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Palmieri et al. J Cancer Metastasis Treat 2020;6:55 I http://dx.doi.org/10.20517/2394-4722.2020.105 Page 3 of 8

A first peripheral blood sample for cfDNA analysis was taken during the genetic counseling visit at the
stage of disease progression. Plasma was used for cfDNA extraction. A second sample for cfDNA analysis
was taken after a mean time interval of 2 months (range 1-6 months).

cfDNA extraction and sequencing
Peripheral blood samples (10 mL) were collected from each patient and placed into a Cell-Free DNA
BCT tube (Streck, La Vista, NE, USA). cfDNA was extracted from 4 mL of plasma using AVENIO ctDNA
Expanded Kit according to the manufacturer’s instructions. cfDNA quality and quantity were verified as
[5]
described in Palmieri et al. . cfDNA sequencing was performed using AVENIO circulating tumor DNA
(ctDNA) Expanded Kit (Roche, Basel, Switzerland) on Illumina NextSeq 550 (Illumina, San Diego, CA,
USA). This technology is able to identify various types of alterations, including single nucleotide variants,
insertions/deletions, gene fusions, and copy number variations present in genes linked to cancer (clinical
actionable mutations) with a reportable range up to 0.05%. The sequencing analysis was performed using
AVENIO Oncology Analysis Software (Roche, Basel, Switzerland).

RESULTS
Patient characteristics
From March 2018 to July 2020, a total of 133 patients with locally advanced or metastatic solid tumors were
enrolled at Azienda Ospedaliera Universitaria Senese, Siena, Italy, and included in the study. The mean age
of patients at the first cfDNA analysis was 56 years (range 2-83 yrs); 48% of patients were females and 52%
were males. Out of 56 patients who did at least a second liquid biopsy, 22% had cancer from breast, 14%
lung, 14% ovarian cancer, 5% colorectal, 6% pancreas, 6% prostate, whereas uterine cancer, retinoblastoma,
cholangiocarcinoma, and gastric cancer accounted for 2% each, and soft tissue sarcoma (including right
infratemporal fossa, oral, pharynx, and larynx), Wilms’ tumor, and glioblastoma accounted for 1% each of
the entire series. The median follow-up time for all patients was 2 months (range 1-6 months).

Mutated gene and tumor type association
Next-generation sequencing (NGS) analysis in 133 patients at the first liquid biopsy identified 86 clinically
meaningful pathogenic variants in 54 genes allowing to pick up key mutations in 67.6% (n = 90) of cases
[Supplementary Table 1]. After a mean of 2 months, a second liquid biopsy was performed and 87.5% of
patients remained/became positive. Table 1 summarizes all the mutated genes resulting from the second
liquid biopsy associated with different tumor types. Mainly, the breast, lung, and prostate cancers were
the tumors types that showed the largest number of mutated genes. The tumor type, grade, and stage as
well as the drug treatments for the entire cohort of second liquid biopsy patients are summarized in the
Supplementary Table 1.

Most frequently mutated genes
At the second liquid biopsy time, the mutated genes decreased from 54 to 38 in 16 different tumor types.
Among these 38 mutated genes, those most frequently represented were the following: TP53 (30%),
PIK3CA (10%), EGFR (10%), KRAS (8%), ERBB2 (8%), FGFR3 (6%), and BRCA2 (6%). Interestingly, these
genes were mutated in 16 different tumor types without a specific prevalence among them [Figure 1].

However, clonal mutation was not confirmed in the entire cohort at the second liquid biopsy. Indeed,
9 patients resulted as negativized, 13 patients had only one mutated clone, and 18 had two clones. One
patient had 5 mutated clones [Figure 2].

The most frequent mutations in our cohort were in the TP53 gene, regardless of the primary tumor
type. TP53 was usually mutated in association with another gene [Figure 3]. The most mutated genes in