There has recently been an explosion of data describing copy-number variation (CNV) in the human genome, thus encouraging a revision of what is considered ‘genomically’ normal Citation[1–3]. Although it appears that the vast majority of inherited CNVs represent benign variation, a number have been associated with a clinical phenotype Citation[4,5]. CNVs can also be acquired through somatic events – it has long been appreciated that acquired duplications/amplifications are part of the genomic landscape in human cancers Citation[6,7]. As the database of somatic genomic events in human cancer expands, it is clear that some of these events make direct and important contributions to the cancer phenotype and may predict the potential response of an individual tumor to specific biological therapies Citation[8,9]. As a result of the association between CNVs and inherited or acquired human disease, there is an increasing demand for the assessment of CNVs in the clinic.
There are a number of challenges to the incorporation of assays of CNV into molecular diagnostics or pharmacogenomics/prognostics. First, what is the sensitivity and specificity of a measured CNV with respect to making a specific diagnosis? How well does a measured CNV predict a response to a specific therapy? These are important questions irrespective of the platform used to assess CNVs. Second (and particularly relevant to whole-genome analyses), what is the relevance of detected CNVs that do not inform evidence-based clinical decision-making but may involve important genes or noncoding RNAs? The third challenge is technical and relates to the quantity and quality of template DNA available to perform the assay. In inherited conditions or certain cancers, such as leukemias, there is generally an abundance of high-quality DNA available through simple phlebotomy, but in many other clinical situations diagnostic biopsies are small and heterogeneous. Furthermore, biopsy specimens are routinely fixed in formalin, which causes DNA degradation and can have a significant impact on the amount and quality of DNA extracted from clinical samples Citation[10–12].
Current approaches to the analysis of CNV
Broadly speaking, the analysis of CNV can be divided into whole-genome analyses and targeted analyses, as have recently been reviewed extensively by Feuk and colleagues Citation[13]. With respect to whole-genome analyses, the current gold standard is probably the high-density oligonucleotide single-nucleotide polymorphism (SNP) array platform, which provides a wealth of data on both CNVs and SNPs Citation[14]. However, arrays require significant quantities of high-quality template DNA. If source DNA is limited, a whole-genome amplification (WGA) protocol prior to array hybridization becomes necessary. Although there are many reports of successful WGA protocols Citation[15,16], there remains a significant risk of introducing bias through differential amplification, and this may be particularly problematic if the DNA is degraded Citation[17].
The massively parallel-sequencing strategies that have recently been developed allow integrated sequencing and CNV analysis of DNA samples Citation[18], although it is premature to consider using this technology routinely on clinical specimens.
The protocols developed for the targeted analysis of CNVs consist of either PCR- or hybridization-based approaches. There are multiple PCR-based protocols described for the assessment of CNVs, including quantitative PCR, multiplex amplifiable probe hybridization Citation[19], quantitative multiplex PCR of short fluorescent fragments Citation[20] and paralog ratio testing Citation[21], but arguably the most successful from a diagnostic perspective has been the multiplex ligation probe assay Citation[22]. This is a robust technique for measuring CNV at up to 45 loci and there are now numerous diagnostic kits available, all based on the attractive strategy that utilizes specially designed probe pairs, each designed so that universal forward and reverse primers flank the probe. There are many advantages to this approach, particularly the ability to interrogate multiple loci in a single experiment, the possibility of analyzing degraded DNA from formalin-fixed, paraffin-embedded clinical specimens and the type of hardware required – thermocyclers and capillary electrophoresis – being relatively commonplace in laboratories worldwide. However, some difficulties remain: the design of probes is complex and requires significant time and experimental expertise, the maximum number of loci interrogated is relatively rigid and, although the DNA template requirements can be as low as 20 ng, in many cases up to 200 ng is required Citation[23].
Single-molecule approach to copy-number counting: molecular copy-number counting
The idea of counting single molecules is attractive because it targets specific loci (molecules) of interest and removes any need to manipulate (amplify/label) DNA prior to analysis. Molecular copy-number counting (MCC) derives from HAPPY mapping, a single-molecule digital-PCR approach to genome mapping devised by Paul Dear Citation[24], and was originally the result of a collaboration between the Dear and Rabbitts laboratories at the MRC Laboratory of Molecular Biology, Cambridge (UK) Citation[25]. It is a simple assay that exploits the ability of PCR to detect single molecules of a target sequence (marker) in DNA aliquots that have undergone limiting dilution. The term ‘digital PCR’ refers to each dilute aliquot being either positive or negative for a target sequence.
In MCC, the number of aliquots that are positive for a target sequence can be counted, and this value compared with those of other target sequences, thus allowing the relative copy-number of multiple sequences in the original DNA sample to be inferred. It was originally used to define and then clone a nonreciprocal translocation using DNA from a renal carcinoma cell line Citation[25]. More recently, a modification of the basic MCC technique in order to analyse CNVs in DNA from archived formalin-fixed, paraffin-embedded (FFPE) specimens was described – microdissection MCC or µMCC Citation[26].
For MCC experiments, the DNA under investigation undergoes limiting dilution so that the target sequence is only present in a proportion of aliquots (commonly a 96-well plate). DNA is ideally loaded at a concentration such that a normal copy sequence will be present in approximately 50% of the aliquots. Raw MCC results are the total number of aliquots that are positive for a specific target sequence. This number is converted into the number of copies per aliquot using a calculation based on the Poisson distribution. The relative number of copies per aliquot of multiple target sequences reflects their relative copy in the original DNA sample. Through the simultaneous MCC testing of multiple target loci, as well as reference loci expected to be at normal copy, the relative copy number of target loci can be inferred. MCC uses a two-phase hemi-nested PCR protocol, full details of which have been published Citation[25].
Key attributes of MCC
There are a number of key attributes of MCC that make it attractive for its exploitation in molecular diagnostics, which will now be described.
Accurate measurement of copy number across a wide dynamic range
The original description of MCC demonstrated the ability to distinguish the 1:2 variation in copy around a nonreciprocal translocation Citation[25]. More recently, we demonstrated that MCC could accurately assess the more significant difference in copy number seen in regional amplification in a cancer cell line. In a single experiment variation in copy number extending over an eightfold range can be assessed Citation[26]. The range of MCC can be extended considerably by repeated experiments using lower amounts of DNA per aliquot. Similarly, the use of greater numbers of aliquots could extend the dynamic range within a single experiment.
Flexibility of MCC allows rapid targeting of regions of interest
Since the design and supply of primer sets is readily achievable, MCC can be rapidly adapted to regions of particular interest Citation[25–27], making it well suited to refining CNV at very high resolution when required, and to the detailed screening of candidate regions, or a set of scattered candidate loci. The design of hemi-nested primer sets is uncomplicated and can be readily achieved using standard approaches and published primer parameters. Although the analysis of FFPE material requires the design of primer sets with uniform external amplimer length, this is generally straightforward. The flexibility of MCC contrasts favorably with MLPA and array-based analyses and means that it may prove very useful in a broad range of diagnostic tests, including the rapid independent development of customized diagnostic tests by multiple end-users.
Assessment of multiple loci in a single experiment
Molecular copy-number counting cannot compete with whole-genome analyses with respect to genome coverage and does not seek to. In fact, in the clinical situation, it could be argued that much of the data generated by whole-genome analyses is both diagnostically redundant and potentially distracting, and that targeted analyses may, therefore, be a better way to accurately assess CNVs with a defined clinical or phenotypic association. Compared with other published methods, MCC offers a step change in the number of specific target sequences that can be assayed in a single clinical specimen. The maximum assayed to date is 192, but up to 1200 markers have been successfully multiplexed using a similar HAPPY mapping protocol. The potential benefits of interrogating more loci in a diagnostic assay are, for example, higher-resolution analysis of the specific site of a deletion or duplication event in inherited disorders or the assay of multiple separate genes of interest in a cancer specimen.
Technical requirements
Molecular copy-number counting has relatively few technical requirements and can be performed in a standard laboratory when only a few markers are interrogated. As it depends largely on PCR, it is amenable to automation in a more high-throughput facility. In the Dear laboratory, a custom-built robot is used to set up PCR reactions, although commercially available liquid-handling robots could readily be adapted to a similar protocol. Otherwise, the major requirements are access to a ‘clean room’ to perform phase 1 PCRs, and access to thermocyclers, both of which are widely available in modern molecular biology facilities. With respect to reading PCR results, there are two options: gel electrophoresis or melting-curve analysis Citation[25]. In our practice, melting-curve analysis is preferred due to the high-throughput nature of the work performed but this is unnecessary when fewer loci are being analyzed.
There are interesting possibilities in adapting MCC to novel technological platforms, including the use of microfluidic devices. The platform that is currently the most advanced is that from Fluidigm Citation[28,29]. At present, there are no published reports of the analysis of multiple loci simultaneously but the platform should be readily adaptable to this end.
Application to formalin-fixed biopsies
With respect to the potential to exploit clinical archives or to analyse formalin-fixed biopsy specimens, the ability to tolerate small quantities of degraded template DNA is a specific advantage of µMCC. An extreme case was used to demonstrate the point, in which an effective total of only 160 haploid genomes (equivalent to 0.5 ng) from a grossly degraded specimen were used to generate a copy-number profile along chromosome 3 Citation[26]. From our experience in analyzing FFPE material, it is anticipated that sufficient DNA could be extracted to perform µMCC from almost all invasive cancer specimens, whether diagnostic biopsies or surgical resection specimens. The adaptation that allowed µMCC to be used on badly degraded DNA was the use of short (100–120 bp), uniform-length external amplimers in phase 1 PCRs.
The demonstration of the successful analysis of multiple loci using limited quantities of material is encouraging for the future development of diagnostic or prognostic applications in any clinical contexts where DNA quantity is limited or when a flexible yet precise method of assessing CNVs is required. There may particularly be an opportunity to exploit MCC in cancer diagnostics and pharmacogenomics. Although somewhat controversial, the copy number of oncogenes, such as HER2Citation[8] and EGFRCitation[30], in diagnostic biopsies is already used to guide therapeutic decisions in breast and lung cancer, respectively. Furthermore, specific resistance to biological therapies can be the result of focal amplification of specific genes Citation[31]. Currently, the technique most commonly used to measure gain/amplification at specific loci is FISH. However, FISH experiments often fail on FFPE material Citation[9,32] and there can be significant controversy and subjectivity in the interpretation of the results obtained Citation[33,34]. It can also be difficult, expensive and laborious to analyze more than a few loci by FISH. µMCC could be an accurate and rapid alternative to FISH with the ability to assay multiple loci. Since the available information on gene dosage in cancer is expanding rapidly, future diagnostic µMCC assays, with their inherent flexibility and the capacity to analyze hundreds of markers, could undergo iterative refinement and be moulded to an end-users specific requirements. Finally, as this is a PCR-based protocol, primer sets could also be designed to perform sequence analysis to test for specific clinically relevant mutations. Thus, the critical genetic and genomic events for specific cancer types could be analyzed simultaneously.
Impact of PCR efficiency on MCC
PCR efficiency is a critical aspect of standard quantitative PCR protocols. MCC makes the assumption that single molecules of all tested loci will be detected if present in an aliquot. This does not require each marker to have the same PCR efficiency, merely sufficient efficiency that over the course of a two-phase hemi-nested protocol, there will be sufficient product to detect. In fact, in spite of standardization of oligonucleotide design, it is recognized that different markers have different efficiency and different band strengths on gel electrophoresis. This is of no consequence as it is the presence or absence of the band that is critical, hence the term digital PCR. To date, our experience, and that of others, indicates that the protocol is robust on cell line and degraded DNA and that approximately only 10% of markers fail using the published design parameters.
Summary
Molecular copy-number counting is a flexible platform with the capacity to simultaneously analyze multiple-target loci, and is highly tolerant of poor-quality and limited template DNA. It should be an ideal way to test for well-characterized CNVs in clinical specimens.
Financial & competing interests disclosure
Frank McCaughan is an employee of the UK MRC, which has filed for a patent for molecular copy-number counting. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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