff (identified previously by Termini et al) but did not reach statistical significance.

In conclusion, CTCs are detectable in about 39% of pts with asymptomatic monoclonal gammopathies, both in MGUS (in 24%) and more frequently in SMM (in 52%). Their presence is associated with increased risk of progression to symptomatic disease, but longer follow up is needed to identify their role in MGUS and the additive information over available risk stratification tools. Longitudinal assessments may provide further prognostic information. CTCs could provide an non-invasive, easy-to-follow biomarker.