Multiple myeloma (MM), an incurable hematological malignancy, is characterized by recurrent cytogenetic and molecular abnormalities. Malignant plasma cells show typical multifocal distribution in the bone marrow (BM) and occasional extramedullary dissemination (1). Considering the spatial heterogeneity of myeloma, analyzing BM aspirates collected from a single site often does not provide comprehensive insights into the genetic profile of tumors. During disease progression, drug resistance and clonal evolution pose major issues (2, 3). Longitudinal evaluation of mutational landscape and tracking tumor burden can facilitate the identification of early signs of treatment resistance and relapse. However, repeated BM aspiration is impractical as the procedure is invasive and causes discomfort. Although BM biopsy is the current gold standard for MM diagnosis and prognostic stratification, tracking malignant clones remains challenging because of the spatial and temporal limitations of BM biopsies; therefore, there exists an urgent need to identify a novel biomarker to monitor disease progression.

The presence of cell-free nucleic acid fragments in human blood was first described in 1948 by Mandel and Métais (4). Cell-free DNA (cfDNA) is believed to have originated as a consequence of cell apoptosis and necrosis, and possibly also active secretion (5). Increased cfDNA levels were first reported in the serum of cancer patients in 1977 (6). According to several studies, cfDNA is of potential diagnostic and prognostic importance in various cancer types, and its levels during treatment are reportedly correlated with outcome (7–9). With the development of molecular methods, mutant DNA fragments, confirmed to be of tumor origin, have been detected in plasma. Mutations in cfDNA can serve as highly specific markers for cancer, and tumor-derived DNA in cfDNA, also known as circulating tumor DNA (ctDNA) (10), detection provides a non-invasive approach to diagnose cancers. ctDNA carries information pertaining to the dynamics of cancer-specific genetic and epigenetic alterations; moreover, ctDNA represents the entire epitome of mutations present in primary and metastatic tumors (11). In comparison to previously used blood-based biomarkers, cfDNA-based detection methods show higher sensitivity, and thus, they seem to have great potential for both quantitative analysis of tumor burden and genetic analysis in case of patients with cancer.

Relative to other cancer types, cfDNA concentration is higher in MM, and there exists high concordance between mutations found in DNA using BM aspirates and those found in ctDNA (12, 13). Although cfDNA analysis for clinical prognosis and to monitor disease burden in MM has been recently reported (14, 15), the results are unclear. Therefore, herein we performed a meta-analysis to systematically explore the clinical relevance of circulating cfDNA in patients with MM.

To date, myeloma remains an incurable disease, and patients often experience a relapse owing to residual tumor cells. MRD has become one of the most important biomarkers for outcome prediction and therapy optimization. However, longitudinal monitoring of BM MRD remains limited as repetitive BM biopsies are invasive to patients. In addition, in case of some patients with extramedullary disease, BM specimens may not accurately reflect the disease burden. Previous studies based on circulating cfDNA for MRD detection in various hematological malignancies have validated its significance for measuring a small number of residual tumor cells (26–28). Unfortunately, most such studies involving patients with MM either assessed only a few patients or lacking of the comparison with BM MRD assessment; therefore, the conclusions remain controversial.

Our pooled data confirmed that ctDNA detection had an obvious advantage in terms of MRD detection specificity (specificity, 0.91; 95% CI, 0.72–0.99). According to the guidelines for area under the SROC value interpretation (29), ctDNA presence in patients with MM has a relatively high diagnostic ability (AUC > 0.9) and can indicate MRD positive ( Figure 2 ). Clonal immunoglobulin gene rearrangements in plasma samples have been detected by NGS or allele-specific oligonucleotide–qPCR to monitor tumor burden (18–20). At the time of MRD evaluation, the results of ctDNA detection using blood samples were compared with those of NGS or MFC using paired BM samples. Mazzotti (18) demonstrated the absence of a correlation between ctDNA and BM MRD detection by NGS using only immunoglobulin gene rearrangements in patients with MM. Further, Vrabel (20) compared ctDNA and BM MFC, a common method for MRD detection, and the sensitivity of MRD detection using ctDNA was only 66.7%. In contrast, Biancon (19) analyzed MRD by MFC using BM samples and found complete concordance with ctDNA data in all cases, and a high level of correlation was detected between ctDNA and BM MFC data (r = 0.5831, P = 0.0044, Pearson’s correlation test). Finally, regarding the sensitivity of MRD detection, ctDNA detection in plasma was not superior over BM assessment as per our analysis (sensitivity, 0.58; 95% CI, 0.43–0.72).

We believe that further studies with a larger cohort of patients achieving CR are warranted to determine the reasons for inconsistency in MRD detection sensitivity using ctDNA in different studies. The three studies pooled for the meta-analysis of MRD detection accuracy all used immunoglobulin gene rearrangements in cfDNA to track residual myeloma cells. However, MM is characterized by multiple recurrent somatic mutations, copy number variants, and structural alterations (30). Thus, given the extensive heterogeneity in MM, a large targeted sequencing panel may prove useful for improving MRD detection sensitivity using ctDNA.

In other cancers, cfDNA analysis has been used to identify mutations related to drug resistance and to predict therapeutic response, which could influence the choice of treatment (31–33). The prognostic value of cfDNA has been explored in myeloma. Although cfDNA is an admixture of normal and tumor-derived DNA (ctDNA), increased concentration of cfDNA signifies higher tumor burden and is indicative of the prognostic value of survival in many cancers (34, 35). In addition, high cfDNA levels at baseline are significantly associated with poor PFS and OS in patients with myeloma (25). ctDNA represents the entire epitome of mutations present in primary as well as metastatic tumors (36). Plasma-derived ctDNA analysis in patients with MM was found to show good concordance with standard mutation analysis using BM samples. Mithraprabhu (22, 23) analyzed plasma-derived ctDNA as an adjunct to BM biopsy for mutational characterization and tracking disease progression and found that in comparison with BM samples, plasma samples showed a higher proportion of TP53 mutations. Besides, Li (24) found a significantly higher detection rate of BRAF, KRAS, and NRAS mutations in plasma-derived cfDNA samples than in BM samples (53% vs 34%). Plasma evidently reflects mutations originating from all focal sites, both at the intramedullary and extramedullary levels. Therefore, assessing both BM and plasma samples should provide a more comprehensive landscape of tumor mutational burden.

The results of the studies included herein indicate the prognostic value of circulating cfDNA. cfDNA levels in MM are reportedly significantly associated with both PFS and OS. In two studies including PFS as the outcome indicator, higher levels of cfDNA were found to be associated with poor PFS ( Figure 3 ) (19, 25). As evident from Figure 4 , higher level of cfDNA was found to be associated with poorer OS. Because ctDNA only accounts for a small proportion of total cfDNA, we also performed subgroup analysis to assess the usefulness of ctDNA quantification to predict OS; we found that ctDNA-positive patients or those with a high level of ctDNA showed inferior OS ( Figure 5 ).

In the analyzed studies, gene mutations, mainly including RAS-RAF and TP53, were detected, which may predict poor prognosis in patients with MM (37). Mithraprabhu (23) reported that high levels of ctDNA are a prognostic factor in case of relapsed/refractory MM patients but not in case of newly diagnosed MM patients. This could be because a higher frequency of gene mutations in plasma was detected in relapsed/refractory MM patients than in newly diagnosed MM patients. However, Li found a significantly higher detection rate of BRAF, KRAS, and NRAS mutations on using plasma-derived cfDNA samples (53%) than on using BM samples (34%) in case of newly diagnosed MM patients, and patients with these mutations showed shorter OS than those without them (24). These two studies used different ctDNA detection methods: Mithraprabhu used NGS, while Li used droplet digital PCR. Although previous studies have verified that these methods show high concordance in terms of tumor genotype (38, 39), the inconsistency in the data reported by Mithraprabhu and Li suggest that further studies are warranted to compare these methods; furthermore, more patients need to be evaluated to confirm the prognostic significance of plasma-derived ctDNA in newly diagnosed MM patients.

This study has several limitations that need to be addressed. First, the lack of currently recognized ctDNA gene targets in patients with MM might contribute to bias. Second, different ctDNA detection methods were used in the included studies. Finally, owing to the limited number of studies on ctDNA detection in MM, the data included is not very rich, which may contribute to further bias.

To the best of our knowledge, this meta-analysis represents the first comprehensive study to investigate MRD detection and prognostic value of circulating cfDNA in patients with MM. We report that ctDNA detection has an obvious advantage in terms of MRD detection specificity; moreover, higher levels of cfDNA were found to be associated with worse prognosis in patients with MM. cfDNA detection is a non-invasive method and thus shows promise as a good alternative to BM biopsies for monitoring clonal evolution and tumor burden so as to guide the treatment of patients with MM. However, before its wide application in patients with MM, accurate ctDNA gene targets and standardized detection methods need to be established.

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

XY conceived and designed the study. XY and WL collected and analyzed the data. LZ and JY analyzed the data. XY wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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