**2. Biological and clinical implications of liquid biopsy analysis in MM**

Liquid biopsy is gaining momentum in MM due to the inherent nature of the disease and the obvious limitations associated with BM biopsy. A summary of the potential utility of CTC and CFNA evaluation are presented in this section (**Figure 3**).

#### **2.1. Determining the tumour genome composition**

Given the clonal genomic heterogeneity that exists within MM, particularly with disease progression, it is theoretically necessary to perform repeated BM biopsies to track tumour

emerging evidence supports the notion that a range of haematologic malignancies including MM are likely to harbour significant sub-clonal and spatial genetic heterogeneity. In MM, levels of cfDNA are significantly higher than in both normal volunteers and non-MM cancers [54,

The presence of extracellular RNA (exRNA) in plasma/serum was also described some decades ago [41, 56, 57] with significantly higher levels in cancer patients compared to healthy individuals [58]. Additionally, tumour burden was shown to correlated with the level of circulating mRNA [59]. The exRNA released into the circulation is surprisingly stable owing to its protection from RNAse-mediated degradation through packaging into exosomes, which are shed into the blood stream [60–64]. Biomarker studies measuring cancer-specific circulating mRNA have identified higher level of mRNA and correlation with disease status in a number of malignancies including nasopharyngeal carcinoma [65], prostate [66, 67], gastrointestinal [68–72], breast [73–76], hepatocellular carcinoma [77], lung [78, 79], cervical [80], lymphoma [81] and thyroid [82]. These findings highlight the potential utility of exRNA in cancer diagnostics, and while promising, it will be critical to assess exRNA in larger and annotated sample sets to establish specific circulating mRNAs as reliable non-invasive biomarkers.

Similarly, extracellular miRNA are found in virtually all fluid compartments of the body including blood where they can circulate bound to proteins, high-density lipoproteins or apoptotic bodies, or within exosomes, thus providing stability against RNases (reviewed in [83]). The levels of circulating miRNA have been described in a number of cancers with one of the earliest reports being in diffuse large B-cell lymphoma (DLBCL) demonstrating a correlation between the level of specific miRNAs and prognosis [84]. Subsequently, several publications of circulating miRNA in malignancy have demonstrated increased miRNA in cancer patients when compared to healthy individuals and a decrease in levels following tumour debulking surgery (reviewed in [85–87]). Circulating miRNA expression profiles are also known to have signatures correlative to disease stage, diagnosis and relapse [88–91]. The origin of circulating RNA subtypes is, however, not as well understood as for cfDNA, as these can potentially be sourced from tumour cells, the supporting micro-environment or immune cells [92]. While mRNA has not been assessed in MM, there are a number of studies of miRNA

55] and the potential utility of cfDNA in MM will be discussed below.

and the prognostic utility of these will be discussed in the next section.

CFNA evaluation are presented in this section (**Figure 3**).

**2.1. Determining the tumour genome composition**

**2. Biological and clinical implications of liquid biopsy analysis in MM**

Liquid biopsy is gaining momentum in MM due to the inherent nature of the disease and the obvious limitations associated with BM biopsy. A summary of the potential utility of CTC and

Given the clonal genomic heterogeneity that exists within MM, particularly with disease progression, it is theoretically necessary to perform repeated BM biopsies to track tumour

*1.2.4. Extracellular RNA (mRNA and miRNA)*

78 Hematology - Latest Research and Clinical Advances

**Figure 3.** Currently in MM, BM-derived tumour cells are utilised for mutational characterisation, biomarker identification and to define disease burden. Peripheral blood can be utilised to obtain both CTC and CFNA. DNA and RNA can be derived from both sources and as both CTC and CFNA are derived from multiple tumour sites they theoretically will provide a more comprehensive profile of the disease in comparison to a single-site BM biopsy.

genomic evolution within any particular patient. Such an approach, however, remains an unattractive proposition as BM biopsies are invasive, not without complications, subject to sampling error and cannot capture the increasing spatial heterogeneity present with disease evolution. Therefore, the capacity to perform sequential mutational characterisation using a more 'holistic' and non-invasive approach would be a highly desirable alternative option. Both CTC and CFNA have been explored for this purpose in MM, however, the field is relatively unestablished with only a small number of publications addressing this concept. One of the earliest reports by Zandecki et al. evaluated CTC and matched BM-MM cells and demonstrated that chromosomal abnormalities were consistent between these two compartments [93]. The recent development of both NG flow cytometry and contemporary sequencing technologies has enabled the analysis of CTC at the single-cell level [27] but to date only small numbers of patients have been evaluated using this approach. Analysis of 9 patients with single cell WGS revealed that all BM-defined mutations were similarly present in CTC [27]. Further analysis was done on CTC in two patients with low disease burden (one with treated MM and one with MGUS). The CTC of the MM patient harboured readily detectable somatic mutations in BRAF, TP53 and IRF4, with reappearance of the mutations when the patient relapsed, indicating that the treatment had not eradicated these particular sub-clones. CTC analysis of the patient with MGUS revealed the presence of a NRAS mutation, also present in the BM. The concordance with BM genetic composition was also confirmed by Mishima et al. utilising NGS when analysing 8 paired CTC and BM aspirates [28]. Clonal, defined as a > 0.90 mutant allele fraction (MAF), mutations were present in >99% of CTC and BM, while subclonal shared mutations (defined as >0.05 MAF) were concordant in >80%, with 16% of subclones (<0.05 MAF) discordant between the two compartments consistent with spatial heterogeneity [28]. Similarly, copy number alternations (CNA) were assessed and found to be 92% concordant. Likewise, RNA-seq at the single cell level also accurately predicted the recognised non-random chromosomal translocations that manifest in MM responsible for overexpression of key MM-associated oncogenes [27]. Overall, for both somatic mutations and CNA, CTC appear to harbour more variances indicating that they are likely sourced from multiple tumour sites and the manifestations of spatial heterogeneity may thus be more accurately captured by this type of analysis.

clonal architecture of the disease during therapy. The prognostic significance of mutational profiling in MM also remains largely unknown, with only a few genes thought to have prognostic significance. Furthermore, it should be recognised that the prognostic impact of certain mutations has been based on studies profiling material sourced from BM aspirates and therefore may not have been representative of the entire tumour genome. This shortfall could be potentially overcome using CTC and/or ctDNA as an adjunct to BM biopsy for

Liquid Biopsy in Multiple Myeloma

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The establishment of single-cell RNA-seq methodology in CTC from MM patients [27] presents an important opportunity for the identification of biomarkers that may define differing sub-sets and stages of the disease. Theoretically, this may represent a technically feasible and hence more reliable strategy than evaluation of exRNA as the RNA would be derived from tumour cells that should overcome some of the stability issues that may be associated with exRNA. Furthermore, CTC-derived RNA should address the issue of spatial heterogeneity and hence provide a more inclusive RNA-signature during disease evolution, as compared to present data based on single-site BM biopsies. To date, the potential of exRNA for differentiating the continuum of plasma cell dyscrasias ranging from MGUS to intra-medullary, EM MM and PCL has been explored in MM, but mainly through the analysis of miRNA and not mRNA (reviewed in [83]). Available evidence would suggest that MM cells contain differing populations of miRNA species at defined disease stages [96–100], therefore an approach identifying miRNA biomarkers non-invasively utilising exRNA would represent a readily accessible and novel approach. However, while circulating miRNA biomarker studies, including patients from different disease stages, have defined relative differences in both the expression of specific miRNAs and the absolute levels of circulating miRNAs as potential biomarkers [101–109] there is widespread discordance between the published studies with respect to the specific miRNAs identified [83]. For example, Yoshizawa et al. found significant downregulation of miR-92a in patients with symptomatic MM when compared to normal subjects, conversely, Besse et al. when comparing serum miRNA profiles between healthy, ND and EM patients, demonstrated that miR-130a expression was significantly decreased in the EM patients, hence a potential biomarker for EM disease, but no correlation with patient outcome was evident [101]. Jones et al., identified that a combination of miR-1246 and miR-1308 could distinguish MGUS from MM patients [106], whereas miR-25 was shown to correlate with conventional serum markers of MM at both diagnosis and at complete response (CR) post autologous stem-cell transplantation (ASCT) [103]. Some of the studies also identified a prognostic correlation with the actual levels of circulating miRNA. Rocci et al. demonstrated that miR-16 and 25 were significantly associated with OS in ND MM [102]. Qu et al. identified miR-483-5p as a potential predictor of MM survival [110]. Hao et al. showed that lower expression of miR-19a predicted poor OS [107], while Kubiczkova et al. concluded that a combination of miR-34a and let-7e can distinguish MM patients from normal individuals, and that patients with lower levels of miR-744 and let-7e had shorter OS [105]. In parallel with these studies, an evaluation of exosome-derived miRNA demonstrated that both let-7b and miR-18a were significantly associated with progression-free survival (PFS) and OS [111]. Unlike CTC, one of the significant shortcoming of assessing circulating miRNA levels is that expression levels might be

mutational analysis and consequently, for prognostic profiling.

**2.2. Biomarkers**

The evaluation of cfDNA for mutational characterisation and monitoring of disease burden in MM has also recently been described [54, 55, 94, 95]. Importantly, and for the first time, spatial and clonal heterogeneity on a large scale in MM was confirmed by our study evaluating paired BM-derived CD138 selected MM cells and cfDNA utilising a highly sensitive targeted sequencing platform consisting of a panel of 96 cancer-associated mutations including mutations of KRAS, NRAS, BRAF and TP53. This demonstrated that 21% of MM patients had mutations detectable only in the plasma and that the prevalence of spatial heterogeneity increased with disease evolution [55]. Numerous signalling pathways are known to be mutated in MM [13, 14], with previous BM WES studies demonstrating that activating mutations of the RAS-MAPK pathway were present in approximately 50% of patients. In contrast, our study demonstrated RAS-MAPK activating mutations in 69% of cases and the co-existence of multiple mutated sub-clones in a significant proportion, with >3 activating mutations in 23% of patients (range, 3–17 mutations per patient) representing a hitherto unrecognised mutational convergence on the RAS-MAPK pathway. This had remained largely undiscovered in single-site BM WES studies owing presumably to the relative insensitivity of the methodologies used and the presence of clonal heterogeneity at sites distant to the BM biopsy site. While it is likely, with high-sensitivity approaches, that there will be a MAF threshold for minor BM sub-clones that enables them to be reproducibly detected in the plasma, this will not be relevant with less sensitive strategies like WES and targeted amplicon sequencing that cannot detect smaller sub-clonal mutations, thus explaining, and consistent with, the 96% concordance between BM and PL demonstrated using NGS technologies [54]. Interestingly, mutations in PIK3CA, which have only rarely been described in MM, were found in a recent study to be present, but only in the plasma, indicating that these could be a feature of EM disease [54]. However, confirmatory studies with larger cohorts of patients and more comprehensive and targeted panels of MM-associated mutations are required to validate these observations. Theoretically, therefore, plasma cfDNA analysis of MM patients, particularly in the case of EM-MM, would not only provide information on the underlying biology of this disease, but could also furnish information on response to therapy through quantitative sequential tracking of plasma-only mutations. Moreover, RNA-seq of plasma cfRNA derived from MM patients is also theoretically possible to provide information on single nucleotide polymorphisms (SNP), although this has not yet been described in the published literature. So, while in the short-term it is unlikely that plasma cfDNA evaluation will replace BM biopsy, as the latter remains necessary in MM for diagnostic purposes, we believe cfDNA analysis as an adjunct to BM biopsy for comprehensive mutational characterisation is likely to become a reality in the near future.

To date, the analysis of CTC or cfDNA with paired BM has largely been performed only at single time-points and has not been comprehensively utilised for longitudinal assessment using sequential specimens that would be critical to identify the evolution of the clonal architecture of the disease during therapy. The prognostic significance of mutational profiling in MM also remains largely unknown, with only a few genes thought to have prognostic significance. Furthermore, it should be recognised that the prognostic impact of certain mutations has been based on studies profiling material sourced from BM aspirates and therefore may not have been representative of the entire tumour genome. This shortfall could be potentially overcome using CTC and/or ctDNA as an adjunct to BM biopsy for mutational analysis and consequently, for prognostic profiling.
