**1. Introduction**

#### **1.1. Multiple myeloma**

Multiple Myeloma (MM) is a multi-focal genetically heterogeneous clonal plasma cell (PC) malignancy that at diagnosis is present at multiple intra-medullary sites within the bone marrow (BM, **Figure 1**). It is the second most common haematological malignancy after lymphomas [1]. MM is preceded by a usually unrecognised asymptomatic clonal PC disorder

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exclusively confined to the BM called monoclonal gammopathy of undetermined significance (MGUS) [2, 3]. During disease progression the PCs evolve the capacity to grow independently of the BM milieu and thus proliferate outside of the BM, manifesting as extramedullary (EM) MM and/or plasma cell leukaemia (PCL), with published observations showing that PCL represents a more genetically abnormal sub-clone that evolves from the original intra-medullary PC population [4]. The incidence of EM soft-tissue plasmacytomas in newly diagnosed (ND) patients ranges from 7 to 18% with an incidence of almost 20% at the time of relapse [5, 6]. The diagnosis and monitoring of MM relies on sequential BM biopsies and the quantitation of biomarkers of disease burden in the blood and/or urine - clonal immunoglobulin (paraprotein, PP) and/or isotype restricted free-light chains (Serum Free light chains, SFLC or Bence Jones Proteinuria) and in 15–20% of patients the MM cells secrete only SFLC, so called light chain MM [7]. Notably, the routine biomarkers of tumour burden are not informative in subsets of patients with oligo-secretory (OS)-MM or non-secretory (NS)-MM, which are subsets of MM that have low or no measureable levels of PP (<10 g/L) and/or SFLC (<100 mg/L), respectively, and constitute about 10% of MM patients at diagnosis (Australian National Myeloma and Related Diseases Registry, [8]). Moreover, patients with secretory MM at the time of initial diagnosis can transform to NS-MM or OS-MM at the time of relapse and patients with advanced disease are significantly more likely to have OS-MM disease compared to ND patients [8]. These low or immeasurable levels of PP prevent a patient's disease from

being monitored using traditional blood and urine tests and as such OS-MM and NS-MM patients are typically ineligible to participate in clinical trials of newer anti-MM therapeutics. Conventionally, therefore, treatment response in these patients is determined via sequential BM biopsies, where available, and whole-body PET/CT or MRI represent the only accepted non-invasive modality for following response in NS-MM. Therefore, despite well-established diagnostic and monitoring modalities, there remains a critical need to address specific subsets

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The treatment of MM has witnessed significant progress with the introduction of proteasome inhibitors (PI) and immunomodulatory agents (IMID), however, the disease remains incurable. MM cells acquire resistance to systemic therapies through the accumulation of mutations that are often not present during the initial stages of the disease [9] with this genetic evolution providing the more resistant clones with a growth and survival advantage (**Figure 1**, [10]). Mutational characterisation of MM utilising whole genome sequencing (WGS) or whole exome sequencing (WES) of single-site BM biopsies predominantly of ND patients have indicated that the MM mutational spectrum is both complex and heterogeneous with recurring mutations in KRAS, NRAS, TP53, BRAF, IRF4, MAX, ATM, ATR, CCND1, CYLD, DIS3, BRAF, FGFR3, RB1, HIST1H1E, EGR1, TRAG3, FAM46C and LTB [11–14]. The MM mutational landscape is dominated by RAS-MAPK pathway mutations (45–50%) and while these mutations can be targeted therapeutically, the biological implication of the presence of these mutations remains controversial. Chng WJ and co-authors observed that the presence of KRAS mutations, but not NRAS mutations, was associated with inferior overall survival (OS) and a more aggressive disease phenotype [15]. However, it has been recently demonstrated that NRAS mutations may confer reduced sensitivity to bortezomib but not dexamethasone, while the presence of KRAS mutations does not appear to correlate with drug sensitivity [16]. Conversely, more recent studies have concluded that harbouring RAS mutations has no significant impact on disease outcome [13, 14]. In contrast, available data suggests that the presence of mutations involving the DNA damage response genes (TP53, ATM and ATR) is associated with a negative impact on survival [14]. Recently, mutational analysis of PCs from 33 MGUS patients indicated that while the number of somatic mutations was significantly lower when compared to MM the spectrum of mutations mirrored that of MM with KRAS, NRAS, DIS3, HIST1H1E, EGR1 and LTB mutations being observed [17]. Importantly, mutations in the DNA damage response genes were not found, indicating that these are secondary drivers accounting for disease progression and drug resistance. Notably, one patient harboured both KRAS and NRAS mutations highlighting that diversification and heterogeneity in this pathway may exist before the development of symptomatic MM [17]. To date the mutational characterisation of MM has utilised single-site BM biopsies, however, it is now increasingly recognised that such an approach may fail to capture the spatial and temporal genetic heterogeneity of this multi-focal disease, self-evidently with EM MM, but also, based on emerging data, in patients with 'typical MM' manifesting intra-clonal heterogeneity [11, 18–20]. Moreover, both spatial and temporal genetic heterogeneity are now recognised as adding to the genetic complexity of the disease as it evolves [21]. This is exemplified by recent observations from comparisons of BM and targeted biopsies of EM disease that sub-clones absent from the BM may be present at EM sites and critically, that these disease foci may respond differentially to treatment (**Figure 2**, [9, 22]). Therefore, despite the pivotal BM-based studies that currently inform our understanding of

of patients where these conventional markers are inadequate.

**Figure 1.** Multiple myeloma (MM) is a multi-focal malignancy manifesting at several sites black within the BM at diagnosis and in EM tissues in more advanced stages. (A) A PET image of a patient with MM is shown here, demonstrating disease foci at multiple sites (shown in the circles) with the potential that the tumour genome at these individual sites may demonstrate clonal heterogeneity. (B) Representation of the potential changes in clonal and sub-clonal fractions (represented in the different sized circles) are shown with the increasing spatial heterogeneity thought to be present during disease progression. Primary translocations of the IgH gene locus and chromosomal aneuploidies occur during MGUS and chromosomal abnormalities along with primary and secondary driver mutations emerge during MM progression.

being monitored using traditional blood and urine tests and as such OS-MM and NS-MM patients are typically ineligible to participate in clinical trials of newer anti-MM therapeutics. Conventionally, therefore, treatment response in these patients is determined via sequential BM biopsies, where available, and whole-body PET/CT or MRI represent the only accepted non-invasive modality for following response in NS-MM. Therefore, despite well-established diagnostic and monitoring modalities, there remains a critical need to address specific subsets of patients where these conventional markers are inadequate.

exclusively confined to the BM called monoclonal gammopathy of undetermined significance (MGUS) [2, 3]. During disease progression the PCs evolve the capacity to grow independently of the BM milieu and thus proliferate outside of the BM, manifesting as extramedullary (EM) MM and/or plasma cell leukaemia (PCL), with published observations showing that PCL represents a more genetically abnormal sub-clone that evolves from the original intra-medullary PC population [4]. The incidence of EM soft-tissue plasmacytomas in newly diagnosed (ND) patients ranges from 7 to 18% with an incidence of almost 20% at the time of relapse [5, 6]. The diagnosis and monitoring of MM relies on sequential BM biopsies and the quantitation of biomarkers of disease burden in the blood and/or urine - clonal immunoglobulin (paraprotein, PP) and/or isotype restricted free-light chains (Serum Free light chains, SFLC or Bence Jones Proteinuria) and in 15–20% of patients the MM cells secrete only SFLC, so called light chain MM [7]. Notably, the routine biomarkers of tumour burden are not informative in subsets of patients with oligo-secretory (OS)-MM or non-secretory (NS)-MM, which are subsets of MM that have low or no measureable levels of PP (<10 g/L) and/or SFLC (<100 mg/L), respectively, and constitute about 10% of MM patients at diagnosis (Australian National Myeloma and Related Diseases Registry, [8]). Moreover, patients with secretory MM at the time of initial diagnosis can transform to NS-MM or OS-MM at the time of relapse and patients with advanced disease are significantly more likely to have OS-MM disease compared to ND patients [8]. These low or immeasurable levels of PP prevent a patient's disease from

74 Hematology - Latest Research and Clinical Advances

**Figure 1.** Multiple myeloma (MM) is a multi-focal malignancy manifesting at several sites black within the BM at diagnosis and in EM tissues in more advanced stages. (A) A PET image of a patient with MM is shown here, demonstrating disease foci at multiple sites (shown in the circles) with the potential that the tumour genome at these individual sites may demonstrate clonal heterogeneity. (B) Representation of the potential changes in clonal and sub-clonal fractions (represented in the different sized circles) are shown with the increasing spatial heterogeneity thought to be present during disease progression. Primary translocations of the IgH gene locus and chromosomal aneuploidies occur during MGUS and chromosomal abnormalities along with primary and secondary driver mutations emerge during MM progression.

The treatment of MM has witnessed significant progress with the introduction of proteasome inhibitors (PI) and immunomodulatory agents (IMID), however, the disease remains incurable. MM cells acquire resistance to systemic therapies through the accumulation of mutations that are often not present during the initial stages of the disease [9] with this genetic evolution providing the more resistant clones with a growth and survival advantage (**Figure 1**, [10]). Mutational characterisation of MM utilising whole genome sequencing (WGS) or whole exome sequencing (WES) of single-site BM biopsies predominantly of ND patients have indicated that the MM mutational spectrum is both complex and heterogeneous with recurring mutations in KRAS, NRAS, TP53, BRAF, IRF4, MAX, ATM, ATR, CCND1, CYLD, DIS3, BRAF, FGFR3, RB1, HIST1H1E, EGR1, TRAG3, FAM46C and LTB [11–14]. The MM mutational landscape is dominated by RAS-MAPK pathway mutations (45–50%) and while these mutations can be targeted therapeutically, the biological implication of the presence of these mutations remains controversial. Chng WJ and co-authors observed that the presence of KRAS mutations, but not NRAS mutations, was associated with inferior overall survival (OS) and a more aggressive disease phenotype [15]. However, it has been recently demonstrated that NRAS mutations may confer reduced sensitivity to bortezomib but not dexamethasone, while the presence of KRAS mutations does not appear to correlate with drug sensitivity [16]. Conversely, more recent studies have concluded that harbouring RAS mutations has no significant impact on disease outcome [13, 14]. In contrast, available data suggests that the presence of mutations involving the DNA damage response genes (TP53, ATM and ATR) is associated with a negative impact on survival [14]. Recently, mutational analysis of PCs from 33 MGUS patients indicated that while the number of somatic mutations was significantly lower when compared to MM the spectrum of mutations mirrored that of MM with KRAS, NRAS, DIS3, HIST1H1E, EGR1 and LTB mutations being observed [17]. Importantly, mutations in the DNA damage response genes were not found, indicating that these are secondary drivers accounting for disease progression and drug resistance. Notably, one patient harboured both KRAS and NRAS mutations highlighting that diversification and heterogeneity in this pathway may exist before the development of symptomatic MM [17]. To date the mutational characterisation of MM has utilised single-site BM biopsies, however, it is now increasingly recognised that such an approach may fail to capture the spatial and temporal genetic heterogeneity of this multi-focal disease, self-evidently with EM MM, but also, based on emerging data, in patients with 'typical MM' manifesting intra-clonal heterogeneity [11, 18–20]. Moreover, both spatial and temporal genetic heterogeneity are now recognised as adding to the genetic complexity of the disease as it evolves [21]. This is exemplified by recent observations from comparisons of BM and targeted biopsies of EM disease that sub-clones absent from the BM may be present at EM sites and critically, that these disease foci may respond differentially to treatment (**Figure 2**, [9, 22]). Therefore, despite the pivotal BM-based studies that currently inform our understanding of

*1.2.1. Circulating tumour cells (CTC)*

ease activity than SFLC ratios [30].

*1.2.3. Cell-free DNA*

*1.2.2. Circulating cell-free nucleic acids (CFNA)*

CTC are intact tumour cells that disengage from tumour sites thus entering the circulation and are a recognised feature of metastatic cancers [23–25]. Historically, CTC were considered to be present only in a sub-set of ND, untreated or end-stage MM patients. Now with the advent of highly sensitive next-generation (NG) flow cytometry techniques the detection of CTC in pre-malignant MGUS and at different stages in MM has become feasible [26–28] and CTC can be detected in the peripheral blood of a substantial proportion of MM patients evaluated in this fashion [29, 30]. Importantly, the numbers of CTC in the peripheral blood in MM patients with active disease is known to be significantly higher than in patients with inactive disease, with higher numbers of CTC being a risk factor for progression to symptomatic MM for patients with either MGUS or smouldering MM (SMM) [29–33]. The number of CTC in ND and RR patients is also known to be an independent prognostic factor for survival [34–37]. Finally, the absolute numbers of CTC was found in one study to be a better predictor of dis-

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CFNA, refers to cfDNA and extracellular RNA (exRNA - includes mRNA and miRNA), and are being widely assessed in an ongoing fashion for their potential utility as blood biomarkers for cancer diagnostics. CFNA are derived predominantly through necrosis, spontaneous or chemotherapy induced apoptosis and active cellular release [38, 39]. Given that CFNA is also present at higher levels in certain physiological conditions and clinical scenarios (reviewed in [40]), it is critical to determine if the nucleic acids released are tumour-specific in cancer patients. In this section the different types of CFNA and their potential clinical utility will be discussed.

The presence of CFNA in body fluids was first described by Mandel and Metais in 1948 [41]. One of the earliest observations in relation to cfDNA was that patients with malignant disease had higher levels of cfDNA in their serum than normal individuals, and that patients with persistently high levels of cfDNA demonstrated a lack of treatment response [42–44]. Mutated RAS fragments and microsatellite alterations in cfDNA were later observed in cancer patients and critically these alterations were not detected in paired tissue biopsies, highlighting the concept, very early-on, that isolated tissue biopsies may not represent the optimal material for defining the tumour genome [45, 46]. The development and utilisation of NGS technologies, WGS and/ or WES of cfDNA containing tumour-derived DNA has identified mutations, tumour-derived chromosomal aberrations and gene rearrangements associated with acquired resistance to therapy without the need to perform sequential tumour biopsies [47–51]. Furthermore, it is evident that secondary mutations are more readily detectable in the plasma than via re-biopsy of primary tumours validating the utility of plasma-based analysis for the characterisation of potentially targetable oncogenes and the identification of resistance-associated mutations that are acquired during disease progression, thus informing therapeutic changes [48, 52, 53]. Available evidence would also suggest that cfDNA may be more representative of the entire tumour genome than the information derived from single BM or nodal/tumour biopsies, as

**Figure 2.** Different sites in a MM patient may respond differentially to treatment. PET image of a MM patient receiving thalidomide therapy taken 2 months apart showing regression of disease at one site (pelvis), while tumour growth is observed at another site (humerus).

the mutational spectrum in MM, the evident shortcomings of these approaches necessitates the adoption of newer and more novel strategies to enable the more comprehensive genomic characterisation of the disease.
