Liquid biopsy by analysis of circulating tumor cells, hormonal and inflammatory biomarkers in plasma cell neoplasms, at Fundación Santa Fe de Bogotá hospital

Abstract

Abstract The detection of circulating tumor cells (CTC) is becoming increasingly important due to growing evidence demonstrating their prognostic value in multiple myeloma (MM), associated with aggressiveness. Given that their evaluation is less invasive, their utilization in clinical practice is gaining significant interest. Due to the low quantity of clonal tumor cells (CPC) in blood, highly sensitive analyses, such as Next-generation flow cytometry (NGF) or Next-generation sequencing, are required. These methods are not readily available in developing countries, and their detection also demands advanced training. Consequently, the performance of these assay in Latin American patients with plasma cell neoplasms (PCN) is unknown. In this study, we evaluated CTC paired with bone marrow (BM) biopsies, using NGF, from precursor to newly diagnosed MM (NDMM), and post-treatment. Additionally, we analyzed cytokine profiles in PCN, at Fundación Santa Fe, Colombia. 206 samples from 103 PCN were studied: MGUS: n=34, smoldering MM (SMM) n=13, NDMM n=28, MM post-treatment (peripheral residual disease (PRD) n=28). Paired blood and BM were evaluated, using EuroFlow NGF for CTC/MRD (FACSLyric and Infinicyt). Median of limit of detection was of 2.5x10-6. Furthermore, hormonal and inflammatory biomarkers were measured in plasma and BM, using a multiplex magnetic-bead based immunoassay (Milliplex Magpix, kits HADK1MAG-61K-01 and HMH3-34K-04). Statistical analysis was performed using R (R Foundation for Statistical Computing, Vienna) and IBM SPSS software. 32% of precursor lesions (MGUS, SMM) had detectable CTC (median 0.02%). 8% had a level ≥ 0.01%. 2.8% had >95% clonal plasma cells (CPC) among total blood plasma cells (PC). All BM biopsies were infiltrated by CPC; 14% had >95% CPC among total BM PC. Upon progression to MM, a significant increase in frequency and level of CTC was identified, rising to 87% of patients, with a mean level of 0.04%. Half of the patients (51%) had a level ≥ 0.01%, which has been associated with unfavorable outcomes. 30% had >95% CPC among total blood PC. There was no correlation between CTC and high-risk cytogenetic abnormalities, evaluated by FISH. The mean of BM involvement was 33% and 52%, using flow cytometry and immunochemistry respectively. 91% had >95% CPC among total BM PC. PRD was detected in 38% of cases; 14% had a level ≥ 0.01%. There was a modest correlation (r=0.045, p=0.001) between the percentage of BM infiltration and CTC, across of total cohort. When comparing immunophenotypic markers, a higher expression of CD81 and CD27 was found in CTC versus BM CPC in precursor neoplasms, and a higher expression of CD138 was found in BM CPC versus CTC in NDMM. Patients with higher CTC levels had more events of infections in all cohort. MM patients with higher levels, there was more hypercalcemia. Stepwise increase in plasma concentrations of interleukin-6 (IL-6) throughout the MGUS SMM NDMM continuum, from 11.3 +/- 1.9 to 21.4 +/- 8.9 pg/mL (p-trend<0.001), with a return to 11.9 +/- 1.9 pg/mL among MM post-treatment. A similar pattern was observed for plasma insulin, and the opposite pattern for plasma leptin. BM glucagon (+46%) and TNF-alpha (+55.3%) were markedly increased (+46%) in MM post-treatment versus NDMM. To our knowledge, this study is the first to show the behavior of CTC in Latin American patients with PCN. The frequency and levels of CTC were associated with disease progression, increasing from precursor lesions to NDMM. A striking finding was the high percentage of NDMM cases with CTCs with a cutoff of ≥ 0.01%, which is associated with inferior PFS and OS. This highlights the importance and feasibility of detecting CTC in the clinical practice for Latin American patients. Furthermore, we confirmed that systemic inflammation is also determining factor in myelomagenesis, as evidenced by the involvement of plasma IL-6 throughout disease progression. The increase in BM TNF-alpha and glucagon only among patients with post-treatment MM could be a manifestation of the local inflammatory and catabolic state triggered by the treatment. Thus, the incorporation of liquid biopsy incorporating CTC's and other metabolic markers is a less invasive strategy for providing personalized medicine based on the biology of the disease that is feasible and reproducible in Latin America, were access to next-generation sequencing and other contemporary risk-stratifying tools are less accessible.