Please note that this presentation aims at summarizing currently available data only without the claim of representing established knowledge. Thus information presented in this course may be subject to change in the future due to the rapid involvement of the field of extracellular vesicles. My name is Lorraine O'Driscoll. I am a member at large of the eyes of board. I will narrate this lecture on EBS as cancer biomarkers, which has been coordinated by myself and contributed to by three other experts in this field, as listed here. Now, let's begin. Based on our work in this field where we have collectively separated and analyzed EVs from a broad range of body fluids as potential cancer biomarkers. Here we come together to outline what cancer biomarkers are, their intended uses. And why particularly those that are relatively easily accessible using minimally invasive or even non-invasive methods are really needed. We highlight different types of minimally invasive biomarkers that are being investigated including of course extracellular vesicles or EVs. We summarize with some examples different model systems that have been used and are being used to generate an understanding of EVs as cancer biomarkers. We then progress to outlining EV quantities and several types of EV cargo as cancer biomarkers considering the merits and the challenges of each approach. Again, examples of studies in this space are summarized. We note some example studies that have progressed to clinical trials. Please keep in mind that the example studies we have included in this study are only examples. We do not believe them to be superior to others that we have not listed but in the interest of space a comprehensive review is not possible or intended here. Finally, we briefly critique the state-of-the-art in this area and what we believe the important future directions will be aimed at, achieving the most from EVs as cancer biomarkers for both societal and economic benefit. So what are cancer biomarkers? A cancer biomarker by definition is a biological molecule produced either by tumor cells or the microenvironment in response to cancer that can be objectively measured and evaluated as an indicator of cancerous processes within the body. In the simplest terms, cancer biomarkers include those that indicate the presence of cancer or provide information about the likely future behavior of a cancer. That is, its likelihood of progression or its response to therapy. So in principal cancer biomarkers may be developed for many purposes including for screening and risk assessment, for cancer diagnosis, for cancer prognosis, for predicting response to therapy, as pharmacodynamic and pharmacokinetic indicators, and as recurrence indicators. Most cancer biomarkers currently in clinical utility are tissue-based and so require invasive biopsies. Arguably the ideal cancer biomarkers are those that are present in body fluids and so can be evaluated using non-invasive or at least minimally invasive techniques. The sampling of body fluids is sometimes referred to as a liquid biopsy. It is estimated that one in every two people will develop cancer at some stage during their lifetime. And we must consider that cancer is the second leading cause of death after cardiovascular diseases. So developing non-invasive or minimally invasive cancer biomarkers is of substantial interest to many stakeholders. First and foremost, the patient, early as possible diagnosis and optimal treatment is key to achieving the best quality of life and best possible outcome from cancer. Clinicians want better, easily accessible, reliable and robust biomarkers to be able to manage every cancer patient in as personalized a manner as possible. Healthcare and the economy also benefit when patients are diagnosed and treated as early as possible. And of course, industry is also irrelevant stakeholder. The potential to develop liquid biopsies for cancer is big business. As explained, liquid biopsies have potential to provide complementary or ideally alternative cancer biomarker approach to traditional solid biopsies. Solid biopsies have many limitations including the fact that they cannot be consistently performed in certain situations or in real time. But despite the obvious benefits of each minimally-invasive liquid biopsy approach, several challenges have yet to be addressed to make this a reality for routine use. Entities in body fluids that are being investigated as cancer biomarkers include circulating tumor cells abbreviated to CTCs, circulating tumor DNA abbreviated to ctDNA. And more recently EVs, both their quantities and their cargo. As summarized in the table here each type of liquid biopsy entity has benefits and limitations. It is conceivable that the best panel of cancer biomarkers will include a combination of these entities. Circulating tumor cells, circulating tumor DNA and EVs. Focusing on EVs, the topic of this chapter, many model systems are being used by researchers in order to identify and understand the potential of the EVs as cancer biomarkers. Immortal cell lines are often used in research as a starting point for studies of cancer. In EV research vesicles released from cells can be separated from the cultured cells condition medium, characterized and analyzed. As with any other application of cancer cell lines and research, their merits and challenges also hold true When included in EV research studies. Decent food, merit, such as simple systems, relatively low cost, relative ease of handling, continuous self-renewal, long-term culture is possible and the suitable for initial assessment of the reproducibility and robustness of EVs as biomarkers. Challenges may include altered cell properties compared to the original tumor. Some cell lines have been genetically manipulated. Genetic drift. Absence of their local environment. No interaction with other cells. The need to culture in serum-free medium or medium with EV-depleted serum and this model does not consider other factors present in a complex in vivo system including EV's released from non-cancer cells. Then following on from medium condition by cultured cells, pre-clinical animal models are extensively used in cancer research including EV research for cancer biomarkers. Some benefits of including animal models are in vivo models, bring the complexity of a living organism necessary to move towards truly understanding EV release and function and have potential to try EV activity in the body in real-time. Some challenges include differences between animals and humans, tumors in mice are somewhat artificial. EVs may be affected by different and inappropriate injection roots and the difficulty to replicate in vivo relative concentrations of EVs that may exist in humans with cancer. However, as exemplified in this table, a range of mouse models have been used so far in the quest to identify and understand EVs and their content as cancer biomarkers in many cancer types. And undoubtedly, while respecting the three hours of reduction, replacement and refinement for ethical and appropriate use of animals, these models will continue to form an important part of future EV research. Of course, appropriate by a food specimens from content and patients and appropriate volunteers as controls are very important models to be used for cancer biomarker identification and validation. And indeed, many research groups are investigating EVs from a range of body fluids, including blood plasma or serum, urine cerebral spinal fluid and saliva for this purpose. As explained, compared to the tissue biopsies, EVs from body fluids are non-invasive or minimally invasive and a relatively easy to access. And so it could be used for ongoing monitoring of disease of its response to therapy of its recurrence, etc. However, considering the heterogeneity of tumors and the heterogeneity of EV populations, we still have much research to do in order to achieve the best and the most appropriate use of these EVs. Added to this is a challenge that the volume of specimens from patients. That is the real world is very limited compared, for example, to condition medium from cancer cell lines grown in the laboratory. So this calls for higher throughput, highly sensitive methods for EV analysis. Characteristics of EVs that may be measured as potential cancer biomarkers include EV concentrations in a body fluid and, or EV carried materials, their cargo, including DNA, RNA, proteins, lipids, metabolites. And cancer biomarker panels could be of a single characteristic or combinations of these characteristics. So what aspects of EVs from bio fluids could be considered as cancer biomarkers? The first consideration is EV concentrations. In theory at least, differential concentrations of EVs could be suitable as cancer biomarkers. However, it must be considered that normal cells release EVs. And so finding the cancer-related EVs may be like finding a needle in a haystack. So there are different opinions on the likelihood of being able to use EV concentrations as biomarkers. The merits of such approach, if possible would be it's the simplest type of analysis of EVsm, at least theoretically. Challenges are lack of standard methods to routinely and reliably count EVs, and quantifying EVs associated with cancer in the midst of EVs from healthy normal cells could be problematic. So far, there does not seem to be any conclusive evidence of EV concentrations being a reliable robust cancer biomarker. EV carried RNA has been quite extensively studied as potential cancer biomarkers. Different species of RNA including messenger RNA microRNA, long non-coding RNA have been associated with EVs and related to cancer. Efforts are made to determine if the RNA is in or on the EVs compared to some molecules. It seems that RNA is quite abundant. And of course, it has an advantage that there are highly sensitive and specific techniques developed for RNA analysis. However, for the EVs RNA reliable references for quantification have yet to be identified and agreed upon. Here in the table, we outline some of the studies that have been performed to date in favor of RNA as cancer biomarkers. But jubilant as a note of caution, it has been suggested that only a small fraction of total micro RNA and plasma is associated with EVS and also conclusions regarding microRNA presence in EVs may be dependents on the EV separation methods used. Specifically the work described in reference 32 indicates a depending on whether or not ultracentrifugation is used to separate EVs clearly affects the conclusions with regards to this and whether or not the cancer patients plasma EVs microRNA Has clinical relevance. EV derived DNA is also being explored as potential cancer biomarkers, although such studies of EV derived DNA as biomarkers of disease have been limited in number so far. However, some have reported the potential of EV DNA in small pilot studies of cancer. DNA found in EVs apparently includes mitochondrial DNA as well as genomic DNA. Analysis of EV carried DNA could be beneficial as theoretically it may represent the entire genome and also reflect mutation status of the parental cells. Such as showing B-raf mutations detected in double-stranded DNA from EV's of patients with melanoma. However, most DNA found to be associated with EVs is apparently sensitive to DNAase, implying that it is on, rather than in, the EVs. Whether this association is specific and correct has yet to be established. Furthermore, and colleagues reported that only a small fraction, about 10%, of EVs carry material consisting of DNA. This highlights that EVs are heterogeneous. And further analysis is required to understand the processes by which DNA could be transported via EVs and if it is relevant as cancer biomarkers. EV derived proteins have been explored as cancer biomarkers for more than a decade. This may be due to the extensive research that has been done to analyze the role of EV derived proteins in other diseases as well as cancer. Also protein analysis is well-established and most laboratories have adequate infrastructure to do protein analysis and are well experienced in this. Many proteins have been suggested as potential EV derived biomarkers, some identified through analysis of EVs secreted by cell lines, some true pilot studies, and others directly from a collection of plasma or serum samples, for example. Similar to other EV derived molecules, proteins identified in these studies are based on the abundance and whether they are relevant to the disease. Challenges in which scientists may face in these studies are whether isolation methods they utilize can provide protein biomarker profiles which are truly EV molecules. Most of the isolation methods have resulted in possible protein contaminants coisolated with EVs. Analysis of EV derived lipids as potential cancer biomarkers are still in their infancy. Few publications have looked into this possibility, and have mainly used limited sample sizes. The limited number of studies so far may be due to the complexity of lipid analysis. As one needs to have access to infrastructure such as mass spectrometry to be able to analyze lipid profiles of EVs. As there are only a few studies of EV derived lipids and how lipids may play a role in EV biogenesis thus far, further studies are needed to investigate EV lipids in a more detailed manner. Similarly to EV lipids, studies on EV derived metabolites are limited in number so far. This may also be due to the need for specific infrastructure to analyze metabolite profiles. However, unlike lipids, analysis of metabolites can be performed using a much more limited amount of starting material. This is useful in particular if the studies are based on clinical specimens, maybe that need to be derived from bio banks. However, currently normalization methods from metabolite analysis have yet to be clearly defined. So as such, further studies are needed to explore the utilization of EV metabolites as biomarkers in cancer. Research in many countries has progressed to clinical trials aimed at evaluating EVs in bio fluids as biomarkers for cancer. With interest in the relevance as diagnostic, prognostic, or predictive biomarkers. This table summarizes examples of such trials. Typically, these are prospective longitudinal studies focused on a particular cancer type or subtype. An example study design is shown where blood specimens are procured for subsequent EV separation and analysis following SOPs. The specimen's been taken prior to treatment commencement at fixed times, then throughout treatment and post treatment. While every study may have a different design depending on the question being addressed, what is important that prior thought is given to planning the study and that all specimens are collected and processed the same to ensure that differences observed are meaningful biological differences, not due to technical differences. The trials most often include analysis of EVs in blood plasma or serum, although for prostate cancer, urine EVs are also quite often evaluated. While some are single center trials, most include multiple institutes. The results of the first study on this table support EV's as cancer biomarkers by showing that high EV tissue factor activity as measured by the fibrin generation test is associated with an increased risk of venous thromboembolism in cancer patients. In particular, in those with pancreatic cancer. Due to the prospective nature of the other studies, results are yet not available. And so something that we must look forward towards in the future. Of course, such translational clinical trials are beneficial for generating proof of principle data on EVs as cancer biomarkers. However to critically evaluate their true benefits and move towards clinical utility, more well powered multicenter international clinical trials will be necessary. To ensure where possible that any such research can routinely and efficiently be progressed from bench to bedside. It's essential that effort is invested from the outset into ensuring that studies are very well designed and that the same standard operating procedures developed will be adapted in all centers involved in any given clinical trial. Because of the many considerations that need to be addressed including those exemplified on this slide, members of the ISF community are currently working together to develop evidence-based guidelines for this purpose. These will build on and complement ISF's minimal experimentation requirements for definition of extracellular vesicles and the functions as published in 2014 and updated in 2018. We would also strongly encourage any researchers working in this field to familiarize themselves with and utilize the knowledge based EV track, which can be found at evtrack.org and which is focused on transparent reporting and centralizing knowledge in EV research. We would also encourage early engagement with regulators such as the FDA, EMA, etc to help ensure that relevant outcomes can be progressed to clinical utility in as timely a manner as possible. So in conclusion, evidence suggests that EVs have potential as diagnostic, prognostic, and predictive non-invasive or at least minimally invasive biomarkers for cancer. And that EV researchers working together throughout the world and to the highest standards possible will maximize use of EVs in as timely a manner as possible for both societal and economic benefit. And finally, here we have included some examples of relevant literature for further reading. We very much hope you enjoyed this lecture on EVS as cancer biomarkers, thank you.
