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Prof Stephen Pennington is a Senior Fellow at the Conway Institute of Biomolecular and Biomedical Research, University College Dublin (UCD). Prof Pennington did his PhD at the University of Cambridge, UK, and has always been fascinated by proteins, and how the structure of proteins controls their function and how they behave and operate inside cells and tissues.
Relatively early in Prof Pennington’s research career, he began working in a new field called proteomics, which involved an attempt to study proteins not as individual molecules, but on a larger scale. The beginnings of proteomics can be traced back about 20 years. While it showed initial promise, it is only now beginning to come to fruition, due largely to major improvements in the technology and techniques that are used to measure and identify proteins.
The early hope for proteomics was that it would provide a new way to separate out proteins using a technology called 2D gel electrophoresis. This technique meant that it was now possible to separate hundreds, perhaps 1,000 proteins, in one go. The challenge that followed was to identify the proteins that had been separated out by gel electrophoresis, said Prof Pennington. The turning point in this respect came when scientists started to use mass spectrometry to identify proteins separated out by gel-based approaches.
There were two main techniques for getting proteins and their sub-units, peptides, into mass spectrometers for analysis. These were so important that the scientists who developed them were awarded the 2002 Nobel Prize for Chemistry: John Fenn and Koichi Tanaka were co-winners of the Prize for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules; and Kurt Wüthrich was the other co-winner for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution.
“What it really meant was… instead of just studying proteins one at a time, we could potentially study them in tens or hundreds at a time,” said Prof Pennington. “The next turning point was the realisation that you didn’t have to put the whole protein into the mass spectrometer, you could break the protein up into fragments or peptide fragments and put the fragments into the mass spectrometer.”
Prof Pennington said further improvements in mass spectrometers has made it possible over the last 10-to-15 years to take a single cell, or a blood serum sample, break the proteins contained in that sample into peptides, and put these peptides into a mass spectrometer that is capable of now identifying thousands, if not tens of thousands, of proteins in one sample.
There were implications for the diagnoses of diseases that arose out of these new technologies, including for prostate cancer, where levels of protein serum antigen (PSA) are currently used as an imperfect diagnostic tool.
“We were fortunate enough to join a consortium, the Prostate Cancer Research Consortium, which is a Dublin-based consortium that eventually hopes to extend itself across Ireland — which enables us to connect with basic scientists, like ourselves, but [also] with clinicians and in particular urologists,” said Prof Pennington.
The urologists were interested in scientists applying the new technologies to identify the mechanism by which prostate cancer progressed, said Prof Pennington. He said they were also interested in scientists trying to identify some new biomarkers, which for prostate cancer would be proteins that might help to improve upon the current diagnostic tests for the disease.
The only ‘real show in town’ when it comes to the early diagnosis and subsequent monitoring of the progress of prostate cancer is by measuring PSA levels. However, the problem is what while PSA is used to screen for this cancer, it is not particularly accurate. “Many men who have an elevated PSA won’t have prostate cancer and some men who have very low PSA will have prostate cancer,” said Prof Pennington. “More importantly, the level of PSA at diagnosis is not necessarily indicative of how aggressive the disease might be.”
Presently, following an elevated PSA test, the patient will have a digital rectal examination where the clinician palpates the gland for any abnormality or growth. That will lead on to an MRI in some cases, but more commonly a biopsy to try and determine prostate malignancy.
There is a clinical need for a means of determining whether a patient diagnosed with prostate cancer is likely to die from the disease or not, said Prof Pennington. He said that what clinicians really need is to be able to intervene at an appropriate time in the progression of this cancer. They want to be more confident in their decisions, and the timing of them, when it comes to doing a radical prostatectomy or monitoring.
At the recent John Fitzpatrick Irish Genitourinary Cancer Conference 2017, Prof Pennington spoke about how proteomics is being applied to better understand prostate disease progression, and, working alongside urologists, to identify proteins that may be able to produce a diagnostic test that could play a big role in guiding treatment decisions for prostate cancer.
Prof Pennington’s team published a review article on this subject in Nature Reviews Urology, which was guided by urologists and identified two critical questions that clinicians face in respect of prostate cancer. “The first was, you would want to know whether a man who had been diagnosed with prostate cancer had the aggressive form of the disease or not — whether he was going to die from it,” said Prof Pennington. “That still is the ‘Holy Grail’ and that is something that a lot of people have tried to work towards.”
Prof Pennington said the other question that you would pose, if the disease was potentially going to need treatment or at least monitoring, would be around whether it was confined to the prostate or had spread beyond it.
With these two key questions in mind, Prof Pennington’s team set about designing protein discovery experiments based on samples taken from men who fell into two categories: Those who had a blood sample taken and who, followed up subsequently, had a radical prostatectomy/had the prostate removed; and those with the less-aggressive form of the disease. The scientists looked at the proteins in the blood from the two groups and found different proteins present.
They used mass spectrometry to measure many different proteins of interest that could potentially be used as biomarkers. They then ran a test based on a mathematical algorithm, which produced something called a ‘confinement score’, which indicated the probability that the tumour was confined to the prostate or had spread beyond it. There are about 15 or 16 protein fragments, or peptides, that are measured as part of this test.
UCD advised Prof Pennington to take a patent out on the test. This was followed up by clinical evaluation studies, which are underway and funded by an Enterprise Ireland commercialisation grant. Once that grant finishes, the intellectual property will be licensed to a UCD spin-out company founded by Prof Pennington and his co-researchers. That spin-out recently received funds from the Horizon 2020 programme that supports SMEs.
The goal is that this new test for prostate cancer will be available for clinical use in three years’ time. Prof Pennington said the test will, at that point, help clinicians make a decision about whether prostate cancer should be invasively treated, or whether an active surveillance regimen would be more appropriate.
The best approach for many patients in respect of prostate cancer is not radical prostatectomy, although this is currently the most effective front-line treatment, outlined Prof Pennington. Active surveillance is a good option for many patients, particularly for those with intermediate-to-low risk disease.
“The problem that they and their clinicians face [is that it is] recognised that there aren’t enough tests that allow that decision is to be made with some degree of confidence or comfort,” said Prof Pennington. “This test would be a part of the picture; it wouldn’t be the whole picture, [it would be] a part of the diagnostic process that would help the clinician and the patient to make that decision.”
Ideally, all men aged from 50 years should get their PSA checked. In private medical screens in Ireland and in the US, many men from the age of 40 are likely to have their PSA checked, said Prof Pennington. He would like to see the new test becoming part of routine care at the time of diagnosis and as they are actively monitored.
“The challenge that we face is that [it] measures a multiple of these proteins and it does it on a mass spectrometry platform,” said Prof Pennington. “That mass spectrometry platform, therefore, needs to be of clinical diagnostic capability and we think it is,” he said. The technology is reaching a turning point, because mass spectrometers are now being used in clinical diagnostic labs in hospitals, he said.
Prof Pennington said there is a need to do further research and this requires samples from more men. The samples currently are sourced through a connection with two hospitals in Ireland and one hospital associated with the Catholic University in Rome. These facilities will provide samples for Prof Pennington’s team to run tests in a new laboratory that will open at the Conway Institute in May.
“In terms of the clinical samples, to continue the evaluation of the study, what we are going to be doing is engaging with some key opinion-leading urologists to design the next clinical evaluation study, which hopefully would provide evidence to allow this to be incorporated into clinical guidelines,” said Prof Pennington.