**2.2. Biomarkers**

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 accu-

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

rately captured by this type of analysis.

80 Hematology - Latest Research and Clinical Advances

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 impacted upon by both the biological characteristics of the disease and the tumour burden, thereby presenting significant challenges when it comes to identifying correlations with prognosis. Moreover, the discrepancy between these studies highlights the lack of concordance between the analytical methodologies, study populations and treatment regimens employed. A recent publication that provided a systematic review and meta-analysis of 7 circulating miRNA studies in MM established that utilising a combination of miRNAs rather than single miRNAs would be more effective in the diagnosis and prognostic classification of MM [112].

Notably, the study detected V(D)J rearrangement in cfDNA in all patients assessed while rearrangements in CTC were only detectable in 70% of patients, indicating that cfDNA, derived from CTC, BM and EM tissue, may represent a better medium for disease evaluation than CTC alone. Long-term monitoring of recurrently occurring mutations in sequential serum samples from 11 patients over a period of 7 years has also been performed in MM [95]. While the majority of patients assessed demonstrated a correlation between PP and the quantifiable levels of specific mutations in sequential plasma samples, there were clearly some patients where analysis of cfDNA was able to identify impending relapse prior to the emergence of clinical relapse, showing the potentially higher sensitivity of cfDNA analysis for disease monitoring. However, a major limitation of sequentially monitoring an initially identified mutant clone is the possibility a sub-clonal mutations not detectable at diagnosis evolving during relapse or being present at an undetectable level initially and subsequently predominating due to selection pressure. Indeed, this appeared to be evident in one of the patients studied, where the initial detection of a BRAF V600E mutation was not maintained despite the continued serologically persisting disease [95]. The evolution of the tumour genome during therapy was also clearly demonstrated in our study, in which 7 patients were monitored sequentially [55]. One patient in particular demonstrated the potential complexities presented by genomic disease evolution. Initially the patient manifested both TP53 R273H and NRAS G13R mutated clones that were responsive to therapy with lenalidomide and dexamethasone (Rd). Subsequently a rapid increase in SFLC consistent with light-chain escape was coincident with the emergence of two new KRAS clones, KRAS G12A and KRAS G12 V. The clonal fraction of both KRAS clones reduced with a switch to Ixazomib-cyclophosphamide-dexamethasone (ICd) therapy coinciding with a serological response. However, the NRAS G13R clone that was responsive to Rd. progressed on ICd in contrast to the TP53 R273H clone that continued to respond,

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**Figure 4.** Line graph represents the clonal fraction of 4 mutant clones (left Y-axis) and lambda LC (right Y-axis) in sequential PL of relapsed patients collected at months 1, 13 and 24 during therapy. Patient relapsed on revlimid and dexamethasone with increase in levels of two mutant clones KRAS G12 V and KRAS G12A at month 13 coinciding with lambda LC, however, TP53 R273H and NRAS G13R were found to decrease. A switch to Ixazomib, cyclophosphamide and dexamethasone (cd) at month 13 decreased levels of KRAS G12A and KRAS G12 V with increasing levels of NRAS

G13R suggesting differential response of mutant clones to treatment. Image reproduced from [55].

#### **2.3. Therapeutic monitoring**

Longitudinal and dynamic monitoring of CTC and CFNA could provide an avenue for detecting loss of response in patients before the emergence of clinical relapse. This could be particularly useful in patients with minimal residual disease (MRD) or when conventional markers are inadequate. MM treatment has witnessed significant progress with the utilisation of novel therapies including PI and IMID with approximately 75% of patients achieving a near-complete response (CR) or CR with front-line therapy. In this context the attribution of MRD negativity has emerged as a paradigm that may be critical in informing treatment decisions. While MM invariably relapses, MRD-negative patients consistently demonstrate more prolonged PFS and may represent a group where therapeutic de-escalation or modification can be safely considered [113, 114]. Consequently, the necessity for highly sensitive assays to detect MRD is critical and this has led to the development and increasing adoption of NGS MRD approaches [115–119]. Currently, MRD status is determined via single site BM biopsy, which clearly has its limitations. Given that 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 and the developments in the detection of CTC in MM, the possibility of assessing the overall numbers/presence of CTC in patients during therapy for MRD assessment is imminent [29–33]. Recently, CTCs were monitored to predict response to in MM patients treated within an open-label, randomised, multicenter phase III clinical trial MM5 of newly diagnosed MM patients with the authors concluding that CTC detection in conjunction with BM for MRD detection [120]. Likewise, cfDNA has been used for dynamic monitoring following surgery/radiotherapy for the detection of residual disease in a number of solid cancers, with persistently high or increasing levels of cfDNA detectable mutations post-surgery/ radiotherapy associated with a lack of treatment response and a much greater risk of relapse [42], while patients with undetectable levels of somatic mutations in cfDNA have demonstrated fewer or no recurrences [121]. The monitoring of MRD using cfDNA in diffuse largecell B Lymphoma has also demonstrated that the persistence of cfDNA mutations identifies patients at risk of recurrence before the emergence of clinical relapse [122]. However, any correlation between the presence or re-emergence of cfDNA detectable mutations and disease progression has not yet been assessed in MM.

Currently, only three studies in MM have utilised liquid biopsy in comparison with conventional markers of disease burden in sequential plasma samples, of which only one study has compared CTC and ctDNA for therapeutic monitoring. All three studies utilised different approaches. Oberle and colleagues assessed clonotypic V(D)J rearrangement in CTC and cfDNA in a cohort of 27 MM patients. Overall, an association between both the presence of cfDNA and CTC V(D)J detectable rearrangements with response was demonstrated [94]. Notably, the study detected V(D)J rearrangement in cfDNA in all patients assessed while rearrangements in CTC were only detectable in 70% of patients, indicating that cfDNA, derived from CTC, BM and EM tissue, may represent a better medium for disease evaluation than CTC alone. Long-term monitoring of recurrently occurring mutations in sequential serum samples from 11 patients over a period of 7 years has also been performed in MM [95]. While the majority of patients assessed demonstrated a correlation between PP and the quantifiable levels of specific mutations in sequential plasma samples, there were clearly some patients where analysis of cfDNA was able to identify impending relapse prior to the emergence of clinical relapse, showing the potentially higher sensitivity of cfDNA analysis for disease monitoring. However, a major limitation of sequentially monitoring an initially identified mutant clone is the possibility a sub-clonal mutations not detectable at diagnosis evolving during relapse or being present at an undetectable level initially and subsequently predominating due to selection pressure. Indeed, this appeared to be evident in one of the patients studied, where the initial detection of a BRAF V600E mutation was not maintained despite the continued serologically persisting disease [95]. The evolution of the tumour genome during therapy was also clearly demonstrated in our study, in which 7 patients were monitored sequentially [55]. One patient in particular demonstrated the potential complexities presented by genomic disease evolution. Initially the patient manifested both TP53 R273H and NRAS G13R mutated clones that were responsive to therapy with lenalidomide and dexamethasone (Rd). Subsequently a rapid increase in SFLC consistent with light-chain escape was coincident with the emergence of two new KRAS clones, KRAS G12A and KRAS G12 V. The clonal fraction of both KRAS clones reduced with a switch to Ixazomib-cyclophosphamide-dexamethasone (ICd) therapy coinciding with a serological response. However, the NRAS G13R clone that was responsive to Rd. progressed on ICd in contrast to the TP53 R273H clone that continued to respond,

impacted upon by both the biological characteristics of the disease and the tumour burden, thereby presenting significant challenges when it comes to identifying correlations with prognosis. Moreover, the discrepancy between these studies highlights the lack of concordance between the analytical methodologies, study populations and treatment regimens employed. A recent publication that provided a systematic review and meta-analysis of 7 circulating miRNA studies in MM established that utilising a combination of miRNAs rather than single miRNAs would be more effective in the diagnosis and prognostic classification of MM [112].

Longitudinal and dynamic monitoring of CTC and CFNA could provide an avenue for detecting loss of response in patients before the emergence of clinical relapse. This could be particularly useful in patients with minimal residual disease (MRD) or when conventional markers are inadequate. MM treatment has witnessed significant progress with the utilisation of novel therapies including PI and IMID with approximately 75% of patients achieving a near-complete response (CR) or CR with front-line therapy. In this context the attribution of MRD negativity has emerged as a paradigm that may be critical in informing treatment decisions. While MM invariably relapses, MRD-negative patients consistently demonstrate more prolonged PFS and may represent a group where therapeutic de-escalation or modification can be safely considered [113, 114]. Consequently, the necessity for highly sensitive assays to detect MRD is critical and this has led to the development and increasing adoption of NGS MRD approaches [115–119]. Currently, MRD status is determined via single site BM biopsy, which clearly has its limitations. Given that 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 and the developments in the detection of CTC in MM, the possibility of assessing the overall numbers/presence of CTC in patients during therapy for MRD assessment is imminent [29–33]. Recently, CTCs were monitored to predict response to in MM patients treated within an open-label, randomised, multicenter phase III clinical trial MM5 of newly diagnosed MM patients with the authors concluding that CTC detection in conjunction with BM for MRD detection [120]. Likewise, cfDNA has been used for dynamic monitoring following surgery/radiotherapy for the detection of residual disease in a number of solid cancers, with persistently high or increasing levels of cfDNA detectable mutations post-surgery/ radiotherapy associated with a lack of treatment response and a much greater risk of relapse [42], while patients with undetectable levels of somatic mutations in cfDNA have demonstrated fewer or no recurrences [121]. The monitoring of MRD using cfDNA in diffuse largecell B Lymphoma has also demonstrated that the persistence of cfDNA mutations identifies patients at risk of recurrence before the emergence of clinical relapse [122]. However, any correlation between the presence or re-emergence of cfDNA detectable mutations and disease

Currently, only three studies in MM have utilised liquid biopsy in comparison with conventional markers of disease burden in sequential plasma samples, of which only one study has compared CTC and ctDNA for therapeutic monitoring. All three studies utilised different approaches. Oberle and colleagues assessed clonotypic V(D)J rearrangement in CTC and cfDNA in a cohort of 27 MM patients. Overall, an association between both the presence of cfDNA and CTC V(D)J detectable rearrangements with response was demonstrated [94].

**2.3. Therapeutic monitoring**

82 Hematology - Latest Research and Clinical Advances

progression has not yet been assessed in MM.

**Figure 4.** Line graph represents the clonal fraction of 4 mutant clones (left Y-axis) and lambda LC (right Y-axis) in sequential PL of relapsed patients collected at months 1, 13 and 24 during therapy. Patient relapsed on revlimid and dexamethasone with increase in levels of two mutant clones KRAS G12 V and KRAS G12A at month 13 coinciding with lambda LC, however, TP53 R273H and NRAS G13R were found to decrease. A switch to Ixazomib, cyclophosphamide and dexamethasone (cd) at month 13 decreased levels of KRAS G12A and KRAS G12 V with increasing levels of NRAS G13R suggesting differential response of mutant clones to treatment. Image reproduced from [55].

highlighting the differential responses of the 4 mutant clones to two different lines of therapy (**Figure 4**). These observations signify that comprehensive mutational analysis in sequential plasma is critical to define therapeutic response in a timely manner. Plasma ctDNA analysis may also represent a novel and informative strategy for disease monitoring in OS-MM and NS-MM patients as demonstrated in our recent publication [55]. Sequential plasma ctDNA analysis in a ND NS-MM patient over a period of 19 months showed that relapsed disease was associated with the reappearance of mutant KRAS G12 V and KRAS G12D clones that had been present at diagnosis in the BM and the emergence of two new clones, NRAS G13D and NRAS Q61K, with the former showing refractoriness to both Thalidomide – dexamethasone, cyclophosphamide, etoposide and cisplatin (T-DCEP) and re-treatment with bortezomib (velcade) – cyclophosphamide – dexamethasone (VCD) and persisting until the patient died shortly thereafter from progressive disease (**Figure 5**). Interestingly the BM biopsy at month 19 showed apparent reduction in disease burden coincident with reintroduction of VCD but droplet digital PCR (ddPCR) of plasma ctDNA showed an increasing clonal fraction of the NRAS G13D clone consistent with VCD-refractory disease distant to the site of BM biopsy. This first-time observation indicates that ctDNA represents a readily accessible non-invasive biomarker of disease burden that may be superior to BM biopsy for monitoring treatment response in NS-MM. Overall, all three studies demonstrated the potential for ctDNA to be used in monitoring MM patients. Clearly, there were limitations due to small sample sizes, lack of homogenous patient treatment and the critical need to be more comprehensive in mutational identification to address tumour genome evolution. Future studies should therefore incorporate sequential cfDNA assessment adopting NGS-based approaches, at diagnosis and subsequent relapses, to quantify previously detected and emergent clones, in larger annotated sample sets, thus enabling the comprehensive tracking of mutations and

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An increasing number of publications have proven the utility of liquid biopsy for cancer screening, diagnosis and disease monitoring. However, for this paradigm to be more widely incorporated into the clinical setting a standardised approach for both the pre-analytical processing and the analytical platforms utilised is necessary. Identification and isolation of MM CTC has been established through the utilisation of ultra-sensitive NGS multicolour flow cytometry techniques [26–28]. Lohr et al. devised a combination methodology that allowed for isolation of CTC using the standard markers (CD138+ CD38+ CD56 variable, CD45 low) and serial dilutions to single cells through fluorescence microscopy to improve the detection of CTC [27]. Currently, these are the only described methodologies for isolation of CTC and

CFNA analysis relies on the standardisation of collection, processing and optimal storage of cfDNA and exRNA, which can now be streamlined with commercially available specialised collection tubes that can stabilise blood at room temperature and preserve the white blood cells to avoid rupture and therefore contamination with cellular DNA in the plasma (Streck, PAXgene, Roche, NORGEN BIOTEK, etc.). Isolation of genomic DNA or RNA from both CTC and CFNA, to a large extent, has also been simplified through the use of commercially available specific isolation kits, although the performance of each of these kits in comparison to other conventional methods is controversial [123]. The choice of the NGS methodologies to address specific questions is however, more complicated and is confounded by the type of nucleic acid being analysed, assay sensitivity and specificity and the heterogeneous content of tumour-derived nucleic acids. A substantial technical challenge to overcome in optimising cfDNA analysis is the level of sensitivity of the various NGS methodologies, as cfDNA may contain low-frequency mutant alleles that might not be readily detectable in WES and targeted amplicon sequencing, which have a sensitivity between 1 and 5%. This is being addressed with the utilisation of unique molecular indices that provide further resolution [48, 124] and such approaches are currently offered by a number of commercial companies

Analysis of exRNA is more complicated as a technology-of-choice needs to be determined for large-scale identification of potential biomarkers. Additionally, the validation and diagnostic implication of circulating RNA sub-types currently relies on 'internal controls' which are often not suited for this type of analysis. This could be addressed using absolute quantification methods, a plausible approach utilising ddPCR, which provides a more robust approach

For CFNA and CTC, unfortunately, to date, few of the described methodologies are as yet validated or standardised, complicating generalisability and inter-study comparisons. Overall, the field would benefit from establishment of certain guidelines for each aspect of sample

by quantitating targets per unit in serum or plasma, as done in cfDNA studies.

assessment of disease burden with correlation to conventional serum biomarkers.

more studies are required to standardise the methodologies described.

**3. Methodologies and challenges**

(Rubicon genomics, QIAGEN, Agilent).

**Figure 5.** Line graph represents the clonal fraction of mutant clones by ddPCR in a non-secretory patient, patient #2. PL was collected at 1, 3, 13, 17 and 19 months post- diagnosis. The proportion of BM MM cells is shown with an increasing clonal fraction of 4 clones coinciding with BM relapse at month 13, only 9 months post-autologous stem cell transplantations (ASCT). At month 19 a BM response to VCD was evident but with an increasing abundance of the NRAS G13D clone. The patient succumbed to refractory progressive disease shortly afterwards. Image reproduced from [55].

at diagnosis and subsequent relapses, to quantify previously detected and emergent clones, in larger annotated sample sets, thus enabling the comprehensive tracking of mutations and assessment of disease burden with correlation to conventional serum biomarkers.
