Extending circulating tumour DNA analysis to ultra-low abundance

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Extending circulating tumour DNA analysis to ultralow abundance mutations: techniques and challenges Andrew Edwin Rodda, Bradyn Jared Parker, Andrew Spencer, and Simon Robert Corrie ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00953 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 16, 2018

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Extending circulating tumour DNA analysis to ultra-low abundance mutations: techniques and challenges 1,2,†

Andrew E. Rodda

1,†

2,3

2,4

*, Bradyn J. Parker , Andrew Spencer , Simon R. Corrie

1

Department of Materials Science and Engineering, Monash University, Clayton, Victoria, 3800, Australia

2

Monash Institute of Medical Engineering, Monash University, Clayton, Victoria, 3800, Australia

3

Myeloma Research Group, Australian Center for Blood Diseases, Monash University, Melbourne,

Victoria, 3004, Australia; Malignant Haematology & Stem Cell Transplantation Service, Alfred Hospital, Melbourne, Victoria, 3004, Australia 4

Department of Chemical Engineering, Monash University, Clayton, Victoria, 3800, Australia

* To whom correspondence should be addressed. Email: [email protected] † The first 2 authors should be regarded as having contributed equally

ABSTRACT Liquid biopsies that analyse ctDNA hold great promise in the guidance of clinical treatment for various cancers. However, the innate characteristics of ctDNA make it a difficult target: ctDNA is highly fragmented, and found at very low concentrations, both in absolute terms and relative to wildtype species. Clinically-relevant target sequences often differ from the wildtype species by a single DNA base pair. These characteristics make analysing mutant ctDNA a uniquely difficult process. Despite this, techniques have recently emerged for analysing ctDNA, and have been used in pilot studies that showed promising results. These techniques each have various drawbacks, either in their analytical capabilities or practical considerations, which restrict their application to many clinical situations. Many of the most promising potential applications of ctDNA require assay characteristics that are not currently available, and new techniques with these properties could have benefits in companion diagnostics, monitoring response to treatment and early detection. Here we review the current state of the art in ctDNA detection, with critical comparison of the analytical techniques themselves. We also examine the improvements required to expand ctDNA diagnostics to more advanced applications and discuss the most likely pathways for these improvements.

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Challenges and Opportunities for cell-free DNA Analysis ............................................................................ 4 Cell-free DNA structure and sample preparation .......................................................................................... 8 Characteristics of ctDNA ........................................................................................................................... 8 Sample collection, extraction and preparation .......................................................................................... 8 Pre-amplification and pre-enrichment ......................................................................................................... 11 COLD-PCR ............................................................................................................................................. 11 SCODA and DISSECT ........................................................................................................................... 14 Assay chemistries: oligonucleotides and enzymes ..................................................................................... 15 Overview of PCR based techniques ....................................................................................................... 16 ARMS ...................................................................................................................................................... 17 Modified nucleotides ............................................................................................................................... 17 PAP ......................................................................................................................................................... 19 Ligation and other enzymatic assays ..................................................................................................... 20 Digital PCR and BEAMing........................................................................................................................... 21 Sequencing-based techniques .................................................................................................................... 24 Advanced detection systems ...................................................................................................................... 27 MALDI ..................................................................................................................................................... 27 SERS ...................................................................................................................................................... 28 Electrochemical Chips ............................................................................................................................ 28 Fluorescently-coded microparticles ........................................................................................................ 29 Insights ........................................................................................................................................................ 29 Sample Preparation ................................................................................................................................ 29 Sensitivity ................................................................................................................................................ 31 Error suppression ................................................................................................................................... 31 Quantification of single molecules .......................................................................................................... 32 Conclusions ................................................................................................................................................. 34 Acknowledgements ..................................................................................................................................... 34


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Vocabulary:

•

(Analytical) Sensitivity: The lower limit of quantity or concentration of a mutated target species that is required to prevent a false negative reading of that species’ presence.

•

(Analytical) Specificity: The upper limit of quantity or concentration of non-target species (e.g. wildtype DNA) that is required to prevent a false positive reading when the mutated target species is absent. Typically given as the ratio of mutated:wildtype species.

•

Clinical Sensitivity/Specificity: Related to false negative/false positive rates for a biomarker in predicting disease state or other clinical diagnosis.

•

ctDNA: Cell-free DNA that is released from tumourous cells.

•

Multiplexing: The ability of an assay to detect multiple mutations in a sample of DNA without subdividing that sample.

Keywords

•

circulating tumour DNA

•

liquid biopsy

•

cell-free DNA

•

cancer

•

assay development

•

multiplex

•

sensitivity

•

specificity

•

multiplex

•

sample-limited


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CHALLENGES AND OPPORTUNITIES FOR CELL-FREE DNA ANALYSIS Circulating cell-free DNA (cfDNA) is continuously released from dying cells and is of interest for a range of 1

diagnostic applications including prenatal genetic testing (fetal cfDNA), monitoring organ transplant 2

rejection (donor-derived cfDNA), and in cancer diagnostics. Cancer is linked to an accumulation of 3

mutations in oncogenes, tumour suppressor genes or proof reading genes in tumourous cells, and ctDNA released from these cells is a direct marker of their mutational state. Circulating tumour DNA (ctDNA), i.e. cfDNA that originated in primary or metastatic tumour cells, is of great interest as a potential biomarker for several applications in cancer diagnosis and monitoring, as summarised briefly in Table 1. 8

4-

It has a range of analytical advantages over other circulating markers such as CTCs or cancer-linked 9

protein biomarkers. Identification of mutations in ctDNA could allow the likely efficacy of treatments and 10-12

potential for adverse drug responses to be evaluated prior to or during treatment,

and identification of 13-14

mutations in cfDNA following apparently curative treatment is likely to presage a relapse. 4-8

Table 1: Principal potential applications for ctDNA analytics. Application (in order of current most-

Principal advantages of ctDNA over solid tissue biopsy

advanced) Genotyping following cancer detection to guide treatment (complementing or replacing tissue biopsy)

• Non-localised sampling (accounts for spatial heterogeneity and secondary clones) • Doesn’t require invasive surgery • Doesn’t require tumour to be accessible

Monitoring for response to therapy

• Potential earlier detection (months to years) than imaging

and/or emergence of resistance

• Regular, repeated sampling possible

mutations and/or residual disease

• Short half-life of ctDNA on bloodstream allows up-to-date

following treatment

sample (compared to once-off resected tumour analysis) • Able to account for spatial heterogeneity

Screening for undiagnosed cancer

• Non-localised sampling (pre-knowledge of tumour location not required) • Non-invasive sampling, potential for automated sampling • Potential for earlier detection • Direct marker of cells’ genetic state

Obtaining a sample of cfDNA is relatively simple and non-invasive, and samples of ctDNA also have several analytical advantages compared to biopsied tissue. Firstly, there is often spatial heterogeneity in 15

mutation profiles both within tumours and between tumour sites, and broader sampling could allow 16

mutations to be detected that might not be sampled by a localised biopsy of a solid tumour. Secondly, ctDNA may still be present and detectable in the absence of a detectable solid tumour, for example if the tumours are metastatic, too small to be observed, or have recently been surgically removed. Finally,


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5, 17

ctDNA has a half-life in vivo of just a few hours,

and thus offers the option of repeated sampling to

examine temporal heterogeneity in mutation profiles, for example, the emergence of resistance mutations. Initially, ctDNA is most likely to be used as a complementary or substitute test for more traditional tumour biopsies. However, while samples of ctDNA and tissue biopsy DNA often show concordance in their mutation profile,

18

ctDNA is a fundamentally different sample, with the ability to

examine spatial and temporal characteristics of the disease, and emulating the results of a tissue biopsy may not always be the end goal for ctDNA assays. With further time ctDNA diagnostics may expand into applications where tissue biopsies cannot be used, to make best use of ctDNA’s unique characteristics (Table 2). Table 2: Analytical and diagnostic considerations: comparison of ctDNA liquid biopsy with biopsied tissue samples.

Sampling procedure

Sample availability

Number of copies sampled

Solid biopsy

ctDNA liquid biopsy

Painful and invasive, often

Minimal pain, requires a routine blood draw.

requiring specialised

Potential for highly standardised routine

surgery.

sampling.

Requires surgical access to tumour site

Plasma ctDNA may be minimal in some cancers 19

e.g. brain cancers.

Samples of other fluids such

as cerebrospinal fluid could be used instead.

20

High

Low

Higher than in cfDNA

Low

Samples only a single site

Broad sample of entire patient. Can be used to

and cannot account for

analyse small tumours and metastases beyond

spatial heterogeneity.

optical detection or tumours at unknown sites.

Results are specific to that

Results cannot easily be isolated to a particular

site.

location, although this may become possible.

No

Yes

Relative abundance of mutated DNA species (vs wildtype)

Sampling of spatial heterogeneity

Potential for repeated monitoring

21-22

The analysis of mutations in ctDNA is complicated by a range of factors (summarised in Table 3). Healthy patients show baseline levels of wildtype cfDNA in their bloodstream from natural cell turnover, while the death of cancerous cells also leads to increased destruction of non-tumour cells from the tumour 6, 23

microenvironment. Non-mutated DNA thus typically significantly outnumbers ctDNA,

and cross-

detection of those wild-type species is a significant problem. Furthermore, cfDNA is usually present at


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low concentrations, and only a small blood draw is typically made, which limits the number of copies of a mutated allele that can be sampled. Unlocking the full potential of ctDNA-based diagnostics will require sample preparation and assay formats that can account for the low quantities/concentrations of ctDNA in early stage patients or those who have recently had tumours resected. Sensitive detection is further complicated because the samples are often highly fragmented. For many potential applications, it is also vital to be able to accurately detect or quantify a range of mutations simultaneously, due to the previously-mentioned spatial and temporal variations in mutation profile and limited sample size. In these cases, multiplexed assays and sequencing-based techniques are the ideal. These partially overcome issues caused by limited sample, where the requirement to analyse larger quantities of DNA (to ensure that the rare mutations are present in all subsamples) conflicts with the maximum blood draw available from patients. It also lessens the work required to run the tests (and hence also the cost). However, for multiplexing to be effective, issues such as cross-reactivity of reagents must be considered, and assay design can be difficult. Sequencing-based ctDNA analysis techniques have the potential to detect arbitrary and unknown mutations, which is potentially even more powerful than multiplexing, where the mutations to be detected must also be chosen in advance. However, extensive sample preparation is required in some techniques, which can lead to loss of sample and thereby loss of sensitivity, and costs can be higher than for many assays. Effective clinical and pre-clinical analysis of ctDNA will rely on assays that address each of these complications. In this review, we will focus primarily on these problems as they apply to single nucleotide variants (SNVs), the substitution of a single base within a DNA molecule. SNVs have a set of properties that makes their analysis both particularly challenging and potentially highly useful. Each SNV has a welldefined chemical structure, and many individual SNVs can be related directly to clinical decisions and treatment outcomes. However, the minimal chemical difference between the wildtype and SNV-containing species often leads to significant difficulties with cross-reactivity, particularly when the wildtype species is present in large excess. We also consider that identifying well-characterised SNVs is an area where there is significant debate regarding the analytical techniques used; for many other types of mutation, sequencing-based techniques theoretically have a significant advantage as these do not require the structure of the mutation to be known in advance. In this review we aim to evaluate the current state of the art techniques in SNV analysis as applied to low abundance ctDNA, as well as several that could potentially be adapted in the future, while discussing the advantages and disadvantages of these different techniques. We will examine a range of techniques that have been or could be used to partially address the current challenges listed in Table 3, with a focus on early-stage patients and those who have recently undergone treatment, i.e. with low quantities of ctDNA.


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Table 3: Distinguishing challenges in ctDNA diagnostics

Major challenges in ctDNA diagnostics Analytical sensitivity

Low copy number and low concentration of DNA extracted. Sample is heavily fragmented and the fragmentation breakpoints are variable.

Analytical specificity

Up to 10,000-fold, possibly higher, excess of wildtype species

Multiplexing

Many cancer-linked mutations have been identified. Many applications will require screening for many different mutations, e.g. examining the emergence of resistance mutations

Cost and complexity

The best current techniques typically require experts to run them, and many are too expensive for repeated or routine use. For clinical application, standardised methods will be required.

CELL-FREE DNA STRUCTURE AND SAMPLE PREPARATION Characteristics of ctDNA Circulating tumour DNA is typically collected from patient blood, although it can also be found in other bodily fluids.

8, 20, 24-25

It is thought to arise primarily from apoptosis, which is supported by observations of

the size distribution of DNA fragments; these are predominantly around 166 bp in length, often with smaller peaks at integer-multiples (i.e. 332 bp, 498 bp, etc). The pattern is ascribed to the length of DNA 5

bound by a nucleosome. Other observations include larger fragments of cfDNA (~10 kbp) that are likely released from necrotic cells,

23

and smaller fragments (~100 bp or smaller) that are suggested to have

higher proportions of cancer-related mutations. double stranded,

28

26-27

The structure of ctDNA may be either single- or

with dsDNA more commonly reported, and prior to purification it may be bound to 29

proteins or encapsulated within vesicles, depending on its origin.

Several studies have also suggested 26, 30-32

that ctDNA was more highly fragmented than non-mutated cfDNA.

There are also recent studies

which suggest that both fragmentation and epigenetic patterns can vary in ways that are specific to the 21-22

tissue from which the ctDNA originated. recent review article by Thierry et al.

Further details on the structure of cfDNA can be found in a

29

3

4

The concentration of cfDNA isolated from patient blood is typically low (10 -10 copies/ml) in healthy or 33-34

early stage patients,

and higher in late stage patients. As the size of the blood sample taken from a

patient is also limited, there are limits on the number of copies of rare mutations that can be obtained, and these are most stringent for those patients (undiagnosed, early stage, or following treatment) where ctDNA-based diagnostics can potentially have the greatest benefit compared to traditional biopsies. The 19

concentration of ctDNA can also vary significantly between different types of cancer. Circulating DNA


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has a short half-life; the exact mechanism(s) of clearance are still poorly understood, but may involve 28, 35

clearance through the kidneys or liver, or degradation by nucleases.

As most ctDNA fragments are very small, it is necessary to be careful in designing assays: for example, it is commonly advised to keep PCR amplicons to 100 bp or less, as larger amplicons have been shown to 26, 36

result in significantly reduced analytical sensitivity.

Similar precautions should be taken with other

types of assays to account for both the shortened length and variable breakpoints of the sample ctDNA, including when PCR is used for pre-amplification prior to other analyses. The observation that ctDNA is more highly fragmented than wildtype DNA may imply that previous studies may have underestimated the abundance of mutant species relative to wildtype. Accounting for the fragmentation of ctDNA is critically important in selecting an appropriate assay (see also general discussion of Analytical Sensitivity later in the text).

Sample collection, extraction and preparation While the major focus of most studies is on new analytical methods, the preparation of cfDNA samples is of prime importance. Appropriate sample preparation can significantly lower the barriers that must be overcome by the detection step of the assay, especially the requirements for analytical sensitivity and specificity. The issues of concern include: standardising patient treatment to ensure consistent sampling; preventing contamination with genomic DNA from lysed cells; ensuring high yields of rare cfDNAs following extraction; concentrating this cfDNA into low volumes for use in the following assay; and 37-38

ensuring the integrity of these cfDNAs is maintained throughout processing and storage.

Not enough is currently known about the biology of ctDNA release into the bloodstream, and so it is uncertain whether different variables (related to medical treatment, lifestyle or sampling variables) might affect the original ctDNA concentrations in patients’ whole blood.

28

For example, it seems likely that

various cancer treatments could momentarily increase the concentrations of cfDNA in the samples 39

through the death of cancerous (and surrounding) cells. Further work is needed to assess the effects of these variables on ctDNA yield and determine whether any sample bias is introduced. A major aim of most techniques is to avoid cell lysis during sampling and processing, as this releases wild-type DNA and further reduces the relative concentration of mutant species. Cell lysis is often observed during the clotting phase of serum preparation, and for this reason plasma samples are generally considered superior to serum.

37

Care should be taken to avoid other situations where lysis

might occur, by processing samples in a timely manner and avoiding excessive agitation. Some researchers have also recommended a slower initial centrifuge cycle to remove cells gently and avoid mechanical lysis.

37

Samples should not be frozen until they are confirmed to be free of cells. For plasma


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preparation, EDTA was previously recommended over heparin or citrate, particularly if processing is likely 40

to be delayed;

EDTA may also be beneficial for preventing nucleases from degrading DNA in the 41

samples, probably because it can chelate metal ion co-factors. However, various studies have shown increased cfDNA yields (from leukocyte lysis) if samples are stored in EDTA tubes for extended periods 37

(a few hours, depending on storage conditions) before processing.

Proprietary blood collection tubes

(e.g. Streck Cell-Free DNA) have recently become available that appear to outperform EDTA in cfDNA applications, seemingly by preventing cell lysis where blood is to be stored for a length of time before processing. These are now becoming the standard for sample collection, processing for up to 1-2 weeks.

42-44

and may be used to delay

45

Degradation of the cfDNA is also a potential problem. DNA in plasma samples may be more susceptible 37

to freeze-thaw cycles than DNA that has been extracted and purified. Other potential sources of degradation are the oxidation and deamination of G and C residues respectively during sample preparation,

46-47

which in some cases can later lead to a misidentified SNV (e.g. C→T due to deamination

of cytosine into uracil). Studies have investigated the use of the enzymes uracil-DNA glycosylase (UDG) and formamidopyrimidine-DNA glycosylase (Fpg) to excise damaged bases during library preparation for next-generation sequencing,

47-48

and shown that this could improve results during later analysis, although

analytical sensitivity would be reduced due to the excluded strands of DNA. DNA samples are typically prepared from plasma by either using commercial DNA extraction kits based on silica membrane binding of DNA, anion exchange or magnetic beads, or by various customised methods, including solvent-based extraction (e.g. phenol/chloroform) and salting-out procedures. While some studies have shown improved results from the custom methods, these results have not been consistent, and these methods are not likely to find extensive use for clinical applications unless they can be simplified and standardised. Conversely, commercial kits, most often based on silica membranes, are easy to implement in a clinical laboratory with minimal training. However they still sometimes require troubleshooting and some may struggle to collect smaller DNA strands. Studies have compared the performance of different methods with varying results.

33, 37, 49-51

More recently, kits have become available

33

that appear to improve the yield of smaller fragments of DNA; recent findings have suggested that these 26

fragments are most likely to contain cancer-related mutations.

Even so, a significant amount of DNA

may be lost during preparation (Figure 1), being either eluted during rinsing steps or permanently bound to silica membranes. Furthermore, most kits also have upper limitations on the amount of sample input and lower limitations in the final resuspended volume (hence the concentration) of the purified DNA. An ideal kit method would take large volumes of dilute sample and concentrate this significantly during the purification processes, so that the sample can be best utilised in the assay.


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33

Figure 1: Purification of cfDNA from a 5 ml blood draw containing 2500 copies/ml.

Loss of sample must

be avoided or minimised by appropriate choice of extraction method and assay design. Calculations are shown as examples, and some extraction techniques can perform better than shown. There is great potential for further development of microfluidics to separate ctDNA from background contaminants, including wildtype sequences, and potentially to concentrate the ctDNA samples to improve analytical sensitivity. New microfluidic techniques for extracting cfDNA from patient samples, such as the work of Sonnenberg et al. using dielectrophoresis, may in future provide improved yield and a 52-53

simpler and faster alternative to the standard kits.

However, this particular technique was

demonstrated using only low sample volumes, whereas the overarching need for sampling of ultra-rare mutations is to increase sample throughput. Lee et al. have also presented techniques based on nanochips

54

and nanowires

55

fabricated from a conducting polymer, whose oxidation state was switched

to capture and then release the DNA. A potential alternative would be an assay that is able to be used with plasma samples (or even whole blood). This is technically difficult due to low concentrations and the range of chemicals that may be present in unrefined samples, which can interfere with reactions and 25, 56-57

detection systems and reduce ctDNA stability, however it is a current area of research.

PRE-AMPLIFICATION AND PRE-ENRICHMENT Where insufficient template DNA is available, pre-amplification may be used to increase sample concentration, often using PCR-based techniques. If pre-amplification is desired, the researcher should recognise that it may also make accurate quantification of mutations difficult. Amplification biases can change the relative numbers of different species,

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and mutations may be introduced due to

misincorporation by a polymerase, which cannot later be distinguished from true mutations in the original


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sample. Introduced mutations can be suppressed by using higher fidelity polymerases, but not completely eliminated. Outside standard PCR, techniques such as blunt-end ligation-mediated whole genome amplification can 60

be used to amplify small apoptotic and larger necrotic fragments. One recent study appeared to suggest 61

that whole genome amplification could be performed without producing a significant bias.

Barcoding

may also be used prior to amplification for sequencing-based techniques, and statistical methods can then be used to eliminate the effects of introduced mutations (see later discussion on “Sequencing-based techniques” for details of these methods). It has also been shown that overlapping small fragments of DNA can “reconstruct” a longer fragment, producing a PCR amplicon longer than the original template 62

DNA.

However it is uncertain whether this can be applied effectively to cfDNA without introducing

biases. Other techniques can enrich mutant sequences compared to more abundant wildtype sequences. This may make quantification of the original allelic fraction difficult, but can significantly improve the analytical sensitivity and specificity of the detection step. Techniques such as COLD-PCR and SCODA combine ctDNA-specific pre-enrichment with well-established standard detection mechanisms such as sequencing, and are reviewed below.

COLD-PCR 63-64

COLD-PCR uses differences in melting temperature to preferentially amplify mutated sequences.

The

original COLD-PCR protocol achieves this by denaturing the homoduplex DNA (containing both wildtype and mutated strands) and re-annealing strands such that the rare sequences are probabilistically incorporated into heteroduplex pairs. These pairs can be denatured at a lower temperature than a homoduplex, and thus can be copied via PCR while the homoduplex sequences are blocked (Figure 2). Several variants exist including “fast,” ice”

66

63

65

“ice” (Improved and Complete Enrichment),

and “enhanced-

COLD-PCR. Fast-COLD-PCR uses a simpler heating sequence, but is only able to enrich mutations

where there is a decrease in melting temperature between the wildtype and mutated homoduplexes (i.e. the mutation is [C or G] → [A or T]). In ice-COLD-PCR, a blocking oligo, matching the wildtype is hybridised with the DNA following the initial denaturing step. The oligo is terminated with a phosphate blocking group that prevents further extension. The temperature is then increased such that the mutant 65

sequence dissociates from the oligo, while the wildtype sequence remains blocked.

One advantage of the COLD-PCR suite of protocols is their ability to enrich unknown or multiple mutated sequences within a locus, which can then be detected using sequencing. This contrasts with other techniques, which require full foreknowledge of the mutated sequence of interest. However, while COLDPCR can significantly improve the detection limits of rare mutations, it is difficult to accurately quantify


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their abundance.

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As with other PCR-based techniques, amplicons should be kept short to account for

fragmented samples.

Figure 2: Selected steps of two variants of COLD-PCR. Adapted from Milbury et al.

68

However, many of the simpler techniques in this series require strict control over the temperature at which the duplex DNA is dissociated (± 0.3 °C or thereabouts). While this is achievable with many modern PCR thermocyclers, the choice of temperature may require several rounds of optimisation. Further 69

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developments (e.g. temperature-tolerant ice-COLD-PCR, enhanced ice-COLD-PCR ) have reduced the precision required for temperature control. Enhanced ice-COLD-PCR replaces the blocking oligo with an oligo based on locked nucleic acids (LNAs), increasing the difference between the respective Tm values and lessening the strict requirements for temperature control, while temperature-tolerant ice-COLD-PCR


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uses a varied thermocycling program. A multiplexed version of e-ice-COLD-PCR has recently been developed into a commercial product by Transgenomic.

SCODA and DISSECT Synchronous Coefficient of Drag Alteration (SCODA) is a recently development microfluidic DNA concentration and purification technique. Sequence-specific SCODA, as used for SNV analysis, utilises an electrophoresis gel that is functionalized with oligos complementary to the mutated sequences, which is then exposed to a rotating electric field. Repeated hybridization/dehydridisation of the sample DNA with the oligos leads to spatial separation from wildtype strands, unrelated DNA and other molecules such as 70

inhibitors, as well as concentration of the sequences of interest into a small volume (Figure 3). This process leads to enrichment (up to 10,000-fold) and concentration (25,000X) of the mutant species without amplification and the associated potential for artefacts, and can process large volumes of sample.

70

Sequence-specific SCODA has been commercialised (Boreal Genomics “OnTarget”), in a

system which Boreal claims can enrich up to 100 sequences simultaneously. The enriched mutated sequences are then analysed via standard sequencing techniques. Applications of this system to a small number of clinical samples of ctDNA showed the ability to detect low-level mutations, with analytical sensitivity for many mutations reaching 0.001% vs wildtype in some cases (typically quantified using 71-73

synthetic DNA samples).

However, it is currently expensive (approximately $1000/sample), and this

impedes its use in studies analysing large numbers of samples or for routine use in clinical applications.

Figure 3: Separation and concentration of a SNV mutant sequence (tagged red) from wildtype sequences (tagged green) using ssSCODA. Reprinted from from Thompson et al.,

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Creative Commons.

In contrast, the DISSECT technique uses magnetic beads conjugated with the complementary wildtype 74-75

sequence to remove wildtype DNA over several repeated extractions.

As in COLD-PCR (which also

originated in the Makrigiorgos research group) temperature conditions are optimised to increase


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differentiation between mutant and wildtype sequences. In combination with a PNA/LNA PCR detection 4

step, analytical specificity of 1 in 10 was achieved in cancer cell line DNA. This technique does not concentrate mutant DNA as SCODA does, however it requires minimal equipment and enriches for all unknown sequences. It also avoids the use of sequence specific PCR amplification, which may lead to biases or errors; excluding any general pre-amplification performed before the technique itself, in both DISSECT and SCODA, the resulting solution contains original sample molecules only.

ASSAY CHEMISTRIES: OLIGONUCLEOTIDES AND ENZYMES All current non-sequencing systems for detecting SNVs in DNA strands rely on the use of DNA oligonucleotides (oligos) or similar molecules to bind DNA specifically at the mutation site. These may act as primers for the polymerisation chain reaction (PCR), or be used in other schemes involving ligation or extension of the bound oligo. While bases in the target DNA bind in a one-one fashion with a matching mutation-specific oligo, non-specific binding can also occur between the oligo and a wildtype strand, lowering the assay’s analytical specificity. Here we examine several techniques to increase the specificity of these oligos and assays towards the mutant species. Typically, the mutation site is placed in one of two positions, depending on the format of the assay: either in the centre of the oligo, which leads to the largest variation in melting temperature between wildtype and mutant species, or at the 3’ end of the oligo, as primers with mismatches at this end are less likely to be extended by a polymerase during a PCR-based assay. Shorter oligos increase the effect of single-base mismatches, but are more likely to cross-react with other areas of the genome. Several types of further modification, particularly at the 3’ end of the primer, have subsequently been used to increase the specificity of binding and extension (Figure 4). Polymerases with 3’ exonuclease activity cannot be used for some of these assays, as they can rewrite the mismatch on the allele specific primer when it is bound to a wildtype strand.


Page 14 of 48

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Figure 4: Comparison of oligonucleotide design strategies and modifications for PCR-based assays.

Overview of PCR based techniques A range of different quantitative polymerase chain reaction (qPCR) assay designs have been investigated for the detection of SNVs, including (but not limited to) allele-specific amplification (ASA), allele-specific blocking (ASB) and restriction fragment length polymorphism (RFLP). The general techniques are well known,

76

and will not be reviewed in detail here.

There are many potential advantages to qPCR-based tests: they are simple to perform, highly costeffective, and when well-optimised, they can detect single copies of target DNA. Both the Cobas (Roche) and Therascreen (Qiagen) testing kits are based on qPCR, and have recently been approved for use as 77

companion diagnostic tests for ctDNA.

However, the amplification of the target that makes it extremely

sensitive also causes the technique to be prone to contamination. Designing useful primer sets can be challenging, especially for multiplexed assays, and most assays are therefore performed in singleplex. Finally, quantification is dependent on a standard curve which may not perfectly match the experiment, and experiments may therefore lack reproducibility. A range of modifications may be made to reagents (particularly primers and/or blocking oligonucleotides) and assay formats to improve the analytical characteristics of the assays, and we discuss a selection of these below.


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ARMS The Amplification-Refractory Mutation System (ARMS) is a cost-effective ASA technique that is theoretically adaptable to any SNV mutation. ARMS-PCR utilizes a primer with a 3’ mutation specific for the tumorigenic SNVs, on top of which additional point mutations are incorporated 1-3 positions further inwards. These further destabilise binding when there is a 3’ mismatch (wildtype template), but in the event of a 3’ match (mutant template) the destabilisation has a smaller effect, and extension is still relatively efficient. The major advantages of ARMS-PCR include the low cost associated with reagents and primers, as well as the short assay times. However, while there are example guidelines for selecting the extra mismatches

78

, in practice primer design may require several rounds of optimisation. This is

particularly the case if a multiplexed assay is desired. Standard ARMS-PCR can often detect down to 1% SNV mutant in a wildtype DNA background, 80

in some cases can reach 0.1%.

78-79

and

Most recently, combinations of ARMS-style primers with other elements

have produced assays with improved analytical specificity or capabilities: Andersen et al. combined ARMS primers with a blocking oligonucleotide within a multiplexed KRAS G12/13 assay, with 1 in 2050,000 specificity;

36

and Stadler et al. demonstrated detection of 3 SNVs with reported 1 in 200,000

specificity using ARMS primers in combination with a modified polymerase (SNPase). This polymerase is reported to show higher extension specificity for 3’ mutations and lacks 5’ to 3’ exonuclease activity. Here it was used in a pre-amplification step before ARMS-qPCR was continued using a second polymerase 81

with 5`to 3`exonuclease activity and dual-labelled hydrolysis probes.

ARMS-type primers were also

used in combination with gold nanoparticles for surface-enhanced Raman spectroscopy (SERS)-based 82

multiplexing (see later section on “Advanced detection systems”). ARMS-PCR has been used to analyse clinical ctDNA samples,

18

and is the basis for Qiagen’s Therascreen kits for KRAS and EGFR

mutants, which detect between 1-6% mutant allele vs wildtype, depending on the specific mutation.

Modified nucleotides Several types of modified oligonucleotide bases have increased affinities for matching DNA (Figure 5). These probes typically increase the melting temperature for perfectly-matched DNA sequences, while at the same time destabilising pairs with a single mismatch. They are typically more expensive than regular DNA oligos, but this may only represent a small increase in the overall cost of an assay. They are commonly used both in PCR and in other assay formats.


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Figure 5: Modified nucleotides that may increase the interaction specificity of oligonucleotides with mutated DNA. Locked Nucleic Acid (LNA) bases are modified RNA nucleotides, having an additional methylene bridge connecting 4’ C to 2’ O atoms (Figure 5). These bases increase the specificity and strength of binding to a complementary sequence.

83

Typical LNA probes are a mixture of DNA and LNA, with each monomer of

LNA increasing the melting temperature of the probe to a matched sequence. LNA can be used as primers for PCR, as well as other uses: Latorra et al. showed that a 3’ LNA base at the mutation site 84

could be used to increase the specificity of allele-specific PCR amplification.

Pinzani et al. demonstrated

an assay for BRAF V600E that could detect 0.3% mutant allele in patients’ circulating DNA, using an 85

allele-specific LNA hydrolysis probe along with allele-specific DNA primers. An LNA-based blocker is also used in the e-ice-COLD PCR platform.

66

Peptide nucleic acids (PNA) are synthetic oligos with an uncharged polyamide backbone, which does not repel negatively charged target DNA and therefore have an increased overall affinity for matched DNA. PNA cannot be used as a primer as the bases are not recognized by DNA polymerases, nor many other enzymes; instead, PNA probes are often used in assays as clamps, binding to and preventing duplication 86

of a section of template DNA. They may also be used as probes with increased binding specificity . They are typically shorter than DNA oligos due to their higher affinity, which further magnifies the effect of a single-base mismatch. PNA probes have often been used in ctDNA assays to specifically block extension of non-target wildtype DNA whilst a separate allele specific primer elongates the target mutant allele. This approach has been used in a range of studies examining SNV detection in ctDNA and other 86-90

samples, with varied specificities going down to 1 in 10,000 in some cases. 75, 91

developed assays with both LNA primers and PNA blocking combined.


Studies have also

Blocking assays do impair

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direct measurement of the wildtype allele, and so require total or wildtype ctDNA to be measured separately in order to estimate the mutant:wildtype ratio. Other, less commonly used modifications are available that can increase the binding specificity of oligonucleotide interactions. These include minor groove binders

14, 92

or Zip Nucleotide Acids.

93-94

For

both of these examples, small molecules are attached at either the 3’ or 5’ end of the DNA oligo, to stabilise binding of the matched oligo to the target via charge-based or hydrophobic interactions. This improves selectivity against a single mismatched base by allowing smaller DNA oligos to be designed, increasing the effect of the SNV mismatch.

IntPlex The advantages of qPCR can be seen in the work of Thierry et al. in the development of the IntPlex 30, 95-97

system.

This method is based on a well-optimised real-time PCR reaction for KRAS mutations,

where the primers are designed to keep the PCR product particularly short (60-100 bp) to fully utilise the small fragments of ctDNA. The primers themselves are also kept short (16 bp) to maximise the effect of the mismatched base, and no primer modifications are used. A phosphate-blocked DNA oligo is used to prevent extension of wildtype product, achieving specificities of around 1 in 20,000 vs wildtype with single molecule sensitivity.

97

Overall KRAS copy number was quantified in a different region to allow the assay

to determine the relative proportion of mutant allele.

PAP Pyrophosphorolysis-activated polymerization, originally developed by Liu and Sommer,

98-99

is a variant of

PCR that uses a dideoxynucleotide at the 3’ end of the primer to block extension. When the blocking nucleotide is perfectly matched with template DNA, it can be removed via pyrophosphorolysis, allowing subsequent extension and amplification (see Figure 4). The selectivity of removal for matched versus unmatched dideoxynucleotides is particularly high, allowing this method to reportedly detect mutated 4

5

sequences at concentrations of approximately 1 in 10 - 10 compared to wildtype concentrations.

100

Removal of the dideoxynucleotide may be inhibited when a mismatch is present within approximately 16 98

bases of the mutation site.

Most studies are performed using a specialty polymerase which increases

the efficiency of pyrophosphorolysis, most commonly a polymerase bearing a F667Y mutation or equivalent.

98, 101

A modified, bidirectional version of PAP (bi-PAP) utilises allele-specific dideoxy- modifications on both forward and reverse primers.

100

This addresses the potential in regular PAP for the polymerase to

misincorporate a nucleotide during extension of the reverse primer, leading to creation of a strand that bears the mutation and a false-positive signal. Using this method, analytical specificity of 1 mutant allele 7

9

per 10 - 10 wild type alleles have been achieved,

102

as good as any technique yet described and far


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better than required for most ctDNA assays due to limited sample. As the length of the amplified product in bi-PAP assays is generally small (the combined size of the two primers, minus one), it is expected that problems analysing fragmented DNA would be reduced. Bi-PAP has been applied to recent studies of ctDNA from patients with uveal melanoma.

103-104

However, the format of biPAP may be difficult to adapt to

multiplex formats with the same specificity, particularly where multiple SNVs are located in close proximity to each other.

Ligation and other enzymatic assays Ligation based SNV assays have not been used directly for ctDNA analysis to our knowledge, however there are a range of SNV assays that use DNA ligation to detect minority SNVs. The mutation site is typically placed at the site of the 3’ hydroxyl of an oligonucleotide, as ligase fidelity is highest on this side of the nick, and mismatches that disrupt the DNA helical structure to a greater extent (e.g. purine-purine) will be more strongly discriminated against.

105

Commonly, ligation is used to attach mutation specific

oligos to micro- or nanoparticles, which function as the reporter and/or be used to aid multiplexing

106-108

.

Many commercial DNA ligases have insufficient specificity towards SNVs for ctDNA analysis, as the analytical specificity of these assays is often solely reliant on the specificity of the enzymatic reaction. Specificity of the ligases can vary significantly depending on the conditions of the assay (buffer composition, temperature, DNA modifications); assays with SNV specificity down to 0.01% vs wildtype background have been reported.

106, 109

Most ligation-based SNV assays described in the literature have poor analytical sensitivity compared to the requirements for a ctDNA assay, requiring fM or higher concentrations (typically between 10k and several million copies of the mutant, depending on reaction size) of the mutant species. This limits the direct applicability of these assays to low abundance ctDNA samples, however there may be scope for them to be used in combination with pre-amplification. Often this relatively poor sensitivity is because the signal is linearly amplified directly from the template DNA. Recent studies by Shen et al. have used the ligase chain reaction (LCR), an analogue of PCR, to exponentially amplify a small section of SNVcontaining DNA.

107-108

This was combined with a colorimetric gold nanoparticle-based readout. Unlike

many ligation-based assays, amplification for LCR is exponential rather than linear. These assays showed sensitivity that was high enough (20 aM in 100 µl ~ 1200 copies) to suggest that they might be adapted further for studies of rare SNVs in ctDNA, and were able to detect approximately 1 mutant per 1000 wildtype species. The sections being amplified are shorter (30 bp) than typical PCR amplicons, which may also be advantageous for analysis of fragmented ctDNA, potentially allowing the smallest fragments of ctDNA to be captured and thereby improving sensitivity. A range of other enzymes and reactions have also been used for novel ctDNA or related SNV assays. Rather than ligation, cleavage of oligonucleotide probes can also be used for signal generation. A recent


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study found that lambda exonuclease, which typically digests DNA with a 5’ phosphate, could also be used on 5’ FAM-labelled DNA if appropriate mismatches were present, and used this phenomenon to design a TaqMan-style probe assay to analyse SNVs in ctDNA.

110

Good analytical specificity was shown

(0.02%) and the assay required only small input quantities of cfDNA. SNV genotyping can also be performed by variants of the Invader assay, which uses a flap endonuclease to cleave oligonucleotide probes.

111

However, this has not yet been adapted for the specific challenges of ctDNA. Polymerase

reactions other than standard PCR can also be used. The Sequenom UltraSeek platform relies on a 112

polymerase performing a single base extension reaction, rather than PCR. 113

isothermal DNA amplification reactions are also available,

A number of different

and of these techniques rolling-circle

amplification (RCA) has favourable characteristics for some assay designs. Many linked copies are made of the target, and because all copies are taken directly from the original molecule, any polymerase incorporation errors do not get a chance to propagate. RCA has been used as a detection technique by 48, 106, 114-116

itself, as well as in preparation of samples for error-corrected sequencing.

DIGITAL PCR AND BEAMING Digital PCR is based on sample DNA being distributed among many compartments; in its simplest form, the sample is diluted such that most compartments contain either a single copy or no copies of target DNA. Typically, compartmentalisation is achieved using either a microfluidic chip (e.g systems made by Fluidigm and Life Technologies) or the generation of an emulsion of homogenously-sized water-in-oil droplets (Bio-Rad, Raindrop). Following PCR, a fluorescent signal reveals whether each droplet is positive/negative for the target sequence, and quantification is by simple counting of positive droplets (Figure 6).


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Figure 6: Generalised concept of digital PCR: DNA is partitioned into large numbers of reaction wells or droplets. Mutated DNA (red) is then efficiently amplified, while the separation of wildtype DNA (black) into partitions reduces its potential to be cross-amplified. The processes of dilution and template compartmentalisation used in dPCR significantly reduce interference from wildtype DNA and from contaminants. When measuring low abundance SNV-containing sequences using traditional PCR, false positive results can arise from the combined effects of large numbers of wildtype sequences interacting weakly with primers; in dPCR, wildtype sequences are also compartmentalised, and thus these effects cannot combine to mimic the results of a mutant strand of DNA. While each individual compartment behaves as a standard qPCR reaction, the overall effect is to reduce interference from wildtype species. Digital PCR also shows an increased tolerance to enzyme inhibiting substances for similar reasons, 118

of the number of copies present.

117

although these substances may still lead to underestimation

Larger numbers of smaller droplets may be used to further disperse

wildtype sequences or inhibitors. Digital PCR thus boasts exceptional analytical sensitivity and specificity, and the detection of single copies of target DNA at 0.01% is common while down to 0.001% has been 119

reported, although not in ctDNA due to limited total DNA in samples.

120-

One major benefit is that dPCR gives repeatable, absolute quantification of mutations including SNVs; 122

it does not require either reference genes or calibration curves to quantify nucleic acids, as the number

of droplets that display fluorescence following amplification corresponds (via the application of Poisson


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statistics) to the absolute number of copies in the original sample. These characteristics have made dPCR the standard reference technique for analysing ctDNA in clinical samples and for standardising/validating new assay techniques.

13, 19, 72-73, 123

Digital PCR is currently more expensive than

traditional qPCR, particularly for the initial purchase of equipment, but significantly cheaper than next generation sequencing and many other techniques, and validated assay kits are available for a number of cancer-related SNVs. BEAMing (Beads, Emulsions, Amplification and Magnetics), an early implementation of dPCR, also splits the sample into large numbers of droplets. In this case they are formed around a set of magnetic microparticles, again with (theoretically) only one copy of template per droplet. PCR is performed on the emulsion, with primers designed such that the amplicons will be bound to the magnetic beads via biotinstreptavidin linkages. Each magnetic bead now contains a large number of copies derived from a single original, which can be assayed by binding a fluorescent label and performing analysis on a flow 124

cytometer.

With the recent advent of commercial dPCR systems, which have essentially similar

characteristics in a more user-friendly, standardised format, only a small number of studies have used 10, 17, 125-126

BEAMing to examine SNVs in ctDNA.

The technique is currently commercialised by Sysmex.

The major technical limitation of dPCR is that multiplexing of assays has been limited to small numbers of 127

species. Simple colour-based multiplexing is possible,

but limited to 3-4 species, due to the difficulties

in distinguishing fluorescent signals with marginally different wavelengths, and in addition, most commercial dPCR instruments report only 2 channels.

128

Recently, methods capable of detecting multiple

mutations using varied combinations of reporter colour and intensity have been described. These assays were based around optimising the individual PCR reactions so that the endpoint fluorescence for each detected target was different, and hence clusters of droplets with different final intensities could be separately identified. Droplets where combinations of sequences are present can also be identified using these methods, as discussed in a recent review of digital PCR multiplexing technology.

128

Several different approaches can be used to implement this scheme, depending on whether mutantspecific probes or intercalating dyes are used as the PCR reporter chemistry. Studies have examined a 129-130

range of mutations using EvaGreen dye and varying primer concentrations or amplicon sizes.

However the fragmented structure of ctDNA makes the implementation of amplicon size methods unfeasible in their current form, as larger amplicons are unlikely to be amplified. In contrast, mutant specific hydrolysis probes can be added to the reaction at different concentrations (Figure 7A), allowing small amplicon sizes to be used throughout. Hydrolysis probes can also be labelled with different ratios of 131

two (or theoretically more) fluorophores for each species (Figure 7B),

and these have allowed dPCR

multiplexing to be expanded up to 6 species per well. For example, Taly et al. used two concurrent multiplex assays (4- and 5-plex) to examine the well-known family of KRAS G12/13 mutations using this


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hydrolysis probe chemistry,

132

while Turner et al. used dPCR assays of up to 6-plex to track the 14, 133

mutational status of a patient’s ctDNA following treatment for breast cancer.

In cases where precise

identification of the mutation is not required, or where it is considered unlikely that a droplet will contain multiple mutant species, multiple detection events may be mapped onto a single region in the colourspace, allowing an increase in the number of mutations that may be examined concurrently, but at the expense of their precise identification (Figure 7C and 7D).

Figure 7: Multiplexing of dPCR using fluorescence intensity-based coding with two colours. Ch1/Ch2 represent different fluorescence channels. Coloured circles show the expected intensity of droplets containing a single mutant species (the majority), while dotted circles show those with multiple species. (A) Amplitude-based multiplexing, (B) Ratio-based multiplexing, (C) and (D) Non-discriminative multiplexing. Reprinted from Whale et al.

128

under Creative Commons licence.

https://creativecommons.org/licenses/by-nc-nd/4.0/

SEQUENCING-BASED TECHNIQUES Traditional Sanger sequencing has very limited ability to detect mutations within a wild type background; 6

various reports estimate it can detect down to approximately 10 %. In contrast to Sanger sequencing, next generation sequencing (NGS; also known as massively-parallel sequencing) is able to report sequences from individual molecules, allowing statistical methods to be used in analysing the large number of reads that cover any particular region. Standard NGS is relatively error-prone,

134

due to

imperfections in the sample processing steps (such as degradation of the template molecules and DNA polymerase errors), as well as read errors during sequencing, and this makes detection of rare variants difficult. Various modifications to standard NGS methods can be used to separate these errors from true mutations that are present in the sample. Many techniques use barcoding or other techniques to group individual reads together with other copies that come from the same original molecule of template DNA; barcodes (also known as Unique Molecular Identifiers, UMIs) are typically short random sequences of


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DNA that are appended to the original sample molecules before PCR amplification takes place. These are added in such a way that it is vanishingly unlikely for two molecules to have both the same endpoint and the same barcode attached. A consensus sequence can then be formed for each original molecule, independent of other molecules from the same region. While these techniques require redundant reads to be generated for each base, they facilitate the detection of SNV-containing sequences with specificities that are orders of magnitude lower than techniques which do not employ barcoding. The major advantage of NGS-based techniques over all others is the ability to detect arbitrary mutations within a DNA sample, rather than being limited to a finite (and typically small) subset of previously-defined mutations. Sequencing can detect a wide range of sequence variants, including insertions, deletions and rearrangements, as well as SNVs. However, these techniques have several practical limits in their current embodiments; they produce large amounts of data, requires specialist analysis, and are often costly compared to other techniques. Cost restrictions may be particularly problematic if deep sequencing is required i.e. to detect low frequency mutations, or in applications where repeated testing is required, such as the monitoring of residual disease following treatment. Sample preparation techniques also restrict the application of NGS, as these may require expert technical skills and may lead to loss of sample and introduction of errors. NGS whole genome sequencing analyses any molecules that are available in the sample, while whole exome sequencing is restricted to protein-coding regions. Both techniques have been applied to 135-136

ctDNA,

however the combined depth and breadth of sequencing that would be required to routinely

analyse rare mutations would lead to enormous quantities of data and prohibitive costs. Targeted sequencing techniques have therefore been developed that focus on regions of interest where SNVs are known or suspected to be linked to therapeutic outcomes. These allow dataset size and costs to be reduced while maintaining sequencing depth, and are the most common format in studies of low abundance ctDNA. The Safe-Seq strategy of Kinde et al. approaches these issues by tagging ctDNA samples with barcode sequences prior to amplification.

134

This was done either by adding externally-generated barcodes via two

cycles of PCR, or by shearing the template DNA in random locations prior to the ligation of sequencing adapters (Figure 8). The entire sample is then amplified, and errors during amplification are detected by comparing reads with identical barcodes to obtain a consensus sequence. Analytical specificity is primarily limited by the error rate of nucleotide incorporation during PCR (dependent on polymerase, as -6

low as ~ 10 ), as any errors that are not made in the first few cycles of amplification will make up an insignificant portion of the total number of reads. SafeSeq, particularly the version that uses external 19, 137-138

barcodes, has been successfully applied to ctDNA samples in several studies.

Ståhlberg et al

described further improvements in the PCR-based barcoding procedure by using a hairpin structure


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within the primers. This was designed to be closed at primer annealing temperatures, preventing nonspecific annealing, while being open during the higher-temperature extension phase of the PCR reaction.

139

Figure 8: Summary of barcoding approaches for high specificity redundant sequencing. A further range of barcoding techniques show high specificity, but are yet to be adapted to ctDNA, primarily due to limited sample. Schmitt et al. extended the barcoding concept by creating Duplex


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sequencing,

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which ligates complementary barcode adapter sequences to both strands of a dsDNA

fragment simultaneously. Library preparation and sequencing is then performed on both strands. Mutations in the sample, which are present on both strands of DNA, can then be separated from errors that occur during PCR pre-amplification steps, which occur probabilistically to a single strand of DNA. Duplex sequencing was shown to be of particular use where DNA samples had become degraded during collection or preparation, leading in particular to the detection of spurious G→T and C→T mutations. 9

-7

Duplex-Seq can theoretically detect mutants down to levels of 1 in 10 , and although only around 10 has been demonstrated, this would be adequate for ctDNA analysis due to limited sample. A minor concern is that the duplex barcodes would not perform error correction for any single-stranded ctDNA, however the 47, 141

technique can revert to single stranded barcoding when paired reads are not available.

A related

technique effectively adds duplex barcodes via PCR for paired reads, but only after initial amplification of 142

target regions.

This improves the usability of the barcoding scheme, but shows reduced analytical

specificity of approximately 1 in 5000 versus wildtype DNA, presumably due to polymerase errors. 48

A different approach is used in the CircleSeq strategy of Lou et al. Here the original single-stranded sequence is self-ligated into a circle for rolling-circle amplification. A consensus sequence can thus be reached by sequencing the linked copies, which removes the need for additional barcodes to be added. In contrast to regular PCR, individual polymerase errors do not get the chance to propagate because each new copy of the sequence is taken directly from the original, and the efficiency of sequencing is also improved. This work was extended by Gregory et al., who ligated dsDNA into circular vectors with complementary tags, combining error-correcting Duplex tags with rolling-circle amplification in a strategy 114

they termed Cypher-Seq.

However, these highly-specific techniques face several technical hurdles in being applied to ctDNA. The most important of these is the extensive work that is required to prepare the sample for testing. This makes them more difficult to translate into regular use, and increases the potential cost of any potential clinical assay. Preparation steps also cause significant loss of analytical sensitivity due to sample losses, damage or errors during preparation, which is of particular concern for rare variants that may be lost altogether, and making their increased specificity of. For example, Newman et al. reported 60 % retention of sample molecules during their barcoding procedure, while most other procedures are likely to be far 47

below this.

If a polymerase is used for barcoding, mutants may not be detected at specificities below the 134

mutation rate of the polymerase, procedures.

while DNA molecules may be chemically degraded during other

48

Other studies have developed techniques that do not require the use of barcoding. The CAPP-Seq platform of Newman et al. uses deep sequencing (10,000X) over a carefully selected subset of the 47, 143-144

genome where mutations were more common and correlated with disease.


Improved library

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preparation methods and ultradeep sequencing allowed the technique to detect individual patient mutations down to 0.1% allelic fraction. Furthermore, the combined data from multiple mutations allowed “total ctDNA” to be quantified down to 0.02%, while keeping sequencing costs and preparation complexity lower than for barcoded sequencing techniques. The platform has recently been acquired by Roche. The Alternatively, Tagged-Amplicon Sequencing (TAm-Seq) uses a Fluidigm automated microfluidic system to amplify ctDNA in targeted loci through multiplex pre-amplification followed by multiple singleplex 123

reactions.

It uses overlapping short amplicons to ensure fragmented ctDNA is effectively amplified. This

technique allows mutations to be detected down to levels of approximately 0.1% as demonstrated in studies of clinical samples,

145

and is commercialised through Inivata. As with the techniques discussed

earlier, the trade-off is lower analytical specificity, but simpler and more efficient workflows. Targeted error correction sequencing (TEC-Seq) does use exogenous barcodes, but incorporates a small number of these into standard library preparation methods, thereby reducing losses seen in other barcoding schemes while achieving around 0.1% specificity following ultra-deep sequencing.

16

ADVANCED DETECTION SYSTEMS A range of novel techniques also exist for reporting the output of an assay; these are primarily used in multiplexed assays to increase the number of species that can be simultaneously detected.

MALDI A range of techniques exist that use MALDI (Matrix-assisted laser desorption/ionisation) mass 146

spectrometry to detect SNVs.

A recent development is the UltraSEEK system commercialised by 112

Sequenom/Agena, and recently reported in the scientific literature.

The system uses non-allele-specific

PCR amplification followed by a mutation-specific probe extension which adds a terminating ddNTP with a biotin label, allowing capture of the extended sequences using streptavidin-functionalised magnetic beads. The purified sequences are then analysed by MALDI, based on the characteristic mass of each extended probe, which allows this method to be multiplexed to high plex numbers. (Figure 9) The analytical specificity of the assay overall relies on the specificity of the primer extension step, which theoretically allows for more flexibility in multiplexing, as unlike PCR-based assays, primer annealing temperatures do not need to be optimal for each mutation. UltraSEEK has been used to demonstrate detection of 0.1% SNVs versus wildtype.

112

However, this high analytical specificity is only found in

assays with lower-level multiplexing, as the technique uses mutant-specific bases in the single-base extension step to achieve high specificity; up to 15-plex per tube is currently claimed by Agena, while 112

Mosko et al. recently described a 4 × 7-plex assay.


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112

Figure 9: Schematic of UltraSEEK detection system. Adapted from Mosko et al.,

copyright (2016), with

permission from Elsevier.

SERS Initial studies have also used surface-enhanced Raman spectroscopy (SERS) to create a multiplexed PCR-based assay. SERS-based detection was first used in combination with a ligation reaction in a 2147

plex KRAS SNV assay, albeit at low analytical sensitivity.

In one study, gold nanoparticles were

functionalized with Raman-reporter molecules (a different molecule, i.e. barcode, for each mutation), and if PCR produced an amplicon, these would aggregate at the surface of magnetic particles and produce a SERS signal. Proof-of-concept was shown via a 3-plex assay using ARMS-based PCR (detection of 0.1% mutant in wildtype was demonstrated), and signal deconvolution could potentially be used to extend this


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number.

82

SERS was also used as the detection step in recent work that used ctDNA to catalyse the

formation of carbon nanotube/Cu nanoparticle complexes that produced and amplified SERS signals. The study used a mutant-specific triple helical DNA probe attached to a gold surface, and interestingly was 56

able to measure the allelic fraction of mutant DNA in a sample of serum. However, this particular technique may be difficult to adapt to multiplex detection.

Electrochemical Chips Detection via an electrochemical chip also has several potential benefits for multiplexing, as well as easeof-use and potential for standardisation. One study recently described a promising electrochemical detection chip, which was demonstrated to detect mutant RNA and KRAS ctDNA at 0.01% relative abundance vs wildtype, and seemingly lower levels appeared possible based on their results.

86

A

multiplexed chip layout was also described that could report the specific mutation present down to at least 0.1% on ctDNA samples (standardised according to a PNA clamping qPCR assay). The ctDNA assay used a PNA wildtype blocker as well as DNA “clutch probes” to bind one half of the double-stranded ctDNA, leaving the complementary strand available for detection via binding to a nanostructured electrode that was functionalised with allele-specific PNA probes. An unrelated study was able to detect EGFR ctDNA on an electrochemical chip in unprocessed plasma or saliva samples with good analytical 25

specificity (0.1% - 1%).

Both platforms were only tested with relatively high quantities and

concentrations of mutant allele (12.5k copies/µl and 20ng in 10 µl respectively of mutant allele), which might be difficult to obtain from early stage patients. However, testing was done without any amplification of ctDNA, which may help avoid artefacts and simplifies processing. The ultimate analytical sensitivity limit may be lower than the samples tested. Blood-based fouling could have problematic effects on these or other assays that use unamplified template.

Fluorescently-coded microparticles While colour-based fluorescent multiplexing is usually limited by broad fluorescence peaks and spectral overlap, there are many companies offering microparticles that are barcoded with multiple fluorescent dyes of different intensities. Commercial kits have been used for detection of a range of cancer-related SNVs in primary tumours,

148

however have not been reported for ctDNA analysis.

INSIGHTS Sample Preparation Sample preparation is vitally important for ctDNA assays: for example, if a mutant is present in vivo at 1 in 10,000, then at minimum 50,000 cfDNA copies must be tested to be statistically sure that at least one 118

mutant copy is present.

33

However, cfDNA is often present at between 1000 and 10,000 copies/ml, and


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so is often the limiting factor, particularly once replicates are included and if samples must be split up for non-multiplexed assays (see Figure 10). In these cases, there is no need for more expensive, more specific detection techniques. Instead, improved sampling and sample preparation techniques are the current priority for work to extend ctDNA’s usefulness beyond late stage patients. One possibility would be to develop new sampling and preparation methods that are able to process larger volumes of sample and/or concentrate the resulting ctDNA more than is currently achievable. New methods will most likely 26-

focus on shorter DNA strands (below 100 bp), which appear to contain a higher proportion of mutations, 27

33

and which many current extraction methods do not collect as effectively as longer strands.

New

methods will need to be cheap and easy to apply, and standardising and validating these methods will be an important part of the process prior to their widespread application. Microfluidic techniques are an obvious option for this application.

149

The influence of microfluidics on analytical techniques for ctDNA can

already be seen in nanowell arrays for dPCR and TAm-Seq and also in SCODA, where ctDNA is separated from other species and concentrated based on binding interactions with a matrix. Another possibility is that assays could be run without extracting ctDNA from plasma or other sources, to avoid significant losses or damage to the ctDNA. However, while this would open up the possibility of point-ofcare ctDNA diagnostics, it brings major technical challenges due to the complexity of bodily fluids and low concentrations of target species in early-stage patients.

Figure 10: Interrelationship of assay specificity, multiplexing and sample collection throughput. Major techniques are shown based on their reported specificity and multiplexing ability (locations of markers are


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an approximate guide and include studies of non-ctDNA samples) and companies with interests in each technology are notes in brackets. Commercialised systems are shown with filled-in shapes, academiconly systems are outlines. Dotted lines A/B/C represent three different thresholds at which higherspecificity assays have no extra utility i.e. the test becomes sample-limited. These lines are based on the assumption of 12,500 genome copies/sample;

33

they are based on triplicate measurements and assume

perfect sampling (under real sampling conditions the curves would move further left). (A) Detection of one mutation of interest e.g. tracking minimal residual disease with a single defined mutation has a hard limit of approximately 1 in 4000 (number of sample molecules divided in triplicate). (B) Detection of a panel of 10 mutations, e.g. for companion diagnostics. With an assay of 10-plex or higher, this can be performed at 1 in 4000 specificity (Marker B1). If a singleplex assay is used, the sample must be split into ten, and higher assay specificities above 1 in 400 are of no extra use (Marker B2). (C) Detection of a panel of 100 mutations e.g. cancer screening. Significant multiplexing is required for even moderately rare mutants to be detectable.

Analytical Sensitivity Currently the only clinically-approved tests for ctDNA are based on quantitative PCR, however digital PCR and sequencing are clearly the most popular in other studies due to their improved ability to detect rare variants. Sequencing-based methods allow greatly increased flexibility by detecting variants without foreknowledge of their exact sequence. In many cases, sequencing studies must be focussed on smaller sections of the genome where mutations are likely to occur (and provide relevant data), due to the high cost and complexity of NGS, particularly those techniques used for low abundance mutations. Nonetheless, NGS techniques have a clear advantage in that they detect variants with little or no a priori knowledge of the mutation presence or position; targeted assays have zero sensitivity to mutations that they are not designed to examine. This advantage is less clear-cut when considering several of the potential applications for ctDNA, such as for companion diagnostics, where the relevant mutations (often 45

SNVs) and accompanying advisories are well-known and limited in number.

In these cases, sequencing

may lose its advantage over targeted assays, particularly if the latter can be performed in a fast, simple and cost-effective manner. This is supported by the current popularity of dPCR in pre-clinical studies of ctDNA, despite dPCR being almost entirely used for singleplex assays. Furthermore, PCR-based detection techniques typically require few preparation steps beyond extraction of DNA into an appropriate buffer; in contrast, some sequencing techniques require separate steps for pre-amplification, attachment of barcodes and tags, and the final sequencing step, each of which may introduce errors and losses. Due to the current limits on sample, dPCR has advantage for analysing samples from early-stage patients and for monitoring residual disease, as it requires minimal sample


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preparation and in some ways works best on dilute samples. If new sampling methods were developed that allowed larger quantities of ctDNA to be sampled from each patient, this could make barcoded sequencing competitive with dPCR in this aspect, by negating the effect of high sample losses. One area which has received little active attention is the effect of fragmentation on assay sensitivity. Many ctDNA fragments are likely to be undetectable to PCR primers and other probes due to overlaps between the probe sequences and breakpoints in the ctDNA. For example, even if PCR amplicons are kept short (e.g. 80 bp) a large proportion of sample molecules would not amplify efficiently, including a majority of the short fragments of less than 100 bp. A purely theoretical, idealised sequencing technique that could analyse individual short molecules would have an advantage here, as each base is detected individually. However current sequencing techniques also rely on PCR pre-amplification, often using larger amplicons, and so many molecules that are unambiguously mutated will not be detected. New analytical and preparative techniques that can detect mutations in shorter fragments of DNA without significant losses would be of exceptional use in this field. Error suppression 86

Most ctDNA assays, with few exceptions, rely on enzymatic recognition at some point in the assay to detect SNVs while rejecting wildtype molecules, interact with ctDNA molecules during sequencing, or to pre-amplify ctDNA prior to analysis. As ctDNA is probed to greater depths, enzymatic error such as polymerase mis-incorporation, extension from a mismatched primer, mismatch ligation, and amplification biases also become a limit. While errors in NGS may be compensated for using more stringent basecalling algorithms,

134

this may result in poorer analytical sensitivity. Similarly, for probe-based assays,

oligonucleotides will often bind with similar affinity to both SNV and wildtype sequences. Errors can be reduced through choice of enzymes with improved properties and through improved oligonucleotide design (including LNA or PNA modifications as appropriate). These approaches offer incremental improvement and may require large investments of time in (re)optimisation. A general strategy for reducing the effect of these errors is the serial coupling of two or more error-prone processes in the design of the assay, where all must occur to achieve an overall detection event. The probability of an overall false positive is thus reduced as the probabilities are multiplied. This is discussed explicitly in reports of the PAP and biPAP techniques, but is also evident throughout a range of the most advanced techniques; for example, SCODA relies on repeated hybridisation and dehybridisation of the target to allele-specific probes, rather than a single pull-down of the target molecule. Barcoded sequencing may also be viewed in a similar way: however rather than coupling multiple events prior to detection to ensure a clean reading, it takes quasi-independent readings of the same molecule and corrects for errors by combining the redundant measurements. This is also the case in other techniques such as rolling circle amplification, where all copies are taken from the original molecule. In contrast, a


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major disadvantage of allele-specific PCR is the fact that a single mistake can create a mutated copy of the allele where none existed in the sample, and this is then amplified as a true copy.

Quantification of single molecules A feature of some of the most promising systems is the ability to interrogate individual molecules. This divide-and-conquer approach takes different forms: spatial segregation of template DNA (dPCR and BEAMing) or barcoding (sequencing techniques). Single molecule analysis not only represents the final frontier of improved analytical sensitivity and specificity, but also provides fully quantitative data rather than aggregated readings that must be calibrated. This change from aggregated signal to individual molecules is the equivalent of moving from analog electronics to digital, and brings similar improvements in information quality. However, these methods require more extensive sample preparation than qPCR, which may increase cost, require skilled workers, and may affect sample integrity and yield. This has been semi-automated in the case of digital PCR, but still requires investment in equipment and training of personnel. Advanced barcoding techniques that use simple, minimal or automated sample manipulation would be of great use in advancing sequencing-based approaches, and advanced sequencing methods such as nanopore or other single molecule sequencing, while they currently have high error rates and are predominantly used for long DNA molecules, may be able to reduce sample losses during preparation and concentration requirements.

150-152

New techniques that can analyse single molecules may emerge

such as super RCA, which is claimed to provide single molecule analysis with a relatively simple workflow.

116

Other techniques could also be adapted, such as recent reports that examine single-

molecule binding events using total internal reflectance florescence microscopy (TIRF), using changes in binding kinetics to differentiate SNV sequences.

153-154

While these would be difficult to adapt to the

particular challenges presented by ctDNA, in particular its randomly fragmented structure, they may represent an alternative way forward.

Summary of analytical capabilities As can be readily observed, further development of analytical techniques is required. For many techniques with the best analytical performance, decreasing costs and standardising and simplifying techniques will be the most likely and most desirable change. For sequencing in particular, the loss of sample during preparation is also a significant hurdle. In the case of both quantitative and digital PCR, improvements to multiplexing would also be of great value, both to lower costs and to make best use of limited sample. Techniques may also be optimised to become more application-specific, with sequencing used for discovery of new mutations and mutation screening, while mutation-specific assays such as qPCR, dPCR and MALDI-based assays are optimised for applications where a smaller cohort of wellknown mutations is examined. Unless a technique is invented that outperforms all others in every facet,


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the current variations in assay capabilities (Table 4) will require an informed choice of analytical technique based on the requirements of the study being undertaken. Table 4: Major advantages and disadvantages of ctDNA diagnostics techniques arranged by family. Additions in brackets are dependent on the exact method used. “Cost” includes equipment setup, perassay reagents and personnel training and labour costs. Technique

Analytical

Analytical

Sensitivity

Specificity

qPCR (standard)

+++

+(+)

+

++++

qPCR (IntPlex)

++++

++++

+

++++

PAP/biPAP

++++

++++

+

+++

+(+)

+(+)

++

++++

++++

++++

+

++

Other enzymatic assays dPCR

Multiplexing

Low Cost

Non-barcoded sequencing

Notes

Lack of multiplexing may reduce sensitivity in studies with multiple targets. Digital PCR can quantify single molecules, while other techniques require calibration. Low sensitivity primarily due to

++

+++

++++

+

sample preparation and stringent base calling algorithms. This can be improved by pre-amplification, at the

Barcoded sequencing

+

++++

++++

(+)

cost of analytical specificity. Barcoded sequencing can quantify single molecules.

SCODA

+++

+++

+++

+

MALDI

Requires a further detection step (typically sequencing) Based on UltraSEEK assay.

++

+(+)

++(+)

+++

Relative cost is best for testing large numbers of mutations.

Electrochemical chips

Few reports. Low sensitivity. +

++

++(+)

++++

Specificity is reliant on the probes used.

Clinical application to patients with early-stage or residual disease A range of further practical restrictions on assay design become important for clinical translation, some of which will vary significantly depending on the exact application (Table 5). Assays will most likely be performed in-house at hospitals or in pathology labs, and will require workflows and analysis methods that do not require years of specialist training. Methods that require specialist equipment will need to be


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widely adaptable to ensure that investment is worthwhile, whereas methods that utilise equipment that is already common have a natural advantage. For any procedures that require PCR, carryover of amplicons between samples is a significant issue.

155

Techniques that can be performed in a single, closed tube (e.g.

such as qPCR) are therefore at a significant advantage compared to assays that require post-PCR sample manipulation. Tests should ideally be designed to incorporate quality control, including internal controls for DNA input quantity and reaction success. Techniques that allow absolute quantification of molecular numbers have an advantage over those that require calibration curves to be generated in allowing consistent application of the tests.

120, 122

Finally, standardisation and validation are also important

steps towards assay translation that may be made easier through good design at the early stages of development. Table 5: Requirements for clinical application of ctDNA Application (in order of

Application-specific notes on clinical implementation

current most-advanced) Genotyping following cancer detection to guide treatment

• •

(complementing or replacing tissue biopsy)

Monitoring for response to therapy and/or emergence of resistance mutations

•

• • •

and/or residual disease

Companion diagnostics: small panel of mutations, usually well-known and related to a potential treatment. Must show advantage over tissue biopsy (alone or in combination) to justify change. This is likely to be most easily achievable in accounting for spatial heterogeneity Sensitivity important so any resistance mutations are detected and appropriate treatment can be chosen; falsenegative means patient undergoes useless treatment

Similar analytical requirements to initial genotyping. Cannot be performed with initial tissue biopsy. Significant advantage in analysing temporal heterogeneity Should be cheap due to repeated, regular testing, or justify expense with improved results.

following treatment Screening for undiagnosed

•

cancer • •

•

As most patients would test negative, requires a very low 156 false-positive rate (high clinical specificity), particularly to minimise effect on patients with no disease. As most patients would test negative, cost must be very low to justify expense Must analyse a larger number of mutations. Where a small number of mutations are of interest (e.g. approximately 50 157 % of melanomas contain BRAF mutations ), simpler targeted methods might also be adapted for screening for at-risk populations. Ideally will give approximate tumour location, which may be 21 22 possible using breakpoint analysis, methylation analysis, 158 or concurrent analysis of protein markers.


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CONCLUSIONS Further development of methods is needed to make full use of ctDNA’s potential for early stage patients and those undergoing post-treatment monitoring. Techniques that allow single molecules to be detected and analysed individually (including dPCR and barcoded sequencing) are likely to dominate in the shortterm, due to the quality of statistical information they can provide compared to aggregate measurements. However further work is needed to either improve and standardise these methods, or to develop new ones with improved characteristics. There should be an increased focus on improving sample collection and purification techniques to ensure that detection capabilities can be fully exploited. While several assay styles already exist that have excellent detection limits, work is required to develop assays that are also highly multiplexed, cheaper, simpler, and able to be implemented outside of academic labs, or to improve analytical sensitivity and specificity of established techniques that already have these characteristics. Finally, it should be acknowledged that this analysis has solely focussed on technical limitations. As in many fields, being able to generate reliable data is a necessary but not sufficient condition. Other matters, such as the clinical sensitivity/specificity of a mutation (used in the diagnosis of a disease state), choice of target mutations, medical interpretation of the results, clinical value and 77

legal/ethical matters must also be dealt with before ctDNA becomes a mainstream clinical analyte.

ACKNOWLEDGEMENTS A.E.R. is supported by Jack Brockhoff Foundation Early Career Medical Research Grant 4227, 2016. All authors were supported by the Monash Institute of Medical Engineering Seed Fund, 2015.


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Extending circulating tumour DNA analysis to ultra-low abundance

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