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Page 6 of 8 Palmieri et al. J Cancer Metastasis Treat 2020;6:55 I http://dx.doi.org/10.20517/2394-4722.2020.105

Figure 3. Eulero Venn diagram. Eulero Venn diagram shows the higher correlation between TP53 with the PIK3CA and EGFR (5 folds
each), with KRAS and ERBB2 (4 folds each), or with BRCA2 and FGFR3 (3 folds each). The figure also shows correlations of other genes
with each other, e.g., ERBB2 and EGFR

is generally characterized by a “driver” clone that is responsible for metastases. The curve in Figure 2,
simulating a Gaussian bell shape (similar to the typical population curve), underlines this concept. Indeed,
it shows that in metastatic patients, a negative selection occurs in the initial number of mutated clones,
reducing them to a maximum of two in comparison to the considerable number of tumor clones in the
early stages of the disease.

In particular, in relapsing patients, the second liquid biopsy showed that 37% had two mutated clones, 26%
only one clone, and 18% more than two clones [Figure 2].

Notably, one of the two mutated genes was often TP53 associated with PIK3CA (20%) or EGFR (20%),
KRAS (16%) or ERBB2 (16%), FGFR3 (15%) or BRCA2 (15%), and other genes with a lower frequency
[Figure 3]. We hypothesize that tumor survival does not depend only on the expansion of the TP53 clone,
but also requires a mutation in other genes for tumor progression. In fact, we have repeatedly found that
in breast cancer, the TP53 mutation is associated with PIK3CA. Indeed, PIK3CA mutations generally arise
[11]
in advanced stages of breast carcinogenesis from dysplasia to carcinoma in situ resulting in a greater
[12]
potential to migrate and invade in vitro, as well as to metastasize . Moreover, in lung cancer, TP53 was
frequently associated with KRAS mutation.

A striking finding deriving from the comparison between the first and the second liquid biopsy in the same
patient has been the constant reduction in the number of mutated genes. Present data suggest that after
the early phase of tumor diffusion, that is characterized by a great number of new somatic mutations, a
selection takes place among the various mutated genes to determine which are the most effective clones for
tumor progression.

In a small subset of patients, we were able to perform a third liquid biopsy, but numbers for the moment
were too small. Data from the third biopsy, in case of further tumor relapse, will better elucidate the trend