The “myeloma cell niche” or its microenvironment in bone marrow comprises both the non-cancer cells and their stroma. Both these components influence growth, metastatic potential and response to therapy of myeloma cells. The stroma or the extracellular matrix is composed predominantly of fibronectin, laminin, collagen type I & IV, and glycosaminoglycans like hyaluronan, heparan sulphate and chondroitin sulfate [37]. The cellular components known to play an important role in myeloma-pathobiology include bone marrow mesenchymal stromal cells (BMMSCs), osteoblasts, osteoclasts, endothelial cells, pericytes, adipocytes, and immune cells including macrophages, dendritic cells, lymphocytes, and myeloid-derived suppressor cells (MDSCs) [38]. The myeloma cells express adhesion molecules on their cell membrane which can bind with the components of the extracellular matrix, like fibrinogen & laminin bind to β1-integrins, hyaluron with CD44 and collagen-I binds to syndecan (CD138) [38].

The interaction of VLA-4 (β1-integrin) with fibronectin is one of the early steps in homing of the plasma cells in bone marrow. The secretion of SDF-1 (CXCL12) from the bone marrow microenvironment contributes to the upregulation of VLA-4 expression on myeloma cells which increases their ability to bind to the extracellular matrix [39]. In addition, adherence to fibronectin also seems to increase chemoresistance amongst the tumor cells, likely via NF-κB pathway. Out of all the cellular components of the “myeloma cell niche”, BMMSCs stand-out for their central role in plasma cell survival and growth. Several sub-types of BMMSCs have been identified, i.e., CXCL12-abundant reticulin (CAR) cells, leptin-receptor (LPR⁺) expressing stromal cells, neural markers NG2 and Nestin expressing stromal cells. These BMMSCs are known to play an important role in regulation of hematopoietic stem cell homeostasis.

Studies have described various MM cell trafficking events which enable the tumor cells to enter & inhabit specific BM niches, and in the course of the disease, egress BM to re-circulate, leading to extramedullary disease. Notably, CXCR4, a chemokine receptor expressed on the majority of plasma cells, interacts with its ligand CXCL12, a chemokine secreted by cells constituting the tumor microenvironment, especially the BMMSCs. CXCL12 has three isoforms: α, β, and γ, all of which can interact with heparin sulfate and extracellular matrix. Its interaction with the extracellular matrix suggests that rather than freely floating, CXCL12 is immobilized in BM. The γ isoform is the most abundant in the BM milieu. It has an extended C-terminus that binds with a higher affinity to heparin sulfate as compared to other isoforms, promoting myeloma cell adhesion [40]. The CXCR4-CXCL12 interaction mediates homing and lodging, as well as retention of both normal and malignant plasma cells into the BM [41–43]. The very initial step of the attachment of myeloma cells to BM microvasculature is contributed by P-and E-selectins and their ligands. P-selectin glycoprotein ligand-1 (PSGL-1), which is expressed on the surface of myeloma cells interacts with the endothelial cells of the BM micro-vessels [44]. This mediates the rolling of plasma cells on the P-selectins expressed on the endothelial cells. Further, the CXCL12-CXCR4 interaction on the surface of MM cells upregulates the activity of the α4β1 integrin, enhancing binding to its ligand vascular cell adhesion molecule 1 (VCAM-1), which is also expressed on the micro-vessels of the BM [45]. This adhesive event is crucial in myeloma cell trafficking into the milieu of bone marrow. In addition to the α4β1-VCAM-1 interaction, the other significant players in myeloma cell trafficking are α4β7 integrin which interacts with MAdCAM-1 and CD44 which interacts with fibronectin [46,47]. The interaction of α4β1 integrin with VCAM-1 and fibronectin triggers MM cell signaling for the production of interleukin-6 (IL-6). The interaction of IL-6 in the BM microenvironment and IL-6 receptor present on myeloma cells stimulate its growth and development. IL-6 also upregulates CD44 expression on the surface of MM cells. Hence, this IL-6 & CD44 loop, leading to higher expression of each other, causes enormous stimulation of MM-growth signals. BMMSCs secrete many other factors which affect MM cell growth, i.e., stem cell factor (SCF), insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and macrophage inflammatory protein-1α (MIP-1α). However, inside the myeloma cell niche, two soluble mediators, i.e., a proliferation-induced ligand (APRIL) and B-cell activation factor (BAFF) interact with B-cell maturation antigen (BCMA) on the surface of MM cells, play an important role in survival and proliferation of tumor plasma cells. APRIL, known to be secreted by immune cells in the BM milieu surrounding plasma cells, has also been studied for its role in PC mobility inside the bone marrow. Animal studies have shown that the plasma cells proliferate and grow in APRIL-rich regions of BM. The increase in PC-numbers causes a decreased availability of APRIL, which in turn causes PCs to move to a separate neighboring BM niche with better availability of APRIL. This process seems to be an important factor for the intermittent movement of PCs inside the BM [43,48–50].

During the late stages of growth of MM, the myeloma cells tend to become independent of growth signals provided by the BM milieu and there are alterations in their interactions with the BM microenvironment. The myeloma cells downregulate the surface expression of CXCR4 and instead start expressing another chemokine receptor, i.e., CCR1, which is known to enhance the egress of tumor cells from BM [51]. The increased surface expression of CCR1 leads to higher interaction with its ligand CCL3. This CCR1-CCL3 interaction inhibits myeloma cell migration towards CXCL12. This prevents interaction of CXCR4 with CXCL12 which further reduces α4β1 (VLA-4) and VCAM-1 activation, promoting myeloma cell exit from bone marrow. Animal studies have shown that blockade of CXCR4 or CXCL12 or VLA-4 contributes to the movement of plasma cells outside the BM. Processes like infection, inflammation and aging also bring about changes in the expression profile of these molecules and contribute to the egress of plasma cells. The exit of tumor plasma cells in peripheral blood circulation causes the extramedullary spread of MM [52,53]. Fig. 1a and b summarizes the main molecular events involved in homing and circulation of CTPCs.

This was the earliest method used for the detection of CPCs by use of light microscopic examination of peripheral blood smear. It is a simple, fast, easily available and cost-effective method present in every diagnostic center. However, the sensitivity of detecting CPCs on peripheral blood film light microscopy is 10⁻² and is much lower than presently available high-sensitivity techniques. In addition, morphological assessment of CPCs is not helpful in distinguishing normal versus tumor plasma cells [51]. Notably, the criteria for diagnosis of plasma cell leukemia, i.e., ≥5% CPCs, is based primarily on morphological assessment of peripheral blood smear.

MFC of peripheral blood is a highly sensitive, affordable and widely available technique. It can be easily used for the assessment of disease burden outside of the BM. This technique is rapid, consistent, and directly quantitative. The panel of markers generally includes antibodies against CD38, CD138, and CD45 for gating plasma cells which are then analyzed for expression of surface markers like CD19, CD56, CD200, CD117, CD81, CD10, CD20, CD28, CD27 [12,54]. A deviation from the normal expression profile of these markers is used to characterize and distinguish normal and tumor plasma cells. The expression of cytoplasmic kappa and lambda light chains provides confirmation of clonality based on light chain restriction [27,36]. One such example of representative flow cytometry plots from peripheral blood of a diagnosed case of MM is depicted in Fig. 2. The usual sensitivity of flow cytometry assay is 10⁻⁴ to 10⁻⁵, however, with next-generation flow cytometry the sensitivity of multicolor flow assay may reach 10⁻⁶. There are certain limitations of flow cytometry-based assessment of CTPCs, like the lack of standardized protocols related to variations in the processing of samples, heterogeneous antibody panels, number of cells acquired for analysis which may also depend upon the volume and quality of the bone marrow aspirate withdrawn, difficulties in the intracellular staining for light chain analysis and expertise in data analysis. All these parameters contribute to the sensitivity of the assay and hence variations in the results across the laboratories [55].

MGUS represents a condition where a monoclonal protein is detectable in serum and/or urine of an individual, however, there is no evidence of MM, amyloidosis, macroglobulinemia, or other related plasma cell or lymphoproliferative neoplasms [62]. MGUS is primarily an asymptomatic state with generally a low rate of progression to symptomatic neoplastic disease. The presence of circulating myeloma cells in individuals with MGUS has been studied, especially over the last two decades, and their burden in peripheral blood has been associated with the rate of progression of MGUS to overt MM.

Kumar et al. [53] studied the presence of plasma cells in the peripheral blood of 325 individuals with MGUS. CPCs were detected in 19.4% of their subjects. In individuals with the presence of CPCs, the median progression-free survival (PFS) was 138 months, whereas the median for PFS was “not yet reached” for those without CPCs. In addition, the individuals with CPCs had a median overall survival of 160 months which was shorter than the median survival of “not yet reached” at the last follow-up for individuals without CPCs. The study also evaluated various other factors like age, hemoglobin level, serum creatinine, serum albumin, serum ß2-microglobulin levels, M protein concentration, type of immunoglobulin, and presence of CPCs. The results of a univariate analysis note that higher M protein concentration, a non-IgG subtype of the involved immunoglobulin, and the presence of CPCs, were the only three factors to be significant predictors for progression. The combination of a load of CPCs with a higher M protein concentration and the M-protein of a non-IgG subtype identified a subset of individuals with MGUS who were at a higher risk of progressing to overt MM. The author suggested a scoring system based on these three variables to stratify the individuals with MGUS for their likelihood of progression to MM. The persons with higher risk of progression required closer monitoring. These individuals might become eligible for clinical studies testing new preventive medicines [53]. The data clearly show that the presence of CPCs was indicative of poor outcomes in terms of both PFS and OS.

SMM is defined as clonal PC infiltration in bone marrow comprising 10% or more of the total cellularity, and/or presence of serum M protein of 3 g/dL or more, in the absence of end-organ damage attributable to the proliferation of neoplastic plasma cells [57]. Due to a lack of data regarding the progression of the disease to the final malignant phase, patients mostly remain under observation without any early therapeutic support. This increases the mortality of around 20% of the patients who are at high risk of getting into the malignant phase within the first 2 years of disease. Also, there is the likelihood of serious end organ damage with malignancy, hence the observational approach may not always be beneficial [24]. The above factors partly contributed to considering the evaluation of CPCs as a prognostic indicator in patients of SMM by many groups.

Witzig et al. [36] studied a cohort of 57 individuals with SMM using slide-based immunofluorescence procedures that identified clonal plasma cells in the peripheral blood by microscopic examination of their morphology and the light chain expression. Within one year of screening for circulating clonal plasma cells, 16 (28%) patients progressed to MM and required treatment, whereas the other 41 patients remained stable. CPCs were noted at baseline in 10 out of the 16 (63%) patients who progressed within 12 months. In contrast, only four out of 41 (10%) of the stable patients had initial documentation of CPCs. Furthermore, the median time to progression to active MM in the patients with CPCs was 0.75 years. The progression rates for one & two years of time-period were 64.3% and 78.5%, respectively. In contrast, patients who did not show CPCs had a median time to progression of 2.5 years, with progression rates of 11.6% & 35.7% within one & two years, respectively. The authors concluded that the detection of CPCs was vital and may aid in the identification of patients having active MM when the rest of the parameters suggest SMM. The absence of CPCs in SMM may indicate a relatively stable disease without immediate treatment requirements [36]. Overall, the frequency of presence of CPCs is higher in patients who progress to active MM. In addition, the load of CPCs could identify the patients with the likelihood of progression in the absence of any indicators of higher risk like end organ damage.

Bianchi et al. [24] studied a cohort of 91 SMM patients. The authors defined a sub-group comprising 15% of patients who revealed a higher burden of CPCs, i.e., >5000 × 10⁶/L and/or >5% CPCs per 100 cytoplasmic Ig-positive peripheral blood mononuclear cells. This group of patients had a substantially rapid progression to active MM in comparison to the patients who had a lower load of CPCs, (12 months vs. 57 months of PFS), and was independent of other known risk factors for progression to MM. Most significantly, those with a high burden of CPCs had a 71% chance of progression during the first two years after diagnosis, in comparison to a 25% chance of progression in the rest of the patients with lower levels of CPCs. Similar findings were observed with overall survival, where the high-burden CPC group vs. the lower-burden CPC group showed OS of 49 vs. 148 months, respectively. The authors concluded that CPCs can be a suitable biomarker that can justify early therapeutic intervention in the absence of any end organ damage. A high burden of CPCs indicates impending active disease and also the presence of radiologically and clinically occult distant disease foci. The authors also observed that CPCs paired with M protein size may build a strong risk stratification model [24]. Overall, this study indicates that the higher load of CTPCs in PB is associated with poorer outcomes in terms of PFS and OS.

Foulk et al. [62] studied a cohort of 85 intermediate/high-risk SMM patients. CPCs were detected in 93.7% of cases at baseline. The load of CPCs at baseline was substantially higher in patients who ultimately progressed to active MM during the study period. The authors found that although the levels of M protein, free light chain ratio, and percentage of plasma cells in bone marrow also indicated disease progression, these parameters did not achieve a level of statistical significance. The study concluded that a well-standardized CPC-assay, in the context of predicting disease progression in patients of SMM, may have direct implications for the management of the disease, such as triggering more advanced diagnostic work-up or starting therapy sooner [62]. The study found that the load of CPCs is a stronger predictor of MM than conventional markers.

MM is an advanced stage of malignancy amongst the spectrum of plasma cell neoplasms. It is characterized by the proliferation of neoplastic plasma cells in the bone marrow which frequently leads to the invasion of adjacent bone, resulting in the typical lytic bone lesions and pathological fractures. The common clinical features include anemia, hypercalcemia and renal failure [57].

CPCs are commonly noted in patients of MM at the time of diagnosis, especially with the use of high-sensitivity assays. There is substantial literature available on significance in patients of MM.

Gonsalves et al. [18] studied a cohort of 157 patients of MM. Multi-colour flow cytometry assay detected CPCs in 54% of their patients at diagnosis and the rest constituted the cohort with absence of CPCs. The follow-up revealed that neither group reached a median overall survival (OS), however, the survival was significantly less for the patients with the presence of “any level” of CPCs when compared with those showing the absence of detectable CPCs at baseline. Patients with CPCs had a 2- & 3-year OS of 76% and 67%, respectively, compared to 91% and 87% for patients without CPCs. The authors also observed that the number of CPCs detected by flow cytometry was an independent prognostic factor in patients with newly diagnosed MM treated with novel agents. Additionally, the study revealed that the presence of >400 CPCs of all WBCs, identified a subset of patients who had a shorter overall survival and also a shorter time-interval to the next therapy. The cut-off of >400 CPCs in their cohort seemed even better in predicting this high-risk group than the traditional prognostic markers like high risk cytogenetic and high ISS stage. The high burden of CPCs did correlate with high-risk cytogenetics as well as increased proliferation. Further, it indicated that this approach improved the ISS staging for the identification of a smaller subset of patients with a poorer outcome. It was concluded that the load of CPCs in newly diagnosed MM patients was a powerful predictor of early relapse from therapy and mortality. As a result, these findings may have consequences for modifications in the present criteria for risk-determination in MM, as well as may need an update in the practice of risk-adapted treatment protocols [18].

Foulk et al. [62] studied a cohort of 166 newly diagnosed (ND) MM patients. The authors used FISH on sorted PCs for analysis of CPCs, which were noted in 98% of their patients of MM. The overall disease burden in patients which included the percentage of plasma cells in the bone marrow, levels of serum M protein and ISS stage, correlated with the CPC counts. The authors found that the CPC counts significantly reduced from the baseline counts, in patients who responded to therapy and achieved remission. Also, patients with clonal plasma cell counts of ≥100 at the time of remission showed reduced survival. The authors also proposed that the stratification of patients according to CPC counts may assist in using remission CPC counts as a measure for minimal residual disease and also may be used as a surrogate endpoint for relapse in clinical trials [62].

Tembhare et al. [55] used MFC to analyze CTPCs in a cohort of 141 NDMM patients at the time-of diagnosis, and subsequently at two time points, that is after completion of three cycles of therapy [PBMRD1 (peripheral blood measurable residual disease 1)] and after completion of six cycles (PBMRD2). The study revealed CTPCs in 76.6% of their patients at baseline. The CTPCs were detectable in 44% of patients at the first post-therapy time-point (PBMRD1) and in 34.4% at the second time-point (PBMRD2). Patients with ≥0.01% CTPCs showed shorter median PFS and OS. However, a longer PFS was noted in patients with detectable CTPCs at baseline but undetectable CTPCs at further time-points of therapy. Patients who showed high-risk cytogenetics, RISS-II/III, R2ISS of intermediate & high-risk, were found to have greater levels of CTPCs. The authors also found that the undetectable combined PBMRD (PBMRD1 and PBMRD2) outperformed the serum-immunofixation-based response. The authors concluded that CTPCs evaluated at diagnosis and further PBMRD are good non-invasive biomarker for prognostication of NDMM [55].

Gupta et al. [54] carried out a sequential assessment of CPCs, at baseline and at 6 months of therapy, in a small cohort of 21 newly diagnosed MM patients, using a ten-color (eleven-antibody) flow cytometry assay. The study quantified CTPCs and circulating normal plasma cells (CNPCs), separately, at both time points. CTPCs were found in 76% and 33% of the patients at baseline and at 6 months, respectively. The load of CTPCs, which included percentage, absolute counts per microliter of peripheral blood, and the proportion of CTPCs out of all circulating plasma cells (CTPCs + CNPCs), were associated with the presence of lytic lesions, plasmacytomas, expression of CD56 and CD81, Chr1p32 deletion, and absence of very good partial response (VGPR). Conversely, the load of CTPCs was lower in patients with concomitant amyloidosis. Interestingly, CNPCs were significantly higher in female patients and were lower in patients with hypoalbuminemia and thrombocytopenia [54].

Gonsalves et al. [63] studied a cohort of 556 newly diagnosed MM. All patients in R-ISS stage I or R-ISS stage II who had ≥5 CTPCs/μL were re-categorized to R-ISS stage IIB. The authors noted that the median time to the next therapy and overall survival in patients re-categorized to R-ISS stage IIB was lesser (21 and 45 months) when compared to patients in R-ISS stage I (40 months and not reached) and R-ISS stage II (30 and 72 months) and was similar to patients with R-ISS stage III (20 and 47 months). The load of CTPCs retained its adverse prognostic significance in a multivariate analysis of the outcomes. Similar results were seen in the study by Xia et al. [64]. The authors sub-categorized their patients of R-ISS stage II into CTPC-low and CTPC-high and found that R-ISS I, R-ISS II with CTPC-low, R-ISS II with CTPC-high, and R-ISS III had median progression free survival of 41 months, 30 months, 19 months and 16 months. The introduction of CTPCs levels into R-ISS demonstrated more robust discrimination of prognosis of newly diagnosed cases of MM, especially in the patients presenting in R-ISS stage-II [63]. The findings of the above two studies clearly indicate that the load of CTPCs could be integrated into existing clinical workflow.

In the last decade, there have been a lot of studies on CPCs that tried to give a cutoff to indicate the favorable or poor outcome. Table 2 summarizes the key results of these studies.

All the above studies in MM indicate that the higher load of CTPCs at diagnosis or any time point during the course of therapy indicate poor outcomes. In addition, higher CTPCs also correlated with many of the conventional prognostic markers. It is also noted that its inclusion in existing scoring systems may enhance their efficacy in identifying patients with poorer outcomes.

Importantly, the prognostic significance of CTPCs is likely to vary depending on the detection method and patient cohort. The load of CTPCs generally increases from the early stages of MGUS to symptomatic MM. Hence, cohorts of different sub-categories of plasma cell neoplasms are likely to provide distinct cut-offs of levels of CTPCs for assessing outcome parameters. A sample bias in studies involving fewer subjects of MGUS and/or smoldering myeloma is more likely because of the asymptomatic nature of these conditions and hence robust cut-offs for these entities require larger screening cohorts and longer follow up durations. For symptomatic myeloma, variations amongst different studies for the cut-off value of the levels of CTPCs at the time of diagnosis are likely to produce variations in the prognostic significance of CTPCs. These variations in values are likely due to different technologies used to detect CTPCs. Morphological assessment of CPCs is easiest but with very low sensitivity as compared to MFC and NGS. A cut off of ≥5% CPCs on morphological assessment is presently used to categorize patients into the poorer outcome entity of plasma cell leukemia as compared to MM. Even for sophisticated techniques like MFC, the sensitivity varies a lot and depends on the methodology of sample processing, the number of cells acquired for analysis, the number and type of antibodies used in the panel, experience and expertise of the flow cytometrist involved in analysis. The above issues are reflected in the results of levels of CTPCs in different studies. In addition, for assessment of CTPCs during the course of therapy, important variables like time points at which the samples are analyzed, type of chemotherapeutic regimen administered, whether patients have undergone hematopoietic stem cell transplant or not, and the duration of follow up of patients, are very likely to influence the prognostic information provided by CTPCs.

Studies have shown that the presence of CPCs prior to ASCT is indicative of both reduced PFS and OS and is independent of the depth of response evaluated at the end of induction [21,70]. In newly diagnosed patients of MM undergoing upfront ASCT, a reduction in load of CPCs by proteasome inhibitor (PI) and/or immunomodulator (IMiD)-based induction therapy, leads to an improved PFS and OS [23] (Table 3).

Dingli et al. [71] studied a cohort of 246 MM patients undergoing ASCT. They found circulating clonal plasma cells in 95 of their patients. Although the complete response (CR) rates after transplantation were similar for their patients with or without CPCs, however, the overall survival and the time to progression were significantly shorter in patients with the presence of CPCs prior to transplant. The authors also noted that the CPCs remained independent of cytogenetic and disease status at the time of transplantation. The authors also proposed a scoring system using both CPC counts and cytogenetics in combination. They concluded that CPCs at the time of ASCT were an independent prognostic factor and in combination with cytogenetic is likely to provide a powerful scoring system that could stratify the patients into risk-groups in a better way and also guide management [71].

Chakraborty et al. [21] studied a cohort of 840 MM patients and utilized a six-color flow cytometry-assay to detect CPCs prior to ASCT. The authors noted CPCs in 162 (19.3%) patients in their cohort. Patients harboring CPCs at the time of ASCT had a greater incidence of having high-risk cytogenetic abnormalities in comparison to those without CPCs. The stringent complete response (sCR) rates after ASCT were significantly lower in patients with detectable CPCs prior to transplant as compared to those without CPCs (15% vs. 38%, respectively). Similar results of significantly poor PFS (15.1 vs. 29.6 months) and OS (41 vs. not reached) were noted in their patients with and without detectable CPCs, respectively. The authors concluded that sequential quantification of CPCs during the course of disease should be incorporated in clinical trials to study the clonal evolution and kinetics of CPCs, and its impact on disease outcomes [21].

Chakraborty et al. [23] studied a cohort of 247 MM patients undergoing ASCT. The authors carried out a sequential evaluation of CPCs at the time of diagnosis and pre-transplant. They categorized patients into three groups i.e., CPCs (−/−) who had no circulating CPCs at both time points (n = 117), CPCs (+/−) had CPCs at diagnosis followed by complete eradication post induction therapy (n = 82), and CPCs (+/+) had CPCs both at the time of transplant and continued presence of cells or emergence of new cells post-induction therapy (n = 48). Post-transplant stringent CR rates were significantly higher in the first group and were found to be 32%, in CPCs (−/−), 30% in CPCs (+/−) and 12% in CPC (+/+) groups. The PFS and OS after transplant were significantly different amongst the three groups with CPCs (−/−) group showing the best outcome. The authors concluded that monitoring for CPCs before induction therapy and prior to transplant was predictive of survival in newly diagnosed MM and should be incorporated into clinical trials [23].