A novel type of cancer biosensor: fluorescent PNA probes.

Cancer is a devastating disease that results from a plethora of factors including age, lifestyle and family history (genetic make-up). The extent of individuals being diagnosed is continuing to grow, with more than 330,000 persons in the UK being diagnosed with cancer in 2011 – that’s equivalent to around 910 people a day [1]. Despite the number of individuals surviving breast, prostate, and skin (melanoma) cancers improving (with five-year post-diagnosis survival rates of over 80%) there is still a staggeringly low percentage of survival among the UK population with lung, oesophageal, brain, pancreatic and stomach cancers (less than 21% living after five-years post-diagnosis) [2].
Poor cancer outcomes are due to the complexity of a tumour’s biological make-up, metastasis (spread of cancer cells around the body from the starting tumour to form secondary sites, or metastases), time of diagnosis (often late) and lack of treatment success (resistance of tumours to selected drugs). Currently, most cancers are diagnosed upon an individual noticing clinical signs and symptoms resulting from the tumour. Tumours of the pancreas, for example, usually do not cause symptoms until they grow large enough to press on nearby nerves or organs, where upon the individual will develop back and/or abdomen discomfort, along with non-specific symptoms such as tiredness. Other tumours may grow around the bile duct and block the flow of bile, resulting in the eyes and skin to appear yellow (termed jaundice). By the time a pancreatic cancer causes signs or symptoms like these, it is usually in an advanced stage, which means it has grown and most-likely metastasised beyond its starting location via the blood circulatory system or lymphatic system. Consequently, this means the survival chances are severely reduced because of the appearance of metastases that may also present new resistance characteristics against therapeutic drugs which makes successful treatment a lot more difficult.
So what is the answer to detecting cancers at an earlier stage? Cancer biomarkers appear to provide a very good starting point. Biomarkers are described as “cellular, biochemical or molecular alterations that are measurable in biological media such as human tissues, cells, or biofluids” [3]. So, what makes a good biomarker? In theory three main attributes need to be considered: first, as described, the marker must be present in peripheral body tissue and/or fluid; second, it must be easy to detect or quantify in assays that are both affordable and robust; and third, its appearance must be associated as specifically as possible with damage of a particular tissue, preferably in a quantifiable manner. When considering a cancer biomarker, the cellular, biochemical or molecular alterations within biofluids are indicative of the presence of a tumour within the body. Effective cancer biomarkers are very promising indeed, due to their potential to reduce cancer mortality rates by facilitating early-stage diagnosis. Research into a particular type of cancer biomarkers, circulating cell-free nucleic acids (cfNAs), is reasonably novel with certain biomarkers including micro-RNAs (miRNAs) being first reported in the past few decades [4]. As the name suggests, the cfNAs are found freely circulating in biofluids; such as blood, urine and saliva, having been actively or passively excreted from cancer cells. The existence of cfNAs in biological fluids of cancer patients has thus raised the possibility of their use not only as diagnostic, but also prognostic and therapeutic markers.
Focusing on miRNAs particularly, these non-coding nucleic acids with short sequences of around 22 bases in length (as opposed to hundreds to thousands of bases found in regular RNA) have been shown to be successfully evaluated in a wide range of tumours and cancer patients, and have the ability to produce a cancer ‘signature’. In other terms, the concentrations of selected miRNAs can be used not only to identify a specific cancer type, for example increased concentrations of miR-21 and miR-92a in blood serum are indicators of an individual with an invasive breast cancer, but also how advanced the disease is – with higher concentrations suggesting a more advanced tumour [5,6,7]. Another advantage of using cfNA biomarkers is that they can be sourced from patients by minimally-invasive means, including blood donation, and saliva/urine collection [8]. These collection methods would reduce the need for current procedures that are necessary for cancer diagnosis, including invasive biopsy procedures which are very uncomfortable, but have also been shown to heighten the risk of cancer spread from the biopsy site due to the localised disruption of healthy tissue surrounding the tumour [9].
miRNAs are currently detected and quantified via conventional means, such as real time quantitative PCR (RT-qPCR), a method commonly used to calculate the concentration of NAs of interest. However, this method is prone to multiple errors and is generally costly, therefore new methods of detection/quantification that display greater consistency and sensitivity need to be considered. One such method focuses on the use of biosensors that produce a measurable ‘signal’ upon reacting with a target. Novel and low cost peptide nucleic acid (PNA) probes are currently being engineered within the clinical biosensors laboratory group, led by Dr Sylvain Ladame at Imperial College London, as part of a non-invasive technology for the detection of specific miRNAs [10]. PNAs are DNA (or RNA) analogues with a more stable backbone (a peptide backbone replacing the sugar-phosphate backbone in DNA/RNA) that allow more sensitive and more specific detection of target NAs by binding to corresponding base pairs in the target via Watson-crick base pair binding (e.g. A with T, C with G). PNAs also offer the advantage of great chemical stability, even in extreme temperatures, and a significantly stronger resistance to enzymes (e.g. DNases and proteases) than traditional oligonucleotides. Furthermore, PNAs are more responsive than DNA when detecting point mutations (individual base substitutions within a nucleotide) making them ideal for specific detection of unique nucleic acid sequences. Upon engineered PNA probes binding to their targets specifically designed chemicals on the ends of probes interact with each other to produce a characteristic fluorescent signal. This fluorescence can then be easily measured to not only confirm the presence of specific miRNAs but also quantify them; with the intensity of the optical signal being proportional to amount of miRNA present. Furthermore, a combination of PNA probes specific to different miRNA strands could help measure the expression of multiple miRNAs in one step from a small volume of biosample collected non-invasively from a given patient. The resulting miRNA profile (also termed ‘signature’) has potential to be used in the clinic to determine a variety of factors including cancer diagnosis, cancer type or metastatic properties, thus suggesting the prognosis of a patient (i.e. how well they are to respond to treatment etc).
With increasing amounts of carcinogenic factors that we are surrounded by and the promise of cancer biomarkers and biosensors becoming clearer, it is only a matter of time before diagnosing cancers at an earlier stage becomes a common routine. The use of novel engineered biosensors such as the one described holds great potential due to their sensitive and highly-selective nature along with low financial costs associated with the technology.

References:
1. Cancer Research UK (2013) “All Cancers Combined Key Facts”. [Online] Available from: http://publications.cancerresearchuk.org/downloads/Product/CS_KF_ALLCANCERS.pdf (Accessed 24.04.2014).
2. Office for National Statistics (2013) “Cancer Survival in England: Patients Diagnosed 2007-2011 and Followed up to 2012”. [Online] Available from: http://www.ons.gov.uk/ons/dcp171778_333318.pdf. (Accessed 24.04.2014).
3. Hulka B.S. “Overview of biological markers”. In: Hulka BS, Griffith JD, Wilcosky TC, eds. (1990), Biological markers in epidemiology, pp 3–15. New York: Oxford University Press.
4. Lee, R.C., Feinbaum, R.L., and Ambros, V. (1993) “The C. Elegans heterochronic gene lin-4 encodes small RNAs with antisense complementary to lin-14”. Cell. 75, 843-854.
5. Redova, M., Sana, J., and Slaby, O. (2013) “Circulating miRNAs as new blood-based biomarkers for solid cancers. Future Oncol. 9, 387-402.
6. Calin, G.A., and Croce, C.M. (2006) “microRNA signatures in human cancers”. Nature Reviews Cancer. 6, 857-866.
7. Gormally, E., Caboux, E., Vineis, P., Hainaut, P. (2007) “Circulating free DNA in plasma or serum as biomarker of carcinogenesis: Practical aspects and biological significance”. Mutation Research. 635, 105-117.
8. Schwarzenbach, H., Hoon, D.S., and Patel, K. (2011) “Cell-free nucleic acids as biomarkers in cancer patients”. Nature Reviews Cancer. 11, 427-437.
9. Loughran, C.F., and Keeling, C.R. (2011) “Seeding of tumour cells following breast biopsy: a literature review”. Br J Radiol. 84, 869-874.
10. Choi, Y., Metcalf, G., Sleiman, M., Vair-Turnbull, D., Ladame, S. (2014) “Oligonucleotide-templated reactions based on Peptide Nucleic Acid (PNA) probes: Concept and biomedical applications”. Bioorganic and Medicinal Chemistry. 22, 4395-4398.

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PhD Researcher based at Imperial College London, UK. Doctoral Research is based on designing and developing novel cancer biosensors via organic chemistry. Previously a Masters degree in Cancer Biology, and Bachelors in Biochemistry was obtained.

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