**3. Cell-free DNA**

Cell-free DNA are short fragments of DNA not associated with a cell and found in PB and other body fluids, such as urine, saliva, breast milk and others [33–35]. The term cfDNA is a general term that includes circulating DNA of both healthy and tumor origin. As circulating tumor DNA (ctDNA) fragments represent only a fraction of total cfDNA, it is necessary to distinguish the origin of fragments during analysis. Physiological levels of cfDNA in PB of healthy individuals are generally low (10–100 ng/ml). This changes, however, in case of various pathological events. Elevated levels of cfDNA were described in patients with inflammation, trauma, sepsis, stroke or heart attack [36–38], but the highest levels of cfDNA were found in cancer patients, where they reached up to 1000 ng/ml [39, 40]. These findings suggest a correlation between levels of cfDNA and tumor burden. Nevertheless, cfDNA levels were not found to be cancer-specific. Stability of cfDNA is variable, ranging from 15 minutes to 2.5 hours; therefore, the amount of cfDNA cannot be used as a diagnostic marker [41].

In the field of MM research, only a small number of cfDNA studies have been published so far. The first pilot study was published by Sata et al. In 2015 [23] they compared ASO-PCR data from peripheral blood mononuclear cells (PBMC), BM mononuclear cells (BMMC), CD20 + CD38− B-cell population in BM and serum cfDNA. Even though the study was quite small and only 20 patients (out of 30 enrolled) were quantifiable, it provided interesting results suggesting further studies and validation are needed. A strong correlation between BMMC and PBMC was found, suggesting circulation of clonogenic PC in PB; PBMC also negatively correlated with treatment as ASO-PCR data from those cells always decreased after treatment. These results suggest a possibility to use PBMC instead of BMMC in monitoring of MRD in MM patients. In addition, DNA sequences found in cfDNA were identical to those found in BM cells in 18/20 cases at diagnosis and 16/20 cases of follow-up samples, while levels of cfDNA remained mostly stable during the course of therapy. Based on these results, the authors assumed that detection of tumor V(D)J rearrangement in cfDNA can reflect presence and persistence of MM clones in patients. However, because of insufficient number of patients who reached complete remission (CR), the potential of cfDNA analysis for MRD monitoring remained unclear [46].

Liquid Biopsies in Multiple Myeloma http://dx.doi.org/10.5772/intechopen.78630 71

In 2017, three important studies on this topic were published [20, 47, 48]. The first by Kis et al. compared cfDNA analysis to BM analysis regarding molecular profiling of disease. This study screened 64 cfDNA samples from 53 MM patients for sequences of all protein-coding exons of *KRAS*, *NRAS*, *BRAF*, *EGFR* and *PIK3CA* genes. This method allowed for detection of tumorrelated fragment of cfDNA at significantly low allele frequencies (0.25%). In 48 cfDNA samples, matching BM data were available. The analysis detected 49/51 (96%) of somatic mutations in cfDNA that were also found in BM; importantly, four additional mutations not detected in BM samples were found in cfDNA (>98% specificity). There were two mutations missed by sequencing of cfDNA samples that were detected during validation by ddPCR in BM samples but not in cfDNA. These outcomes emphasize the potential of cfDNA analysis not only for complex molecular profiling but also for detection of subclones not detected in BM aspirates [20]. The second important study, although once again lacking a larger patient cohort, was conducted by Oberle et al. and focused on detection of clonotypic V(D)J rearrangement in circulating MM cells and cfDNA. A cohort of 27 MM patients with various treatment regimens based on bortezomib, lenalidomide and panobinostat was examined. NGS was used for identification and tracking of patient-specific V(D)J rearrangements. The identification of rearrangements was successful in only 23 out of 27 patients, and these patients underwent further screening of blood samples before and after initiation of therapy. Baseline screening detected patient-specific V(D)J rearrangement in 71% of cases in circulating MM cells and in 100% of cases in cfDNA. However, these values decreased in follow-up samples to 40% and 34%, respectively. The results also correlated with remission status of patients—91% of non-responders/progressors and 41% of responders to therapy had evidence of persistent MM in circulating cells or cfDNA. Interestingly, positivity in circulating MM cells and cfDNA associated with each other (P = 0.042) but disagreed in 30% of cases. This suggests that circulating MM cells are not the only source of MM cfDNA and that cfDNA may reflect tumor burden more comprehensively. All these results indicate that V(D)J analysis from PB samples may be used for evaluation of treatment efficacy and possibly even for MRD prediction [48].

However, validation on a larger cohort is necessary.

Cells can release cfDNA either actively, using exosomes [42], or passively by apoptosis and necrosis (**Figure 1**) [43, 44]. In 2016, however, a study by Bronkhorst et al. proposed that apoptosis and necrosis are not the source of cfDNA and that active secretion is primarily used for cfDNA release [45]. This proposal will require further investigation as it suggests a more active role of cfDNA in cell-to-cell communication. Since cfDNA is released directly from cells, circulating fragments contain the same genetic information as the original cell. In case of cancer cells, this allows detection of cancer-specific genetic and epigenetic aberrations, such as mutations, microsatellite alterations, changes in DNA methylation and others [41].

**Figure 1.** Schematic structure of release of circulating molecules and vesicles into bloodstream. miRNA – Micro RNA, lncRNA – Long non-coding RNA, cfDNA – Cell-free DNA and pre-miRNA – Precursor miRNA.

In the field of MM research, only a small number of cfDNA studies have been published so far. The first pilot study was published by Sata et al. In 2015 [23] they compared ASO-PCR data from peripheral blood mononuclear cells (PBMC), BM mononuclear cells (BMMC), CD20 + CD38− B-cell population in BM and serum cfDNA. Even though the study was quite small and only 20 patients (out of 30 enrolled) were quantifiable, it provided interesting results suggesting further studies and validation are needed. A strong correlation between BMMC and PBMC was found, suggesting circulation of clonogenic PC in PB; PBMC also negatively correlated with treatment as ASO-PCR data from those cells always decreased after treatment. These results suggest a possibility to use PBMC instead of BMMC in monitoring of MRD in MM patients. In addition, DNA sequences found in cfDNA were identical to those found in BM cells in 18/20 cases at diagnosis and 16/20 cases of follow-up samples, while levels of cfDNA remained mostly stable during the course of therapy. Based on these results, the authors assumed that detection of tumor V(D)J rearrangement in cfDNA can reflect presence and persistence of MM clones in patients. However, because of insufficient number of patients who reached complete remission (CR), the potential of cfDNA analysis for MRD monitoring remained unclear [46].

**3. Cell-free DNA**

70 Liquid Biopsy

Cell-free DNA are short fragments of DNA not associated with a cell and found in PB and other body fluids, such as urine, saliva, breast milk and others [33–35]. The term cfDNA is a general term that includes circulating DNA of both healthy and tumor origin. As circulating tumor DNA (ctDNA) fragments represent only a fraction of total cfDNA, it is necessary to distinguish the origin of fragments during analysis. Physiological levels of cfDNA in PB of healthy individuals are generally low (10–100 ng/ml). This changes, however, in case of various pathological events. Elevated levels of cfDNA were described in patients with inflammation, trauma, sepsis, stroke or heart attack [36–38], but the highest levels of cfDNA were found in cancer patients, where they reached up to 1000 ng/ml [39, 40]. These findings suggest a correlation between levels of cfDNA and tumor burden. Nevertheless, cfDNA levels were not found to be cancer-specific. Stability of cfDNA is variable, ranging from 15 minutes to

2.5 hours; therefore, the amount of cfDNA cannot be used as a diagnostic marker [41].

as mutations, microsatellite alterations, changes in DNA methylation and others [41].

**Figure 1.** Schematic structure of release of circulating molecules and vesicles into bloodstream. miRNA – Micro RNA,

lncRNA – Long non-coding RNA, cfDNA – Cell-free DNA and pre-miRNA – Precursor miRNA.

Cells can release cfDNA either actively, using exosomes [42], or passively by apoptosis and necrosis (**Figure 1**) [43, 44]. In 2016, however, a study by Bronkhorst et al. proposed that apoptosis and necrosis are not the source of cfDNA and that active secretion is primarily used for cfDNA release [45]. This proposal will require further investigation as it suggests a more active role of cfDNA in cell-to-cell communication. Since cfDNA is released directly from cells, circulating fragments contain the same genetic information as the original cell. In case of cancer cells, this allows detection of cancer-specific genetic and epigenetic aberrations, such

In 2017, three important studies on this topic were published [20, 47, 48]. The first by Kis et al. compared cfDNA analysis to BM analysis regarding molecular profiling of disease. This study screened 64 cfDNA samples from 53 MM patients for sequences of all protein-coding exons of *KRAS*, *NRAS*, *BRAF*, *EGFR* and *PIK3CA* genes. This method allowed for detection of tumorrelated fragment of cfDNA at significantly low allele frequencies (0.25%). In 48 cfDNA samples, matching BM data were available. The analysis detected 49/51 (96%) of somatic mutations in cfDNA that were also found in BM; importantly, four additional mutations not detected in BM samples were found in cfDNA (>98% specificity). There were two mutations missed by sequencing of cfDNA samples that were detected during validation by ddPCR in BM samples but not in cfDNA. These outcomes emphasize the potential of cfDNA analysis not only for complex molecular profiling but also for detection of subclones not detected in BM aspirates [20].

The second important study, although once again lacking a larger patient cohort, was conducted by Oberle et al. and focused on detection of clonotypic V(D)J rearrangement in circulating MM cells and cfDNA. A cohort of 27 MM patients with various treatment regimens based on bortezomib, lenalidomide and panobinostat was examined. NGS was used for identification and tracking of patient-specific V(D)J rearrangements. The identification of rearrangements was successful in only 23 out of 27 patients, and these patients underwent further screening of blood samples before and after initiation of therapy. Baseline screening detected patient-specific V(D)J rearrangement in 71% of cases in circulating MM cells and in 100% of cases in cfDNA. However, these values decreased in follow-up samples to 40% and 34%, respectively. The results also correlated with remission status of patients—91% of non-responders/progressors and 41% of responders to therapy had evidence of persistent MM in circulating cells or cfDNA. Interestingly, positivity in circulating MM cells and cfDNA associated with each other (P = 0.042) but disagreed in 30% of cases. This suggests that circulating MM cells are not the only source of MM cfDNA and that cfDNA may reflect tumor burden more comprehensively. All these results indicate that V(D)J analysis from PB samples may be used for evaluation of treatment efficacy and possibly even for MRD prediction [48]. However, validation on a larger cohort is necessary.

The last study was published by Mithraprabhu et al., and the subject of this study was mutational characterization of MM. Paired DNA samples of BM PC and plasma derived cfDNA were analyzed for the presence of activating mutations of four oncogenes—*KRAS*, *NRAS*, *BRAF* and *TP53* by NGS. In total, 48 MM patients (33 relapsed/refractory and 15 newly diagnosed) and 21 healthy donors (HD) enrolled in the study. Overall, 128 different mutations were detected in MM patients (cfDNA = 31, BM = 59 and both = 38), while none were found in HD. Interestingly, almost a quarter of all found mutations were detected only in cfDNA samples. These findings proved spatial heterogeneity of MM and showed that cfDNA molecules are derived from multiple tumor sites within a patient's body. This was supported also by majority of cfDNA-specific mutations found in relapsed/refractory patients (30 mutations) in contrast to newly diagnosed patients (1 mutation) as they are more prone to have multiple focal lesions. Moreover, sequences of cfDNA were evaluated by ddPCR in seven patients throughout their treatment, and changes in fractional abundance were discovered, reflecting progression of disease. This proof-of-concept study confirmed the presence of mutations only in cfDNA and proved that genetic composition of MM is complex and evolves during progression of disease. In addition, it proposed that cfDNA analysis could be used as an adjunct to standard BM biopsy for disease monitoring to enable obtaining more complex results [47].

Based on the canonical model of miRNA biogenesis, miRNA genes are transcribed by RNA polymerase II or III into primary precursors, stem-loop structures (pri-miRNA) that contain 5′ end cap and polyA on the 3′ end. Pri-miRNA are cleaved in the nucleus by RNAse II enzyme Drosha and Pasha leading to pre-miRNA [52]. Pre-miRNA are exported into cytoplasm by transport protein exportin 5 [53]. In the cytoplasm, the pre-miRNA molecule is processed by the RISC complex that contains RNAse III Dicer and protein Argonaute 2 (Ago2). RISC complex cuts the molecule into 20–23 nt long double-stranded miRNA duplex with 2 nt overhang on 3′ ends [54]. One of the strands is the so-called guide strand and is complementary to the mRNA sequence. The other ('passenger' strand) is degraded. Which one of these strands is degraded is based on the stability of pairing on the 5′ end of the miRNA duplex [55]. Based on the level of miRNA/mRNA complementarity, the target mRNA is either silenced translationally in case of

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non-complete complementarity or degraded in case of 100% complementarity [56, 57].

mRNA lead to failed translational repression or degradation of target mRNA [60].

in disease progression [61].

*4.2.1. Circulating microRNA in monoclonal gammopathies*

**4.2. Circulating miRNA**

MiRNA regulate a large spectrum of physiological and pathological processes including oncogenesis; they can act as oncogenes or tumor suppressors. Several mechanisms of miRNA role in tumorigenesis have been described: increased expression levels, amplification, epigenetic silencing or loss of miRNA gene that regulates expression of a tumor suppressor gene [58]. On the other hand, deletion and epigenetic silencing of miRNA gene expression that silences oncogene expression have been described as well [59]. Moreover, mutations in target sequences of

In a pilot study of miRNA expression in malignant transformation of PC, increased expression of miR-181a/b, cluster miR-106b-25 (miR-93, miR-106b, miR 25) and miR-21 in MGUS and MM patients in comparison to healthy donors (HD) was found. Interestingly, MM patients showed increased expression of cluster miR-17-92a suggesting a possible role of this cluster

Essentially, all human body fluids (PB, saliva, urine, breast milk, etc.) contain the so-called circulating miRNA [62]. Circulating miRNA are quite stable and resistant to RNases as they are part of protein (Ago2) or lipoprotein (high-density lipoprotein (HDL)) complexes or they are bound inside exosomes—small transport vesicles [63]. It seems that circulating miRNA are involved in cell-to-cell communication as they are exported outside of cells based on biological stimuli. These molecules can also take part in cell processes, such as communication, proliferation, differentiation and in case of tumors also metastases [64]. Specific profiles of circulating miRNA are diagnostic markers differentiating HD from patients, but they also correlate with progression and staging of the tumor [65, 66]. A major advantage of these molecules as potential biomarkers is their simple structure, easy access and measurability by standard laboratory techniques [64].

In MM, circulating miRNA were first described in 2012. In a study by Jones et al., PB serum samples of MGUS and MM patients were analyzed in comparison to HD. They found that

miR-720, miR-1246 and miR-1308 could serve as potential markers of MG [67].

So far, not many cfDNA studies in MM have been conducted; however, the data are exciting and strongly suggest the future role of cfDNA in MRD monitoring.
