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 addict”[
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 environment[
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
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.
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.
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).
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 described in Palmieri
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).
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% (
Mutated genes identified at second liquid biopsy time
Tumor types |
Tot. mutated gene | ||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
breast (11) | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | x | 17 | |||||||||||||||||||||
lung (7) | x | x | x | x | x | x | x | x | x | x | x | 11 | |||||||||||||||||||||||||||
ovarian (7) | x | x | x | x | x | x | 6 | ||||||||||||||||||||||||||||||||
colorectal (4) | x | x | x | x | x | x | 6 | ||||||||||||||||||||||||||||||||
pancreatic (3) | x | x | x | 3 | |||||||||||||||||||||||||||||||||||
prostatic (3) | x | x | x | x | x | x | x | x | x | 9 | |||||||||||||||||||||||||||||
uterine (2) | x | x | x | 3 | |||||||||||||||||||||||||||||||||||
retinoblastoma (2) | x | x | x | 3 | |||||||||||||||||||||||||||||||||||
colangiocarcinoma (2) | x | x | x | x | x | 5 | |||||||||||||||||||||||||||||||||
gastric (2) | x | x | x | 3 | |||||||||||||||||||||||||||||||||||
sarcoma (1) | x | x | x | x | x | 5 | |||||||||||||||||||||||||||||||||
oral (1) | x | 1 | |||||||||||||||||||||||||||||||||||||
pharinx (1) | x | x | x | x | 4 | ||||||||||||||||||||||||||||||||||
larinx (1) | x | x | x | 3 | |||||||||||||||||||||||||||||||||||
wilms (1) | x | x | x | 3 | |||||||||||||||||||||||||||||||||||
glioblastoma (1) | x | x | 2 |
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:
Distribution of the most frequently mutated genes. Among the 38 mutated genes, point mutations in
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
Second liquid biopsy: number of mutated clones. The histogram shows on the absciss axis the number of mutated clones and on the ordinate the number of patients. Most patients have two mutated clones at the time of the second liquid biopsy
The most frequent mutations in our cohort were in the TP53 gene, regardless of the primary tumor type.
Eulero Venn diagram. Eulero Venn diagram shows the higher correlation between
Early dissemination of resistant tumor cells is the major cause of metastatic recurrence in cancer patients[
The higher number of mutated genes occurred in breast, lung, and prostate cancer, irrespective of the number of these tumor types in the present series. Most patients had breast tumor, whereas only three patients had prostatic cancer; number of patients with lung cancer were the same as those with ovarian cancer, but only patients with lung cancer had a great number of mutated genes
In accordance with previous data in the literature, our study confirmed that SNVs (small nucleotide variants) in
In comparison with data from our previous study[
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
Notably, one of the two mutated genes was often
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 in tumor progression, showing further reduction of the number of clones, or a new increase of “random” mutations.
In conclusion, additional study in larger series is required for further confirmation of these preliminary data and suggestions. A greater use of liquid biopsy is recommended for proper detection of genetic mutations and for “the right treatment in the right patient at the right time”.
Authors thank the support from the Regione Toscana - Istituto Toscano Tumori (ITT) (Project “Identification of genetic bases of individual predisposition to lung cancer in non-smokers”) and the ASSO (Associazione per lo Sviluppo della Scienza Oncologica) Onlus. This work was, in part, supported by the MIUR project “Dipartimenti di Eccellenza 2018-2022” to the Department of Medical Biotechnologies, University of Siena”. This work is generated within the ERN-EURACAN (European network for Rare Adult Aolid Cancer).
Performed the experiments, analyzed the data and wrote the paper: Palmieri M
Performed the experiments: Iuso N
Wrote the paper: Cetta F
Analyzed the data: Fallerini C, Tita R
Took care of the clinical part of the study: Fava F, Fabbiani A
Performed genetic counseling and provided patient samples: Baldassarri M, Mencarelli MA
Designed the research strategy, analyzed the data, and wrote the paper: Renieri A, Frullanti E.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
None.
All authors declared that there are no conflicts of interest.
The consent to participate was obtained from patients included in the study.
The consent for publication was obtained from patients included in the study.
© The Author(s) 2020.