Recently published as an invited article in Cellular Oncology, Paolo Nuciforo, Principal Investigator of the Vall d’Hebron Institute of Oncology (VHIO) Molecular Oncology Group, reviews and discusses various factors affecting or determining the inclusion of biomarkers in clinical practice as well as those limiting their use in “routine” diagnosis.
Paolo´s group at VHIO focuses on the validation and development of new biomarkers that could be of potential use in clinical practice. In our interview with Paolo, Vision towards Precision Oncology, we discuss some current factors that affect more biomarkers from successfully entering the clinic.
Paolo, what exactly are biomarkers?
Paolo Nuciforo (PN): A biomarker is a measurable indicator that can be objectively assessed for normal or pathogenic biological processes, or pharmacological responses to a therapy.
And why are they so important in oncology?
PN: Nowadays, determining a biomarker is the key to precision oncology. In the past we only used standard histology techniques, today´s oncology demands the more objective measurement and evaluation of what is occurring in a tumour and how it responds to treatment and, even predicting its response in order to best tailor treatments to the specific behaviour of the tumour at molecular level. We are in the era of precision oncology, a time of more selective treatments and increasingly “tailored” strategies aimed at precisely responding to the vast heterogeneity of tumours and the hugely complex nature of cancer. Each new diagnostic tool reveals tumours to be more complex, each with their own very unique characteristics. This scenario should also translate into a battery of biomarkers capable of selecting specific tumours on the basis of these peculiarities, to allow us to provide treatments that are also more selective and more “tailored”.
What exactly do you mean by precision oncology?
PN: Precision oncology refers to the therapeutic strategies for individual patients or tumours, with treatments becoming increasingly matched based on patient-specific clinical features. It also involves determining which individuals may benefit from one treatment or another. This firstly requires a good understanding of the properties of each tumour and, to do so, requires biomarkers.
Are many biomarkers currently being used in clinical practice?
PN: No, on the contrary. Although a great deal of research has focused on biomarker development, very few are actually currently being used in clinical practice.
Why is that? What are the main obstacles hindering the development and use of novel biomarkers in oncology?
PN: First of all, we have to consider what happens to a tumour sample from the point it is removed from the patient until it is analysed in the search for a possible biomarker. If we look at all the phases the sample goes through, we can group the obstacles into three broad categories: pre-analytical factors — that means everything that happens to the sample and all the processes it undergoes before being analysed, analytical platforms — essentially all the analytical methods the sample may be subjected to, and the sample itself.
Can you go into a bit more detail about each of these three factors?
PN: Of course. The important pre-analytical factors are ischaemia time, which is the period between removing the tissue and fixing or processing it; fixation methods; and fixation time. If ischaemia time is prolonged, it can affect the proteins, their phosphorylation and the stability of nucleic acids. For example, protein phosphorylation levels, which give us an idea of the activation of a biomarker, begin to change the moment the sample is removed from the patient. Similarly, mRNA starts to degrade already within an hour of surgical removal. For these very reasons, we need to have a clear idea of what needs to be done with the patient’s sample in the operating theatre itself since working with biomarkers begins there.
Fixation methods are also very important because each type of fixative has its respective technical constraints and can lead to variations in the detection of biomarkers. We therefore have to assess the most appropriate method for each application and tissue type. Furthermore, the time during which the sample is in contact with the fixative also has a significant impact on the expression of certain biomarkers and the integrity of nucleic acids.
The limitations associated with analytical platforms are specific to each technique, so we have to be aware of these limitations and take them into account when selecting the best one for the study of a specific biomarker in a particular sample.
Lastly, there are a number of limiting factors relating to the sample itself, the most important being its heterogeneity. When we perform a biopsy, within the sample different cell populations coexist, which means that the tumour population in which we are searching for the biomarker is diluted within the sample. In such cases, using a very sensitive technique can help us detect biomarkers expressed at very low levels.
Heterogeneity is also an intrinsic aspect of tumour biology. Biomarker expression and the presence of a mutation may vary in the primary tumours and their metastatic equivalents; we call this inter-tumour heterogeneity. Furthermore, cancer cell populations with different characteristics can also coexist within the same tumor; this is known as intra-tumour heterogeneity.
How do you think these issues can be resolved?
PN: First of all, we have to improve our knowledge and learn to better control the pre-analytical, analytical and post-analytical factors that can affect the end result of a molecular test. A “false positive” or “false negative” result has an enormously important implication for both the patient and for the type of treatment to be received by the patient.
It is therefore important to know what quality standards the laboratories that analyse biomarkers work with. This is particularly important in clinical trials, where, for example, the standardisation and validation of analytical procedures is paramount for studies of biomarkers that predict response to a given drug, and this is an increasingly common demand from those seeking partners of excellence to participate in such trials.
How can the standardisation of these processes be guaranteed? How is it done at VHIO?
PN: Our laboratory meets the technical requirements to cover the entire lifecycle of an analysis, from the pre-analytical phase, including handling and administrative management of tumour samples, to issuing the results report. Last year, under stringent quality parameters, the Spanish Accreditation Body (ENAC) accredited our molecular oncology laboratory for determination of gene amplification by fluorescence in situ hybridisation (FISH) and protein expression by immunohistochemistry under strict parameters, making it one of the molecular pathology laboratories with the highest number of analytical tests with ISO 15189 accreditation. This provides us with a strong advantage over other laboratories in terms of demonstrating the quality of our analytical testing as well as the biomedical research we carry out.
Which healthcare professionals have an important role throughout this process?
PN: Apart from the oncologists, who are responsible for designing the studies and reviewing the methodology, the role of pathologists in cancer research is crucial, particularly when there are samples taken and biomarkers analysed. When dealing with samples, in order to prevent bias during collection and for the interpretation of results, it is very important to establish and use protocols that are developed and standardised by pathologists. Pathologists should form an integral part of trial design committees because it is important that they know how the samples that they will be the first to process will subsequently processed, and how all this will affect the results.
Paolo, tell us a little about your vision towards the future: can you imagine what might be expected of a tumour sample in five or ten years? One wish, what would it be?
PN: In molecular oncology, we have to be able to see ahead into the future. We shouldn’t be asking ourselves what we can do today with a patient sample, but what the patient will need us to do with it five years from now.
My wish would be to have an increasing number of new techniques that are more quantitative and objective, more “resistant” to the influence of pre-analytical factors, and that facilitate the analysis of multiple biomarkers simultaneously using a minimum number of patient samples. These techniques would replace the current qualitative and singleplex techniques. In other words, I would like to have advanced techniques that are capable of squeezing out every last bit of information contained within a tumour sample using only a small part of it.
This would mean changing or adapting what has been regarded as the gold standard for several decades so that patients have access to new treatment options through novel molecular analyses.
This, for me, is the direction in which we really need to go.
Obstacles to precision oncology: confronting current factors affecting the successful introduction of biomarkers to the clinic. Prudkin L, Nuciforo P. Cell Oncol (Dordr). 2014 Sep 4. [Epub ahead of print]