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Page 8 of 10 Finzel et al. J Cancer Metastasis Treat 2018;4:21 I http://dx.doi.org/10.20517/2394-4722.2018.10

This temporal heterogeneity provides interesting information about how the subclonal heterogeneity of a
tumor evolves over the course of treatment, with some clones persisting or disappearing while new clones
appear . Note that this temporal analysis might not always provide useful information about the current
[18]
treatments that would be of clinical benefit to the patient, as the current molecular status of the tumor is the
critical factor in therapy personalization.

On the other hand, 9% of the variants were detected only in the blood, which are a reflection of spatial
heterogeneity. This intratumor heterogeneity occurs either within different regions of the same tumor mass,
or between the primary tumor and its metastases . Moreover, different metastatic sites may also harbour
[4]
different molecular features . Previous studies have reported the existence of mutations that were only
[19]
found in ctDNA but not in the corresponding tissue . Since ctDNA can be shed into the blood from the
[7]
primary tumor and/or the metastases, it can potentially provide tumor information from all cancer sites .
[4]
On the other side, spatial heterogeneity cannot be fully determined with a single-site solid biopsy.
We observed that on average across cancer types, “solid” and “liquid” variants with a demonstrated
functional impact on the protein (damaging) were around 25% of all the mutations found, although the
frequency was different depending on the cancer type. The genes that harboured the highest number of
damaging variants were TP53, KRAS and PIK3CA. TP53 is a key tumor suppressor that responds to several
cellular stress signals by promoting different responses, such as cell cycle arrest, senescence or apoptosis .
[20]
TP53 mutations have been found in almost every cancer type, and its inactivation is a common event in the
tumorigenesis process . KRAS is also one of the most frequently mutated genes in many cancers. It plays
[21]
an important role in the regulation of cell division and activating mutations can lead to cell transformation
because they impair the ability of the KRAS protein to switch between the “on” and the “off” state. Finally,
PIK3CA, another commonly mutated oncogene in cancer , is involved in many cellular processes, such as
[22]
cell growth and proliferation and, when mutated, the increased kinase activity of PIK3CA protein contributes
to cellular transformation .
[23]
What are the clinical implications of analysing the discrepancy between the damaging variants found
solely in the solid and solely in the liquid biopsy? In almost all the cases, these mutations were clinically
actionable, meaning that they provided information about tumor sensitivity or resistance to approved or
investigational targeted therapies. When we took into consideration only the spatial heterogeneity (“liquid”
category), we again found a very high frequency of clinically actionable variants (96%), most of them either
related to approved targeted therapies - for example KRAS mutations in colorectal cancer are associated with
resistance to anti-EGFR antibodies , and MAP2K1 mutations in non-small cell lung cancer are related to
[11]
sensitivity to MEK inhibitors - or to actively recruiting trials of targeted therapies, such as NCI-MATCH
[15]
(NCT02465060). This emphasizes that the addition of ctDNA profiling to the analysis of solid biopsies, the
current gold standard for tumor molecular characterization, can provide valuable extra information for
oncologists, either for the prescription of an approved treatment or for enrolling them in relevant clinical
trials. In fact, the utility of ctDNA analysis for tumor characterization and for guiding treatment choice is
being increasingly recognized, as reflected by the first FDA approval of a blood-based companion diagnostic
to guide targeted therapy in non-small cell lung cancer (cobas EGFR Mutation Test v2).

Limitations of this study include the lack of information about patient follow-up regarding oncologists’
treatment decisions and patient outcomes. Strengths include the number of patients and the cancer types
analysed, as well as having addressed the importance of understanding tumor heterogeneity in the clinical
setting.

In conclusion, this study suggests that the combination of recent solid and liquid biopsies provides the most
comprehensive and therapeutically valuable characterization of the heterogeneity of the patient’s tumor,