*1.2.4. Extracellular RNA (mRNA and miRNA)*

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

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

**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

Liquid Biopsy in Multiple Myeloma

79

http://dx.doi.org/10.5772/intechopen.72652

provide a more comprehensive profile of the disease in comparison to a single-site BM biopsy.
