The Application of MicroRNAs in Cancer Diagnostics - RNA Interference from Biology to Therapeutics - page 282

decision making. Challenges, however, remain for the identification of circulating
miRNA biomarkers. First, the preparation of samples (serum, plasma, urine, etc.)
requires very strict standardization in order to generalize findings from different
patients, research groups, and laboratories. One possible reason for variation is that
thrombocytes present in the plasma fraction are activated upon incubation below
20°C. This will lead to release of small miRNA-containing granula not removed at
20,000 × g centrifugation typically used. Accordingly, any variation in incubation
times across samples before complete processing can lead to variations in the
miRNA expression detected. Second, the quantification of miRNAs is not straight-
forward. Further investigations should clarify the biological half-life of miRNAs,
since these may vary individually, as well as uncover suitable normalization strate-
gies involving identification of ideal “housekeeping” small RNAs.
Moreover, the protocol for isolating exosomes is not standardized. By using
specific exosome surface markers such as EpCAM for affinity purification, it may
be possible to avoid the problem of contaminating exosomes from blood cells.
However, the amount of circulating tumor exosomes is presumably low in the
bloodstream and likely display a heterogenous expression of surface markers. Thus,
using surface markers for exosome isolation will reduce any miRNA signal consid-
erably and introduce bias in the subset of vesicles isolated. It is currently unknown
whether miRNA profiling of crude samples (without discrimination between vesic-
ular or protein complex origin) is better at distinguishing between patient groups,
compared to profiling after specific purification steps. Using a nonspecific proce-
dure (a simple 15,000-20,000 × g spin prior to RNA extraction) will remove large
cellular debris and protein aggregates, but may not be adequate for detection of
small amounts of tumor-derived material. Finally, hemolysis of plasma samples as
well as activation of platelets during sample processing will influence the results
greatly and demands for precise handling of samples to avoid this. If future studies
can successfully overcome these challenges, the distinct expression profiles of miR-
NAs in cancer tissues together with the remarkable stability of circulating miRNAs
should make these new easily accessible candidate biomarkers ideal for translation
into clinical use. Because cancer disease is often diagnosed at a late stage with a
concomitant poor prognosis, development of minimally invasive tests for detection
and monitoring of common solid tumors may significantly help to reduce the world-
wide health burden caused by cancer.
During the past decade, expression profiling studies of microRNAs, and more
recently also of long ncRNAs, have made important contributions to basic, clinical,
and translational cancer research. In order to transfer these findings into actual clini-
cal utility, it will be essential to conduct large multicenter clinical studies for pro-
spective validation of the many candidate biomarkers already identified. Such
studies should also define in which exact clinical setting and in which type of
patients a given biomarker (panel) has true potential for, e.g., diagnosis, prognosis,
prediction of treatment response, or disease monitoring.
The recent availability of new advanced technologies for sequencing of the
human genome, NGS, has already revolutionized biological research and likely will
have similar effects on molecular diagnostics in the very near future. NGS technolo-
gies can be used for discovery of new disease markers with unsurpassed efficiency