Detailed characterization of multiple myeloma circulating tumor cells shows unique phenotypic, cytogenetic, functional, and circadian distribution profile

Abstract

Key Points• Detailed characterization of myeloma circulating tumor cells shows that these represent a unique subpopulation of BM clonal PCs.• Myeloma CTCs are clonogenic, quiescent, and may represent an ancestral clone potentially driven by circadian rhythms.Circulating myeloma tumor cells (CTCs) as defined by the presence of peripheral blood (PB) clonal plasma cells (PCs) are a powerful prognostic marker in multiple myeloma (MM). However, the biological features of CTCs and their pathophysiological role in MM remains unexplored. Here, we investigate the phenotypic, cytogenetic, and functional characteristics as well as the circadian distribution of CTCs vs paired bone marrow (BM) clonal PCs from MM patients. Our results show that CTCs typically represent a unique subpopulation of all BM clonal PCs, characterized by downregulation (P < .05) of integrins (CD11a/CD11c/CD29/CD49d/CD49e), adhesion (CD33/CD56/CD117/CD138), and activation molecules (CD28/CD38/CD81). Fluorescence in situ hybridization analysis of fluorescence-activated cell sorter-sorted CTCs also unraveled different cytogenetic profiles vs paired BM clonal PCs. Moreover, CTCs were mostly quiescent and associated with higher clonogenic potential when cocultured with BM stromal cells. Most interestingly, CTCs showed a circadian distribution which fluctuates in a similar pattern to that of CD34 1 cells, and opposite to stromal cell-derived factor 1 plasma levels and corresponding surface expression of CXC chemokine receptor 4 on clonal PCs, suggesting that in MM, CTCs may egress to PB to colonize/metastasize other sites in the BM during the patients' resting period. (Blood. 2013;122(22):3591-3598) IntroductionIn the late 2000s, 2 pivotal studies elegantly showed that monoclonal gammopathy of undetermined significance (MGUS) precedes multiple myeloma (MM) in most, if not all myeloma patients. 1,2End-organ damage is the most important criterion to classify therapyrequiring MM patients, and the most common CRAB (hyperCalcemia, Renal failure, Anemia, Bone lesions) symptom is the presence of bone lesions detectable on a skeletal survey.3 Extramedullary disease is present in ;10% of newly diagnosed symptomatic patients, but it increases up to 20% in the relapse/refractory setting, 4 and typically anticipates a dismal outcome. Plasma cell (PC) leukemia is one of the most aggressive forms of the disease and even with novel drugs, the outcome is very poor with both short remission and survival rates.5 This landscape does not greatly differ from that of most solid tumors, in which the presence of metastasis is a key prognostic factor. 6 In fact, recent observations suggest that tumor cell dissemination is often an early event, 7 and the clinical sequel of circulating tumor cells (CTCs) has been the focus of extensive research. 8Interestingly, peripheral blood (PB) MM CTCs (morphologic and phenotypically defined as mature PCs) are also a common event throughout the spectrum of MM. [9][10][11][12][13] CTCs can only be detected in a small fraction of newly diagn...

Phenotypic and genomic analysis of multiple myeloma minimal residual disease tumor cells: a new model to understand chemoresistance

“…37 In line with this hypothesis, we recently showed that MM circulating tumor cells represent a singular phenotypic and functional subset of MM tumor cells characterized by lower CD138 expression 22,38 Here, we used new analytic tools developed by the EuroFlow Consortium 20 to simultaneously evaluate the pattern of expression of 23 markers at the single-cell level and longitudinally compare the iPEPs from diagnostic vs MRD clonal PCs. In contrast to the abovementioned hypothesis, our results demonstrate that MM cells with stronger capacity to survive frontline chemotherapy were those with the highest expression of CD138; because MRD clonal PCs are responsible for the disease relapse, these results point out the clonogenic potential of mature CD138…”

“…Then, the iPEP of diagnostic (baseline) and chemoresistant (MRD) cells for all 23 phenotypic markers analyzed plus FSC and SSC was generated for every single clonal PC, after merging of flow cytometry data files and calculation of data. 21,22 First, the merge function of the Infinicyt software (Cytognos SL, Salamanca, Spain) was used to fuse the different data files corresponding to the 4 different 8-color mAb combinations studied per sample into a single data file containing all information measured for that sample. For any single cell in each 8-color mAb combination (aliquot), this included data about those antigens that were measured directly on it and antigens that were not evaluated directly (missing values) for that cell in the corresponding aliquot it was contained in.…”

Inflammatory environment created by fibroblast aggregates induces growth arrest and phenotypic shift of human myeloma cells

“…Therefore, we performed phenotypic characterization of tested myeloma cell lines after exposure to the inflammatory conditions with focus on the RPMI 8226 cells. Overall, we analysed expression of selected 13 cell surface markers which used to be employed for characterization of MM-cell phenotype, e.g., CD19, CD20, CD28, CD38, CD45, CD49e, CD49d, CD54, CD56, CD117, CD138, CD184, HLA-DR [4]. Noticeable differences were detected in expression of three CD markers, specifically CD38, CD138, CD45, and to certain extent CD20, as well.…”

“…Actually, some studies have demonstrated the presence of circulating PCs in more than 70% of patients with MM [3]. Very recent study by Paiva et al [4] elegantly showed that circulating myeloma tumor cells, as defined by the presence of peripheral blood clonal PCs, might be even a powerful prognostic marker in MM. However, in some patients with MM, the PCs proliferation escapes the BM microenvironment influences and it results in extramedullary disease (EMD), which can constitute the most prominent clinical feature of the disease [5].…”

Prognostic significance of bone marrow abnormalities in the appendicular skeleton of patients with multiple myeloma

“…Circulating neoplastic PCs are thought to be independent of the bone marrow microenvironment and have a lower expression of adhesion molecules and integrins. 25 Although circulating PCs did not correlate with an increase in medullary abnormalities in the AS (P 5 .220) ( Table 1) …”