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Page 2 of 8 Palmieri et al. J Cancer Metastasis Treat 2020;6:55 I http://dx.doi.org/10.20517/2394-4722.2020.105

Conclusion: We showed that the clonal evolution of tumors includes a private combination of genes, regardless of
tumor type. In the future, the cancer treatment can be the targeted therapy against specific tumor mutation(s). The
present approach seems promising to both identify key cancer genes and follow clonal evolution over time.

Keywords: Cell-free DNA, liquid biopsy, solid tumors, advanced tumors, private mutations, targeted-therapy

INTRODUCTION
Daily experience shows that different malignancies, such as breast or lung cancers, can be controlled using
standard protocols for many years before tumor relapse. This is possible thanks to the detection of the so-
called “Achilles’ heel” that represents the molecular target for different tumor groups named “oncogene
[1]
addict” . However, only a few tumors have a unique or a small number of mutated genes that could be
[1]
susceptible to target therapy, whereas the vast majority of cancers are “non-oncogene addicted” . Tissue
biopsies from primary tumors usually do not show details of molecular heterogeneity and are even less
informative when obtained from metastases. Additionally, biopsies from metastases are not always easy to
[2]
perform .

Cancer is an evolving microcosm driven by selective pressures due to the environment and drug therapy.
Malignant cells are in competition and/or cooperation with each other and with the surrounding
[3]
environment . Cancer therapy must cope with the cellular complexity of the disease and face its dynamic
[4]
evolutionary aspect . In order to facilitate treatment choice, it is crucial to distinguish between germline
and somatic mutations. New technologies such as the liquid biopsy of cell-free DNA (cfDNA) facilitate
proper detection of somatic tumor mutations, which are the key mutation of specific cancer driver genes.
The technique is non-invasive, is a valuable alternative to physical biopsies, and opens new avenues for
personalized medicine .
[5]

In this study, we show that cfDNA analysis is able to follow, over time, the clonal evolution of multiple solid
tumors. In particular, patients belonging to the same tumor type exhibit different private pairs of mutations.
The most frequently mutated gene is TP53 that we found in combination with PIK3CA, KRAS, EGFR with
a frequency of 71%, or in combination with BRCA1, and ERBB2 with a frequency of 42%, or finally with
PTEN and MYC whit a frequency of 28.6%.

The second liquid biopsy proves to be a powerful tool to understand which pair of mutated genes is specific
for the patient and unique to him, thus leading to a fully personalized treatment.

METHODS
Patients and sampling
Our cohort has been enrolled at the Medical Genetics Unit of the Azienda Ospedaliera Universitaria
Senese, Siena, Italy for diagnostic purposes. The cohort consisted of 133 patients with different solid tumors
who experienced disease progression after standard therapy. Patients were previously treated in advanced/
metastatic settings and most of them were not eligible for curative treatment. Written informed consent
and assent for genetic analysis was obtained from all patients.

Inclusion criteria provided patients with either locally advanced or metastatic solid tumors independent
from the primary tumor site. Patients were excluded if they had early-stage solid tumors and still have to
experience all possible pharmacological treatments of standard guidelines. The main information collected
for each patient includes oncological data, family tree, and cancer history in a genetic consultation setting.