Cancer Biomarkers in Exosomes
A Novel, Sensitive, and Specific Approach to Liquid Biopsy


his month’s Translational Research discussion features exosomes and their important role as a valuable tool to obtain the ever-increasing data establishing the value/utility of liquid biopsies.

This article clearly summarizes the history of progress in this space and the important complementary, sometimes unique, role that exosomes will play with the other tools measuring substrates, such as Circulating Tumor Cells (CTCs) and Circulating Tumor DNA (ctDNA), as biomarkers obtained non-invasively for cancer diagnoses, therapy development, and monitoring. The distinction between micro-RNA (miRNA) and long RNA is discussed, as is the first commercial exosome-based urine test to rule out the presence of a significant number of high grade prostate cancers.

Blood-based liquid biopsies enable easy access to genetic materials from the tumor without the need for an invasive tissue biopsy. Technological advances, including detection of rare mutations in a background of wild-type targets, have generated a boom in the field of liquid biopsies. Tumor-derived materials such as RNA/DNA are generally found in three types of sources: (1) circulating tumor cells (CTCs), (2) circulating tumor DNA (ctDNA), and (3) tumor-derived exosomes and other extracellular vesicles (EVs).

Circulating cell-free DNA (cfDNA) has recently been studied as an origin of tumor DNA for genetic profiling. Continuously shed into biofluids through cell death mechanisms of apoptosis and necrosis (Fig. 1), CfDNA is highly fragmented but can still be used to detect specific gene variants that are telltales of cancer and may inform best treatment practices. For example, NSCLC tumors bearing activating mutations in the EGFR gene respond well to tyrosine kinase inhibitors; however, most patients later become resistant to this therapy due to the EGFR T790M mutation. These mutations can be detected from a tissue biopsy as a companion diagnostic, but the first cfDNA test for these mutations (cobas® EGFR mutation test v2) has now been approved by FDA when tissue is not available.

There are also many laboratory-developed tests (LDTs) for plasma- and urine-based ctDNA analysis of actionable mutations in melanoma, lung, breast, pancreatic, colon, and prostate cancers. However, the vast majority of cfDNA is derived from physiological events in normal tissue and not from the tumor, limiting the sensitivity of these techniques for the detection of oncogenic alterations.

Since tumor cells generally shed a high number of exosomes, the use of exosomes and other EVs as biomarkers for cancer can provide a significantly superior starting biological material. This review will focus specifically on how exosomes can provide a new generation of tools for cancer diagnosis, prognosis, stratification, monitoring, and treatment decisions.

Rich Source of RNA Biomarkers for Liquid Biopsy

Nanoscale in size (30-200 nm), exosomes are part of the EV family of vesicles actively released into biofluids by all living cells including tumor cells, either through the fusion of multivesicular bodies with the plasma membrane or through direct budding from the cells (Fig. 1). The active shedding of vesicles from tumor cells is linked to the MAPK pathway that is upregulated in most tumor cells. It has been reported that a cancer cell can release more than 20,000 of these vesicles over 48 hours. These vesicles carry RNA, DNA, and protein from the tumor, including relevant genetic material and proteins from the carcinogenic process. Exosomes have been specifically implicated in driving key attributes of malignant cell behavior, including stimulation of tumor cell growth, suppression of the immune response, induction of angiogenesis, and establishment of metastases, making them particularly intriguing as cancer biomarkers.

RNA is inherently unstable and cannot survive in a free form in biofluids such as serum or plasma. Prior to the understanding that exosomes contain diagnostically relevant RNA, the liquid biopsy field was limited to CTCs and cfDNA. The toolbox for cancer diagnostics greatly expanded when it was discovered that tumor-derived RNA could be stably isolated from exosomes from the serum or plasma of cancer patients, even after many years of storage in biobanks. The earliest report of RNA in vesicles from cell lines came in 2006 when Ratajczak and collaborators reported the presence of Oct4 and other pluripotency factor mRNAs in murine stem cell-derived vesicles. Subsequent studies confirmed the presence of mRNA and miRNAs in exosomes and other EVs, demonstrated intercellular delivery of exosomal RNA, and proposed a novel medium of communication between cells.

Although exosomal miRNA has long been the main theme for biomarker discovery in exosomes, the exosomal long RNA (mRNA, lncRNA, etc.) has a greater utility in identifying somatic mutations and changes in gene transcription. Long RNAs provide additional opportunities to study disease state or progression processes. Using serum exosomal mRNA and tissue DNA from colorectal cancer patients, analysis of KRAS and BRAF mutations demonstrated a high degree of concordance between these nucleic acids. In addition to the genetic variations seen in ctDNA, exosome long RNAs allowed for the first time the capability to detect mutations on RNA, oncogenic fusion transcripts, and alternative splice variants, as well as the RNA transcriptome profile in a liquid biopsy from serum or plasma. Several studies have linked the presence of miRNAs to lung, prostate, and pancreatic cancer. Together with reported RNA editing and circular RNAs, these diverse exosomal nucleic acids (exoNA) represent advantages unique to exosomes which may not be observable using cfDNA, small RNA, or protein assays.

It is worth noting that, in addition to RNA, exosomes have other potential targets for biomarkers, such as proteins including membrane proteins, lipids, and metabolites. Immuno-pulldowns can be designed in biofluids to enrich or deplete tissue-specific exosomes based on surface protein markers for subsequent biomarker analyses, including for protein analytes. Using plasma, this approach has been shown to significantly improve the clinical correlation of biomarkers with disease states, which is otherwise poor or absent from just analyzing plasma directly.

First Commercial Exosome-based Test to Avoid Unnecessary Prostate Biopsies

After rigorous clinical validations, the first commercial exosome-based test recently became available for prostate cancer. This non-invasive urine test utilizes novel expression signature of three RNA transcripts (two mRNAs and one lncRNA) for the intended use population: men 50 years of age or older, no prior biopsy, and PSA levels of 2 to 10 ng/ml. In one extensive clinical validation involving over 1000 patients, the gene signature within exosomes analyzed from voided urine was indicated as a rule-out test for high-grade (>GS7) prostate cancer with an NPV of 91%, avoiding about 27% of biopsies. The urine gene expression signature assay is derived from genes known to play a role in prostate cancer initiation and progression, highlighting the clinical utility of exosome-derived RNA biomarkers.

Exosome Enhances Detection Limit of Liquid Biopsy

Recent publications on cfDNA analysis reported detection of allelic frequency as little as 0.001%, i.e., 10 mutant copies in a background of 1,000,000 wild-type alleles. While this approaches even the highest fidelity of DNA polymerase, strategies such as unique molecular indexing and bioinformatical background correction have helped improve the performance of Next Generation Sequencing (NGS) assays for mutant alleles at low frequencies in more targets. However, the biological limitation of low mutant copy numbers present in circulation, especially in patients with early-stage cancers, cannot be addressed by the improved methods for allelic frequency discrimination.

The RNA contained within exosomes can serve to increase the total number of mutant copies available for sampling. A recent publication comparing ctDNA mutation detection vs. combined exoNA + ctDNA showed that the combined exosome assay had approximately 10-fold more mutated copies of activating EGFR mutations in NSCLC patients (median of 24 mutant copies/ml plasma on ctDNA vs. 234 copies/ml plasma with the combined approach). In addition, the allelic frequency of the mutations was approximately 3-fold higher when the mutation targets were analyzed by the combined exosome approach vs. ctDNA-only mutation detection by BEAMing. In addition, a recent longitudinal study investigating levels of BRAF, KRAS, and EGFR mutations in exosomes and cfDNA over time demonstrated that the combined exoNA + cfDNA analysis significantly improved the correlation of biomarkers with treatment outcome as compared to cfDNA alone.

For all three types of liquid biopsy targets, the wildtype target is present in vast excess relative to the tumor-derived materials. Detection of a single CTC would require locating that specific cell in a background of 106 to 107 leukocytes. CfDNA and exosomes have similar challenges. A number of factors influence the absolute amount of cfDNA, including exercise and disease state, although there’s no evidence that only wild type and not diseased material is subject to these perturbations. The high background of wild-type nucleic acid and low, sometimes undetectable, amounts of the mutations can lead to false negative results. On the other hand, a positive detection of a mutation, especially when in low allelic frequency, should be interpreted with caution since healthy people, too, might exhibit low allelic frequency of mutations due to clonal hematopoiesis.

Future Outlook on Liquid Biopsies

To facilitate detection of low copy targets in liquid biopsies, there has been a drive to develop and improve downstream analytical platforms. In NGS, for instance, broader panels have been developed with higher sequencing depth. Novel approaches, such as molecular barcoding and background correction, have emerged to resolve low copy/high background issues for NGS solutions. However, patients with low copy targets will continue to be a challenge, because single digit copy numbers will have stochastic detection. It further demands that the front end, including sample acquisition and isolation methods, be efficient, specific, and robust. To this end, validated sample collection and isolation kits for exoNA + ctDNA under cGMP manufacturing can enable robust next generation liquid biopsy diagnostics.

Figure 1.
Exosome release from a living cell and cell-free DNA release from a dying cell into the blood vessel.

Liquid biopsies offer a significant step forward in addressing issues related to conventional biopsies. They also enable a clinician to track disease progression, predict response to treatment, or detect developing resistance to treatment. In these respects, exosomes offer a unique content-rich biomarker tool. Application of increasingly sophisticated techniques for analysis of tumor-derived exosomes should greatly facilitate the development of transformational, robust, content-rich next generation diagnostics.

References available upon request.

About the Authors

Seth Yu, PhD
Exosome Diagnostics, Inc., Waltham, MA

Kapil R. Dhingra, MD PhD
KAPital Consulting LLC

Johan K. Skog, PhD
Exosome Diagnostics, Inc., Waltham, MA