Increased Levels of Circulating Plasma Cells in Patients with Newly Diagnosed Multiple Myeloma Are Independently Associated with Poor Prognosis

The presence of circulating tumor cells (CTCs) has long been suggested as a valuable prognostic biomarker in Multiple Myeloma (MM). Recently, increased baseline CTC levels in patients with newly-diagnosed MM (NDMM) have been associated with adverse outcomes in the GEM2012MENOS65/GEM2014MAIN and FORTE trials. Since January 2017, we have been evaluating the presence of CTCs utilizing Next-generation Flow cytometry (NGF) with a median limit of detection (LOD) reaching 2x10-6. NGF assessment has been performed according to the EuroFlow guidelines for sample preparation, antibodies, cytometer settings and analysis of data. Currently, we have analyzed 525 matched BM and PB samples from NDMM patients and have found clonal plasma cells in 100% of BM and 89.1% (468/525) of PB samples respectively. Patients presented with ISS III/R-ISS III, high-risk cytogenetics, serum β2 microglobulin above median and hemoglobin levels ≤ 8.5 g/dL had increased CTC levels. Inversely, the absence of CTCs or their low prevalence (<10-5) was associated with an increased normal plasma cell compartment within the BM (R2=0.82, P < .0001). No association was detected between the CTC number and the therapeutic response to induction treatment in accordance with previous reports. Nevertheless, over a median follow-up monitoring of 42 months (range: 3-66 months), a running log-rank test with a step increase of 0.1% CTCs, highlighted an optimal cut-off point for PFS risk assessment at the level of 2x10-4. In the multivariable analysis, NDMM patients with CTCs above this cut-off showed a 2.2-fold higher risk of progression which was independent from ISS, induction scheme and baseline cytogenetics (HR: 2.2, 95% CI: 1.24 - 3.28, P< .001). Our large number of matched samples allowed for a phenotypic comparison between BM clonal cells and CTCs. The majority of patients with detectable CTCs (86%) showed a matched phenotypic profile of aberrant plasma cell in the two sites. However, 66 patients showed phenotypic discrepancies based on one of the following patterns: i) all phenotypic subsets were present in both BM and PB but with significantly altered ratios (≥20% of relative prevalence) for at least two concomitant subsets; ii) presence of ≥ 1 phenotypic subsets (with a minimum relative prevalence of 20% of all clonal cells) only in the BM; and iii) presence of ≥ 1 phenotypic subsets only in the PB. Remarkably, patients with phenotypic discrepancies had significantly higher CTC levels than those with a phenotypic agreement on the two sites (P < .001), together with signs of a more diffuse disease pattern, as evidenced by the imaging results. It is likely that these patients may share a patchy BM infiltration (and/or even extramedullary disease) and accumulate circulating clonal cells from various sites (thus leading to increased tumor burden) with a spatial clonal heterogeneity, which, as a total, may differ from a specific phenotype derived from a particular BM site. The biological differences among patients with differential baseline CTCs expand beyond the tumor cells. Performing a deep phenotypic approach for the identification of 32 distinct immune subsets, we analyzed the immune profile of both ΒΜ and PB in NDMM patients differing to their CTC levels by two log decimals. Patients with CTCs ≥ 5x10-3 had substantially increased levels of total T cells (especially CD8+), NK cells, tumor associated macrophages (TAMs) and an elevated ratio of memory to naïve B cells (P < .0001 for all subsets) in their BM microenvironment than those with less than 5x10-5 CTCs. The same differences, though with weaker statistical significance, applied also in PB, where, we could also identify a clear immune signature with characteristics of tumor suppression and T cell exhaustion for NDMM with the high CTC burden. Altogether, our results confirm the negative and independent prognostic impact of increased CTC levels in the NDMM and further imply its relevance in the real-world setting to establish improved risk-stratification models. Moreover, since the liquid biopsy is a better representative of the entire tumor load than a tissue biopsy sample, the analysis of CTCs may serve as the new hallmark for the real-time evaluation of a patient's disease and/or immune status.