The Role(s) of Biomarkers in Precision Medicine  

Abstract

Small, membrane-encapsulated transport vesicles known as exosomes present an overwhelming potential in the field of non-invasive biomarker discovery due to their robust integrity in collection from biofluid samples. This minireview briefly discusses their diagnostic and prognostic contributions in three different pathology domains of high interest: Tumour Metastasis/Cancer Research, Neurodegenerative Diseases, and Drug-Induced Organ Injury. This text also attempts to shed light on outstanding general concerns of the exosomal biomarker approach including uncertainty in the diagnostic performance, heterogeneity, and isolation protocol of these potential candidate biomarkers. At present time, advancements in the ‘omics approaches translate into a high discovery phase of these biomarkers in precision, high-profile medical applications.

Introduction

Tissue biopsies have remained the benchmark in diagnosis and screening of various pathologic states due to the increasing number of validated tissue-variant biomarkers currently on the horizon. Several studies employ methods such as endoscopic biopsy that prove successful in the collection and quantification of potential prognostic biomarkers of cancer progression, angiogenesis, and atypical immune behaviours. However, the invasive nature of this procedure has accelerated the communal interest in cell-free biomarkers that can be selectively obtained through non-invasive means, such as through collection from biofluids and liquid biopsies. Cellular constituents are discovered in high abundance within serum, plasma, urine, saliva, and other biological fluid forms. Fluctuations in the expression of intra- and extra-vesicular targets are currently being explored worldwide as potential high-performance candidate biomarkers for global inflammation and pathological states.

An area of recent interest focuses specifically on exosomes, a sub-class of extracellular vesicles (EVs). Exosomes are small organelles (30-200 nm in diameter) produced via mechanisms of cellular fission from membrane transport vesicles and/or endosome cells. The shedding of these microvesicles permits the release of an array of biomolecules such as lipids, carbohydrates, functional proteins and nucleic RNA subtypes including non-coding microRNAs (miRNAs) that are prone to pathological changes in concentration and expression. Many recent studies attempt to validate the use of such targeted profiling in the minimally-invasive diagnosis of several common, debilitating illnesses including cancer subtypes, neurodegenerative diseases and drug-induced organ toxicity. The main objective within this minireview is assess the advancements and efficacious use of exosome biomarkers within these three pathology domains, while additionally addressing outstanding concerns of overall diagnostic performance, heterogeneity, and exosomal isolation protocol. A critical analysis of exosomes is highly essential to furthering the validation and wide-spread use of these novel candidate biomarkers in personalized medicine.

Cellular fission is a recognizable feature in tumor malignancy. Shedding of tumor exosomes appears to be under mediation of extracellular signals, releasing enhanced concentrations of tumour antigens that are responsible for the suppression of T lymphocyte and monocyte activation. Detection of increasing concentrations of tumour-derived exosomes and circulating tumour cells (CTCs) is positively correlated with clinical outcomes including increasing severity of cancer status, increasing metastasis, and shorter time to relapse.

Further examination of the diagnostic and prognostic sensitivity of tumour-derived exosomal biomarkers in patients with lung adenocarcinoma reveals that there is a significant increase in expression of peripheral exosomes in those with lung adenocarcinoma compared to healthy controls. Findings of similar nature give rise to the future diagnostic potential of these biomarkers, permitting less invasive alternatives for cancer patients. A table obtained from a 2010 review of miRNA biomarkers in cancer research provides an index of circulating serum miRNAs that have been regarded as candidate biomarkers in diagnosis of common cancer subtypes,

Another feature of exosomes and their derived-miRNA profiles are their prognostic capabilities. Identification of miRNA biomarkers of tumorigenesis can further advance screening initiatives in asymptomatic patients with a family history of cancer. A recent study aimed to access the prognostic ability of validated candidate biomarkers in head and cervical cancer patients. Results from a discovery cohort screened prior to and after radiotherapy treatment indicate 8 plasma-derived miRNAs that discriminate between cancer patients and healthy controls, with miR-186-5p demonstrating the highest sensitivity of therapeutic-response. Studies of similar nature display findings of miRNA profiles consistent with biopsy results of tumour tissue, which further validates the clinical efficacy of biofluid analysis in the future cancer screening protocol.

Exosome biogenesis contributes to the transport and intracellular communication of proteins, which can occur under physiological and/or pathogenic conditions. When conditions of multivesicular body fusion with lysosomes are unfavourable or limited, release of exosomal contents into extracellular space serves as a secondary, alternative pathway to protein degradation, promoting the enhanced modulation of function in recipient neighbour cells.

This presents a non-selective, opportunistic function of exosomes whereby, amyloid precursors can be engulfed within an exosome and released extracellularly in pathogenic cellular conditions as amyloid deposits or plaques. This feature of non-selectivity is observed in Alzheimer’s Disease (AD) where the global spread of amyloid deposits in the brain is non-specific to a particular set of cells or area of interest, while Parkinson’s Disease (PD), on the contrary is a highly specific disease down to the type of brain tissue affected and amyloid protein. In both neurodegenerative states, there is an increasing amount of evidence suggesting an inherent link between exosomal miRNA responses and pathogenesis.

A study published in 2015 examines the diagnostic potential of an exosomal miRNA profile in cerebrospinal fluid (CSF), determining their association and discriminative ability of neurodegenerative diseases, PD and AD. This particular study uses flow cytometric analytics and electron microscopy to identify and observe exosomal structures by monitoring the presence of phenotype surface protein CD63 within the sample. 16 CSF-sourced exosomal miRNAs are significantly upregulated while 11 are suppressed in PD patients compared to age and sex-matched healthy controls. From this panel, analysis of 6 specific miRNAs reveals the high diagnostic sensitivity of miR-409-3p, miR-153, miR-10a-5p in PD patients with values 0.97, 0.92, and 0.90 respectively obtained through receiver operator characteristic (ROC) analysis.

Another publication released in the same year focuses its attention to the role of exosomal-derivative proteins in AD, assessing their biomarker sensitivity and association with clinical severity in a matched case-control study. Blood exosomal proteins are isolated from patients in the case (AD) group and control (AC) group, revealing higher levels of tau proteins and A that are significant among AD cohorts. Sensitivity assessment via ROC and area-under-the-curve (AUC) analyses determine that P-T181-tau protein display the highest diagnostic performance (AUC = 0.991) followed by P-S396-tau (AUC = 0.988), A1-42 (AUC = 0.987), and total tau protein (AUC = 0.731). Assessment of these proteins at the time of diagnosis as well as at timepoints 1-10 years post-diagnosis reveal that A1-42 levels are significantly elevated in Alzheimer’s patients in their second blood analysis versus at their time of diagnosis. The findings in this study allude to the possibility that exosomes are directly involved in the pathogenesis of neurodegenerative development, correlating severity and development of AD status to the presence of exosomal-derived amyloid-beta concentrations.

Drug-induced organ injury can be described as damage to organ tissue or its’ functional integrity attributable to the use of diagnosable medications. Injury is highly variable and dependent on the organ as well as heterogeneous patient lifestyles and concurrent medical histories.

One particular subtype, drug-induced liver injury (DILI), is currently of interest in the scope of biomarker discovery as the age of metabolomics and proteomic technology progresses. DILI is a potentially adverse outcome of use of hepatotoxic agents – carbon tetrachloride, d-galactosamine – as well as active agents – acetaminophen, isoniazid – in commonly diagnosed medications. DILI progression following drug administration is concerning for reasons besides clinical outcome; drugs that are suspected causative agents in hepatoxicity are often stripped of their FDA approval until otherwise proven efficacious in patient treatment intervention, and in this instance, under strict conditions of review. Therefore, development of prognostic biomarkers for DILI is essential to both early screening protocol and to treatments of other liver diseases in guiding heterogeneous dose-adjustments and efficacious utility of common drugs including acetaminophen in patients.

In a recent clinical trial using animal models exposed to acetaminophen, assessment of a selective exosomal miRNA panel as biomarkers in DILI is one of several endpoints in this study. MiRNAs, miR-122, miR-155, and miR-192, are selected based on previous performance within organ-injury studies as liver-specific candidate biomarkers. qPCR results reveal altered expressions of the panel both in the presence and absence of treatment with antioxidant, N-acetyl cysteine (NAC treatment). No NAC treatment shows significant increases in panel expression while NAC treatment results in down-regulated expression of these liver-specific exosomal microRNAs.

Such findings demonstrate that liver-associated miRNA profile displayed high, independent expressional changes that correlated with increasing hepatocyte damage in the absence of treatment. Presence of treatment interventions allows for enhanced monitoring of biomarker capabilities as it corresponds to decreasing target expression as antioxidants repair hepatocyte damage. Moreover, concurrent findings in this area are suggestive of the high precision ability of miRNA targets to detect drug-induced organ injuries with high tissue-specificity.

While employment of exosomes and their constituent biomolecules are well-defined their approach and applications to high-profile, precision medicine, outstanding concerns of isolation protocol, overall diagnostic performance and heterogeneity still remain to be seen. Various studies report issues in the standardization of exosome isolation protocol from biofluids and tissue-biopsy, due in-part to variation of types of biofluids and requirements of different pathologies. While serum and plasma sampling is functionally advantageous to exosomal shedding in tumorigenesis and cancer detection, CSF is the primary biofluid-type assessed in neurodegenerative cases. Another important issue highlighted in exosome isolation is the resolution of indistinguishable extracellular material based on size overlap of exosomes and other structures. This also relates to uncertainty in the overall diagnostic/prognostic capabilities of biofluid biomarkers as a whole. Many reports attempt to validate biofluidic molecules expressing high yields and associations which could likely be the result of sampling error due to lack of rigour in isolation protocol.

Also in question is the reliability of exosomes and their miRNA performance among heterogeneous distributions. While alterations in expression of exosomal data can be attributable to the general inflammation and/or immune stress of the body within a pathological state, the specificity in miRNA profiles within each pathological domain provides further insight to this concern. Findings within the literature of drug-induced organ injury suggest that selective miRNA profiles will perform independently of other targets as their function is conducted in a highly-ordered manner of tissue-specificity.

Future analyses should maintain focus in the analysis of exosomal size and standardized isolation protocol associated with differential disease diagnoses. As well, secondary or follow-up research could expand on plausible hypotheses and supporting literature concerning the link between exosome biogenesis and general inflammation in the body which was not included in this focused critical minireview.

Conclusion

In summary, this minireview discusses the primary implications and advancements of exosome biomarkers in precision medicine, addressing the efficacious nature of this research and additionally providing insight to the future diagnostic credibility of these targets.