Are there enough clues for us to find the killer?
One of the hot topics surrounding the advancement in cancer management is the integration of liquid biopsies in routine cancer treatment and care. We know that cancers occur due to mutations and other modifications in our genes that control cell survival, growth, and proliferation. We also know that cancer tumours shed their cells, their proteins, and their genetic materials into our blood stream. So, wouldn’t it make sense for us to look into our blood to find if there are traces of ‘bread crumbs’ that a cancer tumour leaves behind, and consequently detect the cancer early and treat it before it harms us?
Before we examine the answer to that question, let’s first understand the difference between the classic tumour markers, tissue biopsy, and liquid biopsy.
Exploring cancer scenes
You might have come across the term ‘tumour markers’ during your annual health check. Most likely, the tumour-marker testing that you get done today during your checkups is not the liquid biopsies we are talking about here. Although also a blood test, the classic tumour marker tests look for proteins that tend to elevate in levels in the body when one has cancer. However, many of the same proteins could also be elevated if one has other non-cancerous disease or abnormal physical conditions. Hence, tumour markers are non-specific and cannot accurately indicate whether one has a certain cancer or not. What they do is they signal to the doctor that certain cancer-associated proteins are high and further testing should be performed. As non-specific as the tumour markers may seem, certain tumour markers do tend to be associated with certain cancers. For example, PSA is associated with prostate cancer, CEA with colorectal, lung, and breast cancers, and CA125 is associated with ovarian cancer.
Currently, the gold standard for diagnosing cancer is through tissue biopsies. It requires physicians to first locate the suspected tumour. A piece of the suspected tumour tissue is to be removed from the body using tools such as a brush, a needle, a knife, or a puncture instrument. The tissue sample is then sent for processing and staining so a pathologist can examine the cells to determine whether cancerous cells exist, and what their origin and characteristics are. Tissue biopsies can be painful and invasive and are sometimes difficult to obtain. Up to 25% of lung cancer biopsies fail to obtain enough tissue for assessment. Moreover, the complete ‘picture’ of the tumour is often limited as the molecular properties can differ within a single tumour or within a single patient with multiple tumours, hence the tissue that is being taken out of the tumour may show only one piece of the puzzle. Also, the tissue obtained at the time of initial diagnosis, like a photograph, may not reflect the disease condition throughout the course of the disease and treatment.
So, is liquid biopsy the new savior to cancer detection and management? Perhaps it is worthwhile to review the past and the present of liquid biopsy.
Finding cancer clues
Liquid biopsy can be defined as the non-invasive approach to molecular profiling of cancers. In other words, to study and understand the genetic characteristics of a person’s tumour so the right diagnosis and treatment can be applied. It obtains cancer information from a person’s blood or other bodily fluid, such as urine and saliva, by targeting one of the following: circulating tumour cells (CTC), which are cells shed by the primary tumour into the blood stream/bodily fluid; circulating tumour DNA (ctDNA), which contains cancer related mutations and comes mainly from dead tumour cells; and exosomes, the vesicles released by both tumour and non-tumour cells that are used to transport genetic instructions between cells. Theoretically, all this information can present at once to provide a complete picture of the cancer and be assessed throughout the journey of the disease.
Surprisingly, the discovery of ctDNA is not new. In 1948, Mandel and Metais detected non-cell-bound nucleic acids (cell-free DNA) in the bloodstream of individuals with cancer. Subsequently almost 30 years later in 1977, Leon and co-workers reported a higher amount of cell-free DNA in patients with cancer than in individuals without the disease, which influenced further research to be done. The reason why it took more than half a century to be able to apply liquid biopsy clinically like we can today was due to the limitation in technology. The analysis of tumour material obtained by liquid biopsies requires highly sensitive tests because ctDNA is fragmented and highly underrepresented compared to DNA from normal cells, and only a limited number of CTCs can be isolated from a blood sample.  . It is only within the past 5 years  that we have seen sufficient advances in sequencing and PCR (Polymerase chain reaction) technologies to allow us to pick up the minute signals that tumours leave behind in blood.
Hearing from the investigator
Today, liquid biopsy has the great potential to determine the genomic profile of patients with cancer, monitor treatment responses and relapse, and assess the emergence of therapy resistance.
One figurehead in this field is Professor Alberto Bardelli. Bardelli is a Full Professor at Dept. of Oncology, University of Torino, Director of the Laboratory of Molecular Oncology and deputy Director of the Candiolo Cancer Institute-IRCCS, Torino, Italy, President Elect of the European Association for Cancer Research (EACR). He has dedicated his work to developing precision medicines for cancer patients, and has recently received the 2017 ESMO Translational Research Award for his liquid biopsy work in the field of colorectal cancer. He also performed the first comprehensive mutational profile of kinases in colorectal cancers and translated these findings into clinical practice by discovering the molecular landscape of response and resistance to EGFR, HER2 and NTRK1 blockade in colorectal cancer. These findings have found clinical applicability and were translated in liquid biopsies that are presently used to monitor patient’s response during treatment.
“Cancer is a very complex disease and the potential of liquid biopsy is far reaching. Their wide-ranging clinical applications are only starting to emerge.” said Professor Bardelli. “We now know that there is what we called ‘clonal evolution’, which is continuous evolution of the genome of tumour cells as they are subject to stressful conditions including targeted therapies. In other words, this is the effect of Darwinian selection on cancer cells, where therapy selects mutant cells (clones) which survive and proliferate. Liquid biopsy allows us to track clonal evolution at multiple points before, during, and after treatment, providing us with important insights into mechanisms of resistance, and can guide therapeutic decision-making. This is something that is rarely feasible with tissue biopsy due to its invasive nature and limitation on tissue accessibility.”
Another area where liquid biopsy could provide great value for cancer patients is how it can enable doctors to determine cancer relapse. “Imagine a patient, who’s had his tumour removed by surgery. Today, this patient would often have to go through chemotherapy after the surgery to help ensure all his cancer cells (including those that might have colonized other organs), are killed. Imaging (that is detecting tumours with sophisticated cameras) can’t tell us whether there are still small traces of the killer in his body after the tumour mass is removed. There is a great potential that we can, with liquid biopsy.” said Bardelli.
Minimal residual disease refers to a small numbers of cancer cells that remain in the patient during or after treatment when the patient shows no symptoms or signs of cancers. It is the underlying cause of relapse in cancer.
“If we could perform blood tests with the patient post-surgery to monitor (this) minimal residual disease, we could potentially save the patient from the burden of chemotherapy, which is toxic and has its multiple side-effects.” continued Bardelli. “Guiding intervention and circumventing cancers is, where I see, the near-future application of liquid biopsy. Here I believe liquid biopsies have a unique opportunity to change clinical practice in the next five years.”
Finding the killer
If killers exist no matter what, the best thing we can do is to catch them before they start killing. This is precisely where we are moving forward with liquid biopsy – cancer screening and diagnosis.
A recent study in Hong Kong that analysed the circulating cell-free DNA of Epstein–Barr virus (EBV) has demonstrated the potential of liquid biopsy in head and neck cancer screening. 
This holy grail, however, cannot be attained alone with research and advancement in knowledge of cancer and its mutations, technology and data science play an extremely crucial role here for detecting, deciphering, and interpreting the clues in liquid biopsy.
The free-floating DNA circulating in our blood that is not bound to cells has a relatively small and highly variable fraction of tumour DNA present in patients with cancer. As such, a testing technology with high sensitivity is required to pick up these signals. Digital-PCR-based technologies with a high level of analytical sensitivity and specificity have been developed. Next generation sequencing (NGS) is another technology with increased sensitivity to analyse liquid biopsies. Using NGS, millions of ctDNA sequences can be produced in a single reaction, and are subsequently aligned and compared with a normal reference DNA obtained from the same patient making it possible to identify mutations relative to the reference DNA sequence. In the future, digital PCR and NGS will probably both be used complementarily in liquid biopsy analyses. The former approach enables dynamic profiling of individual mutations, but requires us to first know what the mutations are or what to look for, whereas the latter technique enables the discovery of new mutations.
In addition to testing technology, machine learning and data science can expedite the advancement in deciphering and interpreting the “clues” captured in a liquid biopsy. There are still many challenges ahead. Getting enough data and aggregating them is enormously complicated. Having robust computer algorithms to recognize patterns and interpret data into meaningful and actionable day-to-day clinical practice requires many checks and balances to ensure accuracy and applicability. Having said that, based on the watershed we are witnessing in artificial intelligence and data science, we may not be too far away from seeing its wider deployment.
Liquid biopsies can be exploited for diagnostic purposes, to identify and track tumour-specific alterations during the course of the disease, and to guide therapeutic decisions. To fully integrate it into routine clinical practice, however, will require standardized procedures, large validation studies, guidelines and regulations.
About the Author
- Yu, M., Stott, S., Toner, M., Maheswaran, S. & Haber, D. A. Circulating tumour cells: approaches to isolation and characterization. J. Cell Biol. 192, 373–382 (2011).
- Diaz, L. A. & Bardelli, A. Liquid biopsies: genotyping circulating tumour DNA. J. Clin. Oncol. 32, 579–586 (2014). ?This review is 3 years old, you can quote this one if you prefer: Integrating liquid biopsies into the management of cancer. Siravegna G, Marsoni S, Siena S, Bardelli A Nat Rev Clin Oncol. 2017
- https://www.esmo.org/Press-Office/Alberto-Bardelli-Recognised-with-the-ESMO-Translational-Research-Award About ESMO: ESMO is the leading professional organisation for medical oncology. With 17,000 members representing oncology professionals from 150 countries worldwide, ESMO is the society of reference for oncology education and information. ESMO is committed to supporting members to develop and advance in a fast-evolving professional environment.
Dr. Stefan Zimmermann
Head, Innovative Therapies Unit, Immuno-oncology ION Oncology Department
Dr. Stefan Zimmermann is a medical researcher and a practicing oncologist, having published many peer-reviewed papers, especially in the area of lung cancer, in well-established clinical journals including Annals of Oncology, the Journal of Clinical Oncology, and The Oncologist. He has also contributed to various books and medical textbooks, namely: 2 editions of Side Effects of Medical Cancer Therapy, Springer Editions, 2013, and the Oxford Textbook of Oncology. Oxford University Press, 2016. He is the ESMO Press and Media Committee co-chair.