Real-time polymerase chain reaction

Detecting Cancer with Digital PCR.

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Could dPCR be a diagnostics dark horse?

Detecting Cancer with Digital PCRDigital PCR can carry out a single reaction within a single sample, but the sample is separated into a large number of partitions, and the reaction is carried out in each partition individually. [© anyaivanova – Fotolia.com]

  • Over the past few years, scientists from a variety of disciplines have applied digital PCR (dPCR) as a potentially useful tool for breast cancer screening, measuring latent HIV reservoirs in patients, and diagnosing hospital-acquired and sexually transmitted infections, among others. Digital PCR and variations on it offer a new approach to nucleic acid detection and quantification.

    Similarly to real-time quantitative PCR (qRT-PCR or qPCR), dPCR carries out a single reaction within a single sample. However, in dPCR the sample is separated into a large number of partitions, and the reaction is carried out in each partition individually. This separation, proponents of the technology say, allows a more reliable collection and sensitive quantitation of nucleic acids.

    And a recent advance in dPCR, droplet digital PCR (ddPCR™), can measure absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. The technology is said to offer advantages in the field of liquid biopsies, enabling circulating nucleic acids (cfDNA) and circulating tumor cells (CTCs) to be measured in blood. The technique can also detect rare tumorigenic mutations in a high background of “normal” DNA, routinely down to 0.01% and often further.

    But although simple in theory and principle, the technique’s implementation was not, as it was carried out in commercially available 384-well plates with five microliters per partition, requiring large volumes of reagents.

  • The dPCR Arena

    Advanced nanofabrication and microfluidics technologies have now been incorporated into systems that produce hundreds to millions of nanoliter- or even picoliter-scale partitions, but to date, few companies have jumped into the dPCR arena, offering instrumentation platforms that perform dPCR, or both qPCR and dPCR in various configurations.

    In 2006, Fluidigm became the first company to commercialize dPCR. Currently it offers two systems that mix samples with reagents, partition the reaction mixture, and perform thermocycling and read results within each partition. The systems use chips containing microfluidics and valves that partition samples into about 800 reactions, with either 12 or 48 samples per chip.

    Life Technologies, through its 2009 acquisition of BioTrove, now offers two machines that can be used for both digital PCR and qPCR, the OpenArray and QuantStudio 12K Flex. These mix samples with reagents, load mixtures into reaction chambers, run amplification cycles and monitor reactions as they occur. The machines rely on plates that are roughly the size of microscope slide boards with nano-sized holes; capillary forces and careful placement of hydrophilic and hydrophobic surfaces hold samples in place.

    Life Technologies has also introduced the QuantStudio 3D Digital PCR System. This system, which began shipping in June 2013, is built around a high-density, nanofluidic silicon chip that enables up to 20,000 data points. The system’s chip-based approach is meant to simplify workflow, decreasing the number of hands-on steps needed to begin experiments, and reducing the risks of sample contamination and loss of DNA.

    Companies like Bio-Rad and RainDance now market instruments with many more partitions than previously possible using plates with nano-sized holes. In droplet digital PCR, reaction chambers are separated not by the walls of a well but by carefully titrated emulsions of oil, water, and stabilizing chemicals. Samples are put into a machine where they are mixed with all the necessary reagents and dispersed into tiny droplets. The droplets for each sample are transferred into tubes that can be placed in a thermocycler for PCR. Afterward, the tubes are transferred to a droplet-reading machine, which functions like a flow cytometer to analyze each droplet for whether or not a reaction has occurred.

    RainDance, for example, has developed a patented way to put reagents inside of picoliter-sized droplets to encapsulate biology one droplet at a time. Currently, single nucleic acids are placed inside of the droplets. This creates a single-plex PCR reaction inside of each droplet and, the company says, droplets can be generated using one of RainDance’s commercial instrument systems at up to 10,000 per second.

    In an email to GEN, George Karlin-Neumann, Ph.D., director of scientific affairs at the Digital Biology Center, Bio-Rad clarified distinctions between qPCR and his company’s QX200 Droplet Digital PCR™, explaining that either system can quantitate DNA or RNA targets with either Taqman 5’ nuclease assays or fluorescent DNA-binding dyes (SYBR for qPCR, and EvaGreen for ddPCR) run in suitable Master Mixes.

    But, he explained, ddPCR divides a sample reaction into many thousands of small, uniformly sized droplets where each may or may not contain a target template of interest. After thermocycling to endpoint in a standard 96-well plate and thermocycler, the droplets in each well are read and counted in a droplet flow cytometer (or reader) to determine which droplets have the target (“positive” droplets) and which do not (“negative” droplets). The fraction of positive droplets reflects the number of target molecules in the reaction volume, thus yielding the concentration measurement sought.

  • dPCR and Cancer Detection

    And researchers have adopted dPCR for numerous applications including for analysis of several parameters in cancer patients. In study results published in Clinical Cancer Research last March, researchers from the Royal Marsden Hospital in London described their adaptation of ddPCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number.

    Because HER2 copy number in digital PCR is assessed relative to a reference gene, the investigators used EFTUD2, a gene within the ERBB2 locus found not to co-amplify with HER2 and not subject to normal copy-number variations.

    Using the Bio-RAD QX100 ddPCR system, the researchers found that 64% of patients with HER2-amplified cancers were classified as digital PCR HER2-positive and 94% of patients with HER2-nonamplified cancers were classified as HER2-negative by the assay, giving a positive and negative predictive value of 70% and 92%, respectively.

    The authors concluded that “digital PCR of plasma DNA has high accuracy in the determination of HER2 status,” and that the approach of analyzing of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. “This approach could potentially be adapted to the analysis of any locus amplified in cancer,” they concluded.

    And last September, scientists working at Fred Hutchinson Cancer Research Center, demonstrated that ddPCR technology could be used to precisely and reproducibly quantify microRNA (miRNA) in plasma and serum over the course of different days, potentially allowing further development of miRNA and other nucleic acids as circulating biomarkers.

    Under active study as blood-based biomarkers for cancer and other diseases, miRNA measurements in blood samples have been plagued by unacceptably high interday variability, obviating their use as reliable blood-based biomarkers.

    “In the field of circulating microRNA diagnostics, droplet digital PCR enables us to finally perform biomarker studies in which the measurements are directly comparable across days within a laboratory and even among different laboratories,” said Muneesh Tewari, M.D., Ph.D., associate member in the Human Biology Division at the Fred Hutchinson Cancer Research Center and lead author of the study.

    And Dr. Karlin-Neumann says that ddPCR is “en route to being introduced into clinical practice in a number of areas.” Though, he notes, the “only CLIA lab I know of that currently offers a ddPCR-based test is the University of Washington’s Clinical Laboratory, which offers a ddPCR-based test for detection of chromosomally integrated HHV-6 virus in transplant patients.”

    Other labs, he says, that are in the process of developing clinical tests for detection of residual disease in leukemia patients with BCR-ABL translocations include that of Alec Morley, M.D., a pioneer of digital PCR. Dr. Karlin-Neumann also cites the work of Hanlee Ji, M.D., who is measuring copy-number variations by ddPCR in FFPE and cell-free plasma DNA to assess whether gastric and other cancer patients have amplifications in oncogenes that would make them amenable to one of a growing number of targeted therapies.

    Importantly, Dr. Karlin-Neumann pointed out that it’s still too early, regardless of the platform used, to be attempting to detect cancer in naïve patients not already known to have cancer since “we do not have the clinical experience to know what changes to look for and what thresholds are meaningful.”

    And he notes, until recently, there have not been technologies that allowed us to detect and quantitate below ~1% mutant abundance in either mixed tissue biopsies or in cfDNA in plasma or serum. ddPCR is demonstrating that it is capable of lowering this limit to as low a ~0.01% in a single ddPCR reaction well, and where more material is available, this can be lowered further by use of multiple wells. Similarly, fractional changes in oncogenic amplifications and deletions can be tested with ddPCR in both solid tumors and in cfDNA.

    And a team of scientists at the University of California, Berkeley says it has developed a bead-based, microfluidic digital PCR technology and demonstrated its ability to quantitatively measure cancer-related translocation mutations at extremely low levels and subsequently sequence single mutated clones.

    The scientists believe that their technology has advantages over commercial emulsion-based droplet digital PCR platforms, such as those offered by Bio-Rad and RainDance Technologies, because it enables downstream sequencing analysis following the digital PCR analysis step.

    But is this capability in demand? A RainDance spokesperson told PCR Insider that the company’s RainDrop digital PCR system currently does allow for emulsions to be broken following thermal cycling so the amplicons can be rescued and subsequently sequenced. However, RainDance said, it is “just starting to see requests for this kind of thing but it is not a commercial solution on offer at this point.”

    But technology will get continue to get piled higher and deeper, as modifications to PCR continue to accrue and scientists figure out how best to use them.

 

Diagnostic Accuracy of Quantitative PCR (Xpert MTB/RIF) for Tuberculous Meningitis in a High Burden Setting: A Prospective Study.

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Background

Tuberculous meningitis (TBM) is difficult to diagnose promptly. The utility of the Xpert MTB/RIF test for the diagnosis of TBM remains unclear, and the effect of host- and sample-related factors on test performance is unknown. This study sought to evaluate the sensitivity and specificity of Xpert MTB/RIF for the diagnosis of TBM.

Methods and Findings

235 South-African patients with a meningeal-like illness were categorised as having definite (culture or Amplicor PCR positive), probable (anti-TBM treatment initiated but microbiological confirmation lacking), or non-TBM. Xpert MTB/RIF accuracy was evaluated using 1 ml of uncentrifuged and, when available, 3 ml of centrifuged cerebrospinal fluid (CSF). To evaluate the incremental value of MTB/RIF over a clinically based diagnosis, test accuracy was compared to a clinical score (CS) derived using basic clinical and laboratory information.

Of 204 evaluable patients (of whom 87% were HIV-infected), 59 had definite TBM, 64 probable TBM, and 81 non-TBM. Overall sensitivity and specificity (95% CI) were 62% (48%–75%) and 95% (87%–99%), respectively. The sensitivity of Xpert MTB/RIF was significantly better than that of smear microscopy (62% versus 12%; p = 0.001) and significantly better than that of the CS (62% versus 30%; p = 0.001; C statistic 85% [79%–92%]). Xpert MTB/RIF sensitivity was higher when centrifuged versus uncentrifuged samples were used (82% [62%–94%] versus 47% [31%–61%]; p = 0.004). The combination of CS and Xpert MTB/RIF (Xpert MTB/RIF performed if CS<8) performed as well as Xpert MTB/RIF alone but with a ~10% reduction in test usage. This overall pattern of results remained unchanged when the definite and probable TBM groups were combined. Xpert MTB/RIF was not useful in identifying TBM among HIV-uninfected individuals, although the sample was small. There was no evidence of PCR inhibition, and the limit of detection was ~80 colony forming units per millilitre. Study limitations included a predominantly HIV-infected cohort and the limited number of culture-positive CSF samples.

Conclusions

Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals from a tuberculosis-endemic setting, particularly when a centrifuged CSF pellet is used. Further studies are required to confirm these findings in different settings.

Discussion

Although the utility profile and accuracy of Xpert MTB/RIF has been well characterised in sputum samples, there are hardly any data to guide its utility and implementation for TBM. This is critical as the rollout of Xpert MTB/RIF means that quantitative PCR is now available in many high burden settings, and data are urgently required to guide appropriate and relevant usage of this technology in biological fluids other than sputum. That Xpert MTB/RIF performs poorly in fluids from some compartments, e.g., the pleural space, highlights the need for such data [27]. The key findings of this study were as follows: (1) Xpert MTB/RIF is likely a good rule-in test for the diagnosis of TBM in HIV-infected patients; (2) centrifugation of the sample improved sensitivity in this context to almost 80%; (3) among HIV-infected patients, Xpert MTB/RIF performed significantly better than the widely available same-day alternative tests, i.e., smear microscopy, which suggests that prompt diagnosis of TBM is potentially achievable in the majority of patients in this setting; (4) the diagnostic value of Xpert MTB/RIF for HIV-infected patients is clinically meaningful given that it performed significantly better than hypothetical decision-making based on clinical characteristics and basic laboratory data (the CS); and (5) when combined with the CS, Xpert MTB/RIF test usage could be reduced by only a modest ~10% whilst retaining similar sensitivity and specificity compared to using Xpert MTB/RIF alone. This last finding informs clinical practice in resource-poor settings. Finally, we quantified the limit of detection of the assay, its relationship to bacterial load, and the impact of PCR inhibition. These data require reproduction in HIV-uninfected and non-TB-endemic populations.

There are limited data about Xpert MTB/RIF performance in TBM [28]. Published data include only small numbers of microbiologically proven TBM cases (range of 0 to 23) [29]–[32], often in a case-control design with a non-uniform reference standard, and often CSF-associated data were published as part of a laboratory-based evaluation of extrapulmonary TB samples, usually including samples from countries with low TB prevalence. Furthermore, there are no studies from high burden settings, and technical performance evaluations, including bacterial load studies, threshold level of detection, and impact of PCR inhibition, have hitherto not been undertaken.

Xpert MTB/RIF sensitivity was as high as 80% when a centrifuged CSF sample from an HIV-infected patient was used. This suggests that Xpert MTB/RIF, at least in an HIV-endemic environment, represents a possible new standard of care for the diagnosis of TBM. Sensitivity was considerably better than in previous studies using commercially available or non-standardised PCR tools [9],[32]–[34]. The ostensibly better performance is likely related to a combination of centrifugation (and hence concentration of bacilli) and technical aspects, including a more efficient standardised extraction protocol, fractionation of mycobacteria by a pre-sonication step, and a nested PCR protocol, thus maximising amplification. However, possibly higher bacterial loads in HIV-infected patients may have also played a role. Our findings have practical relevance because they imply that at least 3 ml of CSF should be set aside and centrifuged, and re-suspended in phosphate-buffered saline, before being run on the Xpert MTB/RIF. This high-sensitivity and potentially rapid diagnosis in most cases is likely to benefit HIV-infected patients suspected of having M.tb., as diagnostic and treatment delay is associated with higher mortality [35]–[37]. Impact-related studies are now required to verify this hypothesis. It is noteworthy that a second sample improved sensitivity minimally. These data suggest that, at least in an HIV-endemic setting, using a second cartridge is unlikely to give further benefit. However, larger studies are required to confirm this possibility.

Similar to the findings when using sputum, the level of detection of Xpert MTB/RIF was between 80 and 100 colony forming units per millilitre. This explains the sub-optimal sensitivity of Xpert MTB/RIF compared to culture, where the detection threshold is as low as 1–10 organisms per millilitre [38]. We did not find a correlation between TTP and Xpert MTB/RIF CT values, as has been shown in sputum [39]. In contrast to previous PCR-based studies [40],[41], we found that CSF had a minimal inhibitory effect on the PCR reaction when compared to sputum. This may be due to the wash step incorporated into the assay that removes extracellular debris. We did not find a difference in TTP between the Xpert MTB/RIF–positive samples from centrifuged versus uncentrifuged CSF. This may be due to a type two statistical error, as the sample numbers were small.

There were three patients who were culture negative but Xpert MTB/RIF positive, i.e., Xpert MTB/RIF positive in the non-TB group. Our previous work has shown that such cases (Xpert MTB/RIF positive but culture negative) are likely to be true TB positives, and this is corroborated by high specificity obtained in large sputum-based studies where a significant minority of the patients had had previous TB [11]. If these culture-negative Xpert MTB/RIF–positive individuals are hypothetically designated definite-TB cases, then the overall case detection rate improves by a further ~10%.

The proper and meaningful value of a test lies in its ability to influence patient management through its incremental value over pre-test probability, or to have an impact on decision-making based on logical clinical judgement (based upon clinical features and basic laboratory parameters). We therefore derived a CS, hitherto unavailable for HIV-endemic settings, to evaluate Xpert MTB/RIF utility in clinical practice. Xpert MTB/RIF had significantly better performance outcomes than the clinical prediction rule (using a rule-in cut point, so appropriate comparisons could be made). Furthermore, hypothetically combining the CS with Xpert MTB/RIF resulted only in a modest ~10% reduction in test usage, but still maintained high sensitivity and specificity. These data suggest that clinical algorithms or scoring systems to limit test usage are unlikely to be significantly useful in resource-poor settings.

There are several limitations of our study. We could not determine the impact of Xpert MTB/RIF (time and proportion of patients initiated on treatment) compared to a smear microscopy/empiric treatment-based strategy given our study design and the fact that management decisions were not based on Xpert MTB/RIF results. However, this was because Xpert MTB/RIF had not yet been endorsed by the World Health Organization when the study commenced, had not been validated for use in CSF, and had been used as a research tool only (thus, for ethical reasons, study samples were evaluated only several weeks later). Although the confidence intervals of some of our estimates are wide (because of limited sample numbers), this is to our knowledge the largest diagnostic study undertaken in TBM (based on the number of microbiologically proven TBM cases; n = 59). This reflects the challenge and difficulty in performing such studies in resource-poor settings. It is possible that the Xpert MTB/RIF performs much better in HIV-infected individuals because of a possibly higher bacterial load, and thus our findings need to be confirmed in other settings. Given the small number of HIV-uninfected patients, we were unable to meaningfully compare this sub-group. The CS was developed to assess only incremental value above basic clinical and CSF parameters. The CS and the combination of CS plus Xpert MTB/RIF need prospective and independent validation. The non-significant difference in sensitivity between the paired centrifuged and non-centrifuged samples may reflect a type two statistical error, as the number of culture-positive paired samples was limited. Lastly, there were nine patients who could not be categorised within our defined groups and were excluded from the analysis.

In conclusion, Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals in a TB-endemic setting, particularly when a centrifuged CSF pellet is used. A second Xpert MTB/RIF test had minimal incremental benefit. Smear microscopy and the CS, when combined with Xpert MTB/RIF, only modestly minimised test usage in a resource-poor setting. Further studies are now required in non-HIV-endemic settings, and using validated scoring systems, to evaluate the impact of Xpert MTB/RIF on diagnostic accuracy, and morbidity and mortality in patients with TBM.

Source:PLOS