Hunting for the ultimate liquid cancer biopsy - let the TEP dance begin

Cells and extracellular vesicles

The first category of candidates are the by now ‘classical’ circulating tumor cells (CTCs) [6, 7] and circulating DNA fragments (ctDNAs) [8, 9]. They are currently explored in several dozen clinical studies (see e.g. ClinicalTrials.gov for most recent details). Probably the most exciting new development in this area is the very recent finding that ctDNA seems to have a major role to play as a prognostic marker of surgery success of stage II colon cancer resections [10].

In addition, we are witnessing at present the surfacing of a whole zoo of novel molecule and vesicle classes that promise to provide information about cancer origins and subtypes. Some of them will be briefly introduced here (see also Fig. 1 for selected examples).

Extracellular vesicles come in many forms, sizes, shapes [11, 12] and their nomenclature and definition is still somewhat fuzzy, since universal exosomal markers are still missing [13–15]. Several curated databases like ExoCarta [16] and EVpedia [17] are collecting information on their molecular components and characteristics. Exosomes are endocytic secretions that can be generated by many cell types, at least in vitro, and represent, at least for now, the most prominent extracellular vesicle group. They are detectable in many body fluids, form a diverse bunch of membrane vesicles ranging in size from ca. 30 to 150 nm and can be isolated by several different methods, including immunoaffinity capture and ultracentrifugation [18–20]. Composed of proteins, various RNAs and lipids, exosomes carry inside and on their surface a rich load of information that can be deciphered by molecular readout methods like RNA sequencing and proteomic approaches. Exosomes have just been published as suitable leads for the early detection of pancreatic cancer and the cell surface proteoglycan glypican-1 has been proposed as a cancer exosome marker [21]. Moreover, exosomes are believed to contain double-stranded DNA from their generating cells, which has been christened exoDNA [22, 23], but this finding requires further validation and exploration before a possible role of this DNA as biomarker can be considered. Fairly recently, exosomes have been implicated as major players in cancer development, for example, in driving organ-specific metastasis [24, 25]. It is thus to be expected that exosomes will be a major focus of biochemical and functional cancer studies in coming years.

Oncosomes, originally described 2008 in gliomas as vehicles for intercellular transfer of an oncogenic EGFR variant [26], are much larger in size (ca. 1–10 μm) and differ considerably in their composition from exosomes and other extracellular vehicles [15, 27]. They are believed to be generated by membrane blebbing in late stage cancer disease. In prostate cancer cells, oncosome production was reportedly stimulated by EGFR activation and elicited in recipient cells an increase in tyrosine phosphorylation and Akt signalling, thereby altering the tumor microenvironment and contributing to disease progression [28]. A new study has further defined the generation, density, composition and cargo of oncosomes [15]. It documents the enrichment of cytokeratine 18 (CK18) as a molecular marker of oncosomes and links enhanced oncosome shedding to silencing of the cytoskeletal formin protein DIAPH3.

A flourishing superfamily of non-protein coding RNAs

The realization that our genome is not mostly composed of ‘junk DNA’ and that this term predominantly reflected our own ignorance, has led to a profound shift in research activities of scientists focusing on how genomes and their products are regulated. Non-coding RNAs have consequently gained a very solid foothold in most of cell biology research and particularly in human disease studies [29–31].

MicroRNAs (miRNAs), small ribonucleotide oligomers typically composed of ca. 22 nucleotides, are the most intensely studied group of these non-coding RNAs. They serve as repressors of mRNA translation into proteins [32] in a plethora of biological contexts. Many recent reviews have described their generation and functions in considerable detail (for two recent examples see [33, 34]). miRNA involvement in the manifestation and progression of most diseases, ranging from neurological disorders to autoimmunity, inflammation, infections, cardiovascular problems, metabolic pathologies and malignancies is also very well established [35–40]. Over 1900 miRNAs with critical functions have been currently described [41]. miRNAs can act as oncogenes or as tumor suppressors, depending on the specific tissue and cell type context and miRNA polymorphisms have been associated with cancer development risks. Their utility as clinical diagnostics has, however, so far been severely limited by poor data reproducibility. The generation of data in the wet lab and the bioinformatics data processing urgently require optimization and better standardization [41].

Since miRNAs have become a major area of attraction for researchers at the beginning of this millennium [42] over 40,000 publications have investigated the many facets of these fascinating regulatory systems and miRNAs are still getting the lion share of attention until now; but a flurry of additional families of non-protein coding RNAs are emerging in the more recent literature.

Small nucleolar RNAs (snoRNAs) have long been viewed as ‘boring’ housekeeping components of the cell. Hundreds of them have been detected so far. As their name indicates, they live in the nucleolus, the ribosomal RNA producing factory of the cell, form complexes with proteins and aid in the processing of rRNAs [43]. Interestingly, in the last years various groups have reported additional functions of snoRNAs, which link them to the control of cell fates and tumorogenesis [44]. Some snoRNAs have been detected in plasma and could have potential as diagnostic as well as prognostic circulating biomarkers, for example in non-small cell lung cancer (NSCLC) [45, 46]. Larger studies that confirm and extend the initial reports are still needed to validate certain promising snoRNA candidates.

Long non-coding RNAs (lncRNAs), are a highly diverse group of thousands of RNAs implicated in cancer invasion and metastasis, as well as many other diseases [47–49]. For an RNA to be entered into this group, a somewhat arbitrary size threshold of more than 200 nucleotide in lengths has been set. At least a few lncRNAs have been detected in human plasma so far and may have relevance for diagnostic or prognostic assays [50, 51], but clearly much more work is needed to gain a robust understanding of the potential and limits of lncRNAs for cancer detection, classification and treatment.

PIWI protein-interacting RNAs (piRNAs) are single-stranded molecules composed of ca. 26–31 nucleotides and found throughout the animal kingdom [52]. A major, if not the major, function of piRNAs is the immobilization of transposons, particularly in the context of spermatogenesis [53]. Whether piRNAs are really drivers of cancer development or ‘passengers’ still remains to be determined [54]. Although they can be detected as a small fraction of the various RNAs isolatable from plasma-derived exosomes [55], it is far from clear whether they will have any diagnostic value in clinical settings.

Only last year, a new group of RNAs has shot onto the scene and gained significant attention immediately: sex hormone-dependent tRNA-derived RNAs (SHOT-RNAs). Generated from mature tRNAs, they are constitutively present in sex hormone-dependent breast and prostate cancer cells [56]. The ribonuclease angiogenin has been implicated in their biogenesis and sex hormones and their receptors were shown to enhance the generation of SHOT-RNAs. Their potential as diagnostic and/or therapeutic targets, if any, remains to be explored.

Developing analyses of circulating macromolecules, vesicles and cells towards clinical routine applications

CTCs from solid cancers have proven fairly difficult to work with, for all but a few specialist laboratories. CTCs are usually exceedingly rare and their vitality at the time of isolation is often uncertain. Despite nearly two decades of increasing scrutiny ([57, 58], methods to actually culture them ex vivo after isolation are just being developed [59]). Nevertheless, successful RNA sequencing from single CTCs of prostate cancer patients has been reported [60], implicating non-canonical Wnt signaling in mediating anti-androgen resistance. This indicates that it may be possible to omit the ex vivo amplification step for CTCs, at least for some types of analyses. CTC plasticity is, however, another significant worry and clearly needs further elucidation. Along these lines, EpCAM-positivity cannot be considered a robust feature of CTCs anymore [61].

Whole exome and whole genome sequencing experiments of DNA from tissue biopsies was and is certainly enormously helpful to gain much deeper knowledge about the molecular mechanisms and heterogeneity of virtually all cancer types. These are by now also possible using single CTCs [62–64]. CTCs could thus, at least in principle, provide some useful information on the (sometimes changing) polyclonality of tumors. Multiple standard biopsies that are otherwise required to analyze the clonality of tumors, are difficult or impossible to obtain for certain tumor types, so alternative options would be most welcome. Until now, however, it is not really clear how well tumor polyclonality is really reflected in CTCs and whether the current methods of CTC analysis are developed far enough to make this a practical option.

Alternatively, cancer-relevant miRNAs in liquid biopsies, the so called ‘Oncomirs’, [40, 65] might be analyzed. But even there, the required technology is just being developed and still far from being ready for daily routine usage [66, 67].

In general, it can probably be expected that costly ‘-omics’ approaches will not be the main road forward for new diagnostic cancer tests, at least when it comes to broad clinical implementation, even for large comprehensive cancer centers (CCCs). While ‘-omics’ technologies clearly very important for basic and translational research, it is highly desirable to generate more focused analyses, no matter what the material (s) for the analyses will eventually be. Limited sets of molecular markers, analyzed by simple, low cost array type (micro-) chips and automatically read and evaluated by standardized, automatic machines would seem to be the method of choice to lead the way into daily routine application.

Tumor educated platelets (TEPs)

It might be at first counterintuitive to assume that highly chaotic entities like tumor cells could ever be ‘educators’ of anything. Obviously this term is used quite loosely here and a slightly better one for the recently observed phenomenon of tumor-influenced platelets [68–70] might be ‘tumor-conditioned platelets’.

It is, however, also undoubtedly clear that cancer cells impact very significantly on several other cell types, ranging from fibroblasts (carcinoma-associated fibroblasts (CAFs), tumor-associated fibroblasts (TAFs)) and various immune cells, like tumor- associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) to endothelial cells. Molecular studies of the relationships between different cell types within a tumor mass or a metastatic lesion are currently under way and provide evidence for complex, formerly unknown relationships at a rapid pace [71–73].

By comparison, analyzing effects of tumor cells on platelets has been somewhat neglected until now. After all, platelets are merely sheddings of megakaryocytes in the bone marrow that do not contain a nucleus and possess a reduced repertoire of proteins, RNAs etc. Their main normal function is hemostasis, i.e. plugging vascular holes after physical injuries.

However, cancer progression can be accompanied by platelet activation and venous thromboembolisms (VTEs). In addition, platelets can secrete growth factors that can influence cell growth and angiogenesis. They are also able to attract other blood cells like monocytes and granulocytes, which may help to create a pro-metastatic microenvironment [74–76].

The latest TEP study by Best and colleagues [70] is highly intriguing. Based on extensive platelet RNA analyses of more than 280 individuals (ca. 22 million reads per sample) they were able to distinguish healthy persons from cancer patients with more than 95 % accuracy and could even provide some information on likely tumor locations for some major tumor types. Five-thousand non-coding and protein-coding RNAs were detectable in pictogram quantities of RNA, corresponding to platelet numbers that can be found in a single drop of blood. The global RNA deregulation observed was massive, with over 2000 changes observed.

Interestingly, prominent tumor-driving mutations (EGFR, Her-2, c-Met, H-Ras, PI3KCA) were also accurately reflected in the results obtained from platelet RNA sequencing and bioinformatics processing of the resulting data. This finding could have major implications for future clinical trials and therapy regimens [77].

While the tumor type was the predominant factor for the actual platelet conditioning, tumor metastasis did not significantly impact on them when compared to samples from patients without metastasis, which is somewhat surprising.

The exciting initial work described by Best and colleagues is bound to be followed up soon by studies of larger cohorts of individuals with specific tumor types, patients with tumors of different sizes etc.