**3. Methodologies and challenges**

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,

84 Hematology - Latest Research and Clinical Advances

**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].

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 more studies are required to standardise the methodologies described.

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 (Rubicon genomics, QIAGEN, Agilent).

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

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 analysis to allow for comparison of studies of similar kind. These limitations not withstanding, the analysis of ctDNA has gained significant momentum is the past couple of years, with numerous commercial companies offering 'liquid biopsy' testing. Such analysis is now frequently integrated into clinical trials and plasma DNA EGFR mutation testing for non-small cell lung cancer has recently been approved by the FDA [125–127]. Further commercialisation of these 'liquid biopsies' as diagnostics is rapidly evolving, but currently is largely limited to informing treatment choices in late stage cancers.

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