Multiple myeloma (MM), one of the most common hematological malignancies, is caused and characterized by clonal proliferation of malignant plasma cells (1). Clonal plasma cells are predominantly distributed in the bone marrow (BM), mainly due to their adhesion to the BM microenvironment (2, 3). Along with the technical improvement, it has become clear that clonal plasma cells not merely reside in the BM but can also passage into the circulation and subsequently home to intramedullary or distant tissues, a process that allows clonal plasma cells to circulate and reside in the peripheral blood, and these cells are named circulating plasma cells (CPC) (3).

The rise of multi-parameter flow cytometry (MFC) has brought reliable methods for diagnosis and minimal residual disease (MRD) monitoring in MM, and, at the same time, detection of minimal CPC in the peripheral blood (4, 5). Moreover, detection of CPC by MFC could eliminate the need for repeated invasive BM biopsies and possess higher sensitivity than conventional slide-based methods (6). Elevated CPC has been shown in previous studies to suggest a poor prognosis (7–9), while most studies on CPC were conducted in Caucasian populations. Given the racial and therapeutic heterogeneity, the prognostic implication of CPC quantification, especially its dynamics, has not been fully elucidated in Chinese population.

The mutational landscape is currently one of the most intensively studied fields in MM. It was reported that an average of 1.6 mutations were detected per Mb in MM (10). High-throughput DNA sequencing, also known as next-generation sequencing (NGS), has been widely used in MM for the past few years, and mutations like KRAS, NRAS, TP53, FAM46C and DIS3 were observed recurrently. Yet, mutational landscape was heterogeneous in different studies (11–13), and the relationship between mutations and CPC levels in Chinese population has not been well explored (14–17). Previous studies have shown high concordance in gene expression between bone marrow plasma cells (BMPC) and CPC by NGS technology (14, 16), suggesting that CPC are mainly derived from tumor cells in the BM. In this work, NGS was applied to molecularly characterize the BM myeloma cells to determine the association of CPC levels with the mutational landscape.

Here, we used MFC for CPC quantification and NGS technology for mutational landscape mapping to verify the implication of CPC in risk stratification, and to further elucidate the relationship between genetic alteration and CPC quantification. To the best of our knowledge, this is one of the most extensive studies in Chinese population to date focusing on the clinical features and the underlying genetic characteristics regarding CPC.

The past decade has witnessed an ever-increasing focus on CPC, and the extensive application of MFC in CPC detection allows easy and non-invasive examination of tumor cells (6, 7, 28). To investigate the implication of CPC on tumor burden and its predictive value for efficacy and prognosis, we conducted this clinical study involving 301 patients, one of the most extensive clinical studies to date. As a result, we found that higher CPC was associated with lower hemoglobin and higher β2-microglobulin and LDH. The β2-microglobulin, LDH and hemoglobin are well-documented biomarkers of tumor load in MM, thus suggesting the presence of CPC to be a result of high disease burden. BMPC is one of the most established parameters for the diagnosis and monitoring of MM, which involves invasive and painful procedures. Korthals et al. (29) reported that the MRD level of peripheral blood was 40-fold lower than their BM counterparts when detected by IgH-PCR. A non-linear correlation was found between absolute CPC count and BMPC percentage in another study using MFC (30). In the present work, we validated that higher CPC was associated with elevated BMPC by both morphological and MFC method. We also observed a significant positive correlation between BMPC and CPC (both by MFC). Therefore, detection of CPC could be used to monitor tumor load without the need for repeated BM biopsy.

It has been addressed that CPC quantification were correlated with the prognosis of NDMM (7) and relapsed/refractory MM (8, 9). In this work, we found that high levels of pre-therapeutic CPC suggest poor response and adverse outcome in NDMM. It is noteworthy that the presence of post-therapeutic CPC indicated a low response rate, with up to 80% of patients having a response of PR or worse. In contrast, for patients with undetectable CPC after treatment, 75.8% achieved remission of ≥VGPR. Similarly, the remaining CPC portended a dismal prognosis with a median OS of only 11.5 months. Hence, the dynamics of CPC may be a promising biomarker to identify high-risk MM.

MM is a heterogeneous disease with diversified outcomes (31). The R-ISS is the most recognized system for prognostic categorizing (32), but up to half of the patients are classified as R-ISS II, and the prognosis of these patients varies widely. A study from Mayo Clinic showed a similar prognosis between R-ISS I/II patients with ≥5 CPCs/μL and R-ISS III patients (33). When the CPC quantification was introduced into the existing R-ISS, we were excited to observe a more robust risk stratification for the large number of patients with R-ISS II disease, signifying the potential role of CPC in more accurate prognostic anticipation.

Interestingly, we discovered a higher proportion of light-chain subtype in CPC-high patients than the CPC-low ones, which, to the best of our knowledge, is the first report to date describing the subtype pattern regarding different CPC levels. This phenomenon was highly similar to that observed in plasma cell leukemia (PCL) (34, 35), which represents one of the most aggressive forms of MM and typically denoted a dismal outcome even in the era of novel agents (34). It is known that a complete immunoglobulin is composed of heavy and light chains. The immunoglobulin heavy chain (IgH) is encoded by the IGH gene located on chromosome 14q32 (36), and the translocation of IgH, considered the primary genetic event of MM, occurs in 45% of MM patients (26) and more than 80% of PCL patients (35). In this work, we observed a higher rate of IgH translocation in CPC-high MM than the CPC-low MM. Since IgH translocation could yield the deletion or silence of the IGHV gene necessary for IgH assembly (37), we speculated that the higher incidence of IgH translocation in CPC-high MM and PCL potentially lead to the impaired heavy chain synthesis, and hence, the higher rate of light-chain subtype. Additionally, we noted that del(17p) occurred more frequently in CPC-high MM than that in CPC-low MM (20.5% vs 8.3%); and at the same time, Gundesen et al. (34) demonstrated a higher incidence of del (17p) in PCL that in MM (40% vs 11%). Tumor cells in PCL are generally considered to express higher levels of CD19, CD20 and CD45 than those in MM (38, 39); similarly, upward expression of these biomarkers were observed in CPC than their BM counterparts (30). The elevated expression of these antigens may represent, according to the phenotypic transformation of B-cell development (40), a poorly-differentiated cell population that is believed to suggest inferior survival in MM (41). A Chinese study by An et al. (42) illustrated that MM patients with a small amount of CPC had similar prognosis to PCL. Accordingly, the presence of CPC≥0.105%, a threshold represents even a minimal amount of CPC, represents a relatively aggressive MM, and we speculate that the similar phenotypes and cytogenetic characteristics exhibited by CPC-high MM and PCL may partly explain their congruent subtype pattern and adverse outcome.

To further elucidate the association of CPC level with genetic features, NGS was employed to characterize the tumor cells. To our knowledge, this is the first time that NGS has been used to molecularly characterize the tumor cells in a study of CPC in Chinese population. As a result, while more aggressive myeloma is thought to have a high mutational burden (43), we found no significant differences in mutational burden between the CPC-low and CPC-high groups. When CPC levels were compared between patients who carried WT and mutant genes, patients with mutations involving TP53, BRAF, DNMT3A, APOBEC3C, ASCC3 and TENT5C, etc., tended to have significantly higher CPC levels. We have noticed that TP53 mutation, one of the most studied mutations in MM, was related to a higher level of CPC, in line with the superior frequency of del(17p) in CPC-high patients by FISH method. TP53 mutation was reported to be associated with the migration of MM cells from the BM into peripheral blood, thereby facilitating the development of PCL (44, 45). Lee et al. (45) reported that mutation of TP53 was more frequent in PCL than in MM. These findings emphasize the role of TP53 in prompting the MM cells to migrate into the blood.

It is interesting that mutations of IL6ST, EGFR and STAT3 suggested enhanced CPC levels. A latest investigation for functional high-risk (FHR) MM, which refer to patients with suboptimal response to induction therapy or early relapse, demonstrated increased mutations in the IL-6/JAK/STAT3 pathway in FHR patients (46). Therefore, elevated CPC may portend the possibility of FHR MM, providing a simple alternative for the identification of high-risk MM.

To unveil the underlying pathways involved in the egress of MM cells towards the circulation, we performed enrichment for the mutant genes with the highest CPC levels. As a result, pathways involving the regulation of chromosome as well as adhesion and junction were significantly enriched. It has been reported that the CPC represent a subset of myeloma cells with downregulated integrins and adhesion molecules and thus tended to become a BM microenvironment independent subpopulation (14, 47); our results validate the role of adhesion and junction in CPC formation at the genetic level. The regulation of chromosome involves plenty of physiological processes, of which chromosomal instability is most extensively studied in MM. Chromosomal instability can result in copy number and structural changes of chromosomes, and act as a critical element in the development and progression of MM (43, 48), whereas the relationship between CPC and chromosomal instability as well as other procedures involved in chromosome regulation remains poorly understood. Our findings proposed a suggested mechanism of CPC formation, and additional efforts are still warranted.

There are still some limitations in this study. The tumor cells subjected to NGS were sorted by the positive expression of CD138. Although CD138 is well recognized to be expressed on MM cells (49), it has been recently reported that few MM clones lacks the expression of CD138 and these CD138-negative cells may represent a group of cancer stem cells with a higher migration capacity (50, 51). Therefore, the application of NGS in combination with highly purified fluorescence-activated cell sorting (FACS) based on the specific aberrant phenotypes of patients or single-cell RNA sequencing may provide a more accurate method to characterize MM cells. In addition, limited to current technologies, the sensitivity of MFC is insufficient to detect CPC in a small half of the patients; hence, the detection of BM aspirates is still the gold standard for MRD monitoring, especially for patients with preferable responses. NGS technology has been proved to be extremely sensitive in CPC detecting (16), but the prohibitive cost limits its application in disease assessment. Therefore, more sensitive flow cytometry, as well as less expensive sequencing techniques, are still warranted.

To conclude, we adopted the emerging technologies, comprising MFC and NGS, to analyze the correlation of CPC with clinical characteristics and gene mutations. We demonstrated that CPC level can effectively reflect the tumor load, and high level of CPC at diagnosis or after induction therapy can predict high-risk MM that indicate poor treatment response and adverse outcome. We present the phenomenon and mechanistic speculation of an elevated proportion of light-chain subtype in patients with higher CPC. Analysis of the mutational landscape revealed that TP53 mutation and pathways involving chromosome regulation and adhesion may be the underlying mechanism of CPC formation. Overall, while bone marrow examination remains the gold standard in MRD detection for most MM patients, the detection of CPC by MFC may provide a less-invasive, convenient and, more importantly, reliable approach for risk stratification and disease monitoring. Large-scale clinical research and further in-depth mechanism explorations are required for a better understanding of CPC, thus, shed light on promising therapies to overcome the adverse outcome associated with CPC.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethical review and approval was obtained from the institutional review boards of the First Affiliated Hospital of Nanjing Medical University Ethics Committee (No. 2020-SR-589). Informed consents were obtained from all patients before enrolment.

YX designed the study. YX and NS collected the data. YX and MX analyzed and interpreted the data. YX drafted the manuscript, YJ and LC revised the manuscript. All authors contributed to the article and approved the submitted version.