1. Introduction
Generally, the term ”biomarker” refers to any biological indicator whose
variations are related to the definition of the pathological condition,
to the definition of prognosis, to the follow-up or to the definition of
the results of the therapeutic response. The recent discovery of genetic
alterations that cause, in a not neglected part of tumor patients,
neoplastic disease encouraged the formulation of molecular target
therapies that arise from the evaluation of precise molecular targets.
In the context of predictive molecular pathology, liquid biopsy is a
venous blood sampling on which molecular analyzes can be performed to
search for clinically relevant mutations in the EGFR gene in
patients with non-small cell lung cancer (NSCLC). This is usually
performed when it is not possible to have a tissue sample representative
of the neoplastic lesion at baseline or to monitor any resistance
indicator or mutations acquired after a first line of treatment with
tyrosine kinase inhibitors (TKI) .1 In particular,
when it is not possible to perform the analysis molecular on tissue
sample (difficult access to the lesion, comorbidity , especially in
elderly patients, refusal by the patient, etc.) liquid biopsy can be a
valid diagnostic solution. The liquid biopsy cannot replace the tissue
biopsy because, to our knowledge, finding a certain mutation in the
blood does not necessarily imply that the mutation is expressed at a
structured clinically relevant frequency within the selected tumor
(passenger mutation). Even the limited amount of circulating tumor DNA
can be a boundary that can lead to false negative
results.2
In the last five years, the liquid biopsy has been used for different
purposes in oncology. Today the new challenge in lung cancer is
represented by the implementation of some screening tests o, although
this requires a phase of test harmonization and sensitivity
assessment.3,4
The molecules that can be tested in a blood sample in the context of
predictive molecular pathology are different: circulating tumor DNA
(ctDNA) which represents a fraction of the total free DNA (cfDNA), cells
released into circulation by the tumor through active mechanisms and
passive and exosomes which also represent a precious source of nucleic
acids of tumor origin.5 The cfDNA represents, to date,
the specimen of greatest interest because of its immediate availability
with respect to tumor DNA extracted from tissue samples; however due to
the low concentration and the scarce bioavailability in blood torrent,
new approaches are being optimized in order to recover tumor nucleic
acids starting from the lysosomal cargo.2,6 Clinical
response data have shown that in addition to the sensitivity mutations,
there is a ’wide range of different mutations that fall within exon 18,
19, 20 and 21 leading the patient to benefit from the target
therapy.8
Thus they have a predictive potential and for this reason,
new-generation gene sequencing (NGS) represents a valid analytical
platform for the analysis of the mutations present in the
cfDNA.9 However, the analysis spectrum for NSCLC
patients does not only exhaust the EGFR gene; the search for oncogenic
activating genomic translocations such as ALK, ROS, RET has become
clinical practice, a routine that makes the patient eligible for
treatment with TKIs starting from tissue samples.10This possibility represents the direction towards which scientific
research is moving to optimize patient clinical
management.11
Tumors never reach complete molecular stability and this issue means
that, over time, they can develop resistance to target
therapies.12, so it is a crucial decision-making step
to identify what it is happening in the tumor. The presented study is
aimed at assessing the diagnostic and predictive potential of liquid
biopsy into management of lung cancer path of care.
2. Methods
From 2014-2019 liquid biopsy specimens were analyzed
for EGFR sensitive and resistance mutations in Predictive Molecular
Pathology (Department of public Health, University of Naples Federico
II). For each patient 5 ml of plasma and serum sample were withdrawn and
collected in Vacutainer tubes (BD, Plymouth, UK) by a dedicated nurse.
Plasma and serum were immediately separated by two centrifugation steps
at 2300 r.p.m. for 10 min, then supernatant (where nucleic acids were
carried out) was stored at -20C® . Cell-free DNA (cfDNA) was purified by
using the QIAsymphony robot (Qiagen, Hilden, Germany ) and the
QIAsymphony DSPVirus/ Pathogen Midi Kit, according to the manufacturer’s
instructions, finally cfDNA was eluted in a final volume of 35 µl ofnot DEPC water (Ambion). Libraries were produced on the
Ion Chef (Thermofisher, Waltham, Massachusetts ) by using DL-8 kit
and serum and plasma of 4 patients were processed for a total of eight
specimens for run. After library amplification step performed by 22
cycles for cfDNA amplification and 6 cycles for library reamplification
after barcoding, purified libraries derived from eight cfDNA samples
were diluted to 60 pM and combined with eight additional cfDNA-derived
libraries to obtain a 16 Ion Code pooled library. The two-pooled
libraries were re-loaded into the Ion Chef instrument, and templates
were prepared using the Ion S5 520-530 chef kit ( Thermofisher,Waltham, Massachusetts ). Finally, templates were loaded into the
520 chip and sequenced on S5 platform. Signal processing and base
calling were evaluated by adopting the default base-caller parameters on
Torrent Suite [v.5.0.2]. Results were visually inspected with the
Golden Helix Genome Browser v.3.0. (Bozeman,MT, USA). Statistical
analysis were carried out by adopting a binary logistic regression model
on SPSS and IBM software system. Age and sex parameters were added as
covariates.
Sample related to 5 years of activity regarding the molecular
diagnostics performed with liquid biopsy in the predictive diagnostic
laboratory of the AOU Federico II were analyzed. These patients came
from the oncological network pathways related to lung cancer and refer
to the HUB center of the AOU Federico II. The samples of 900 patients
enrolled in the pathway were collected. The availability and
epidemiological interest was verified.
After database review, 515 plasma or blood samples were included in the
analysis (Fig 1). Descriptive analyzes were performed (Tab1). To verify
the differences with respect to the report of liquid biopsy between
patients with basal or progressing tumors, non-parametric analyzes were
performed for 2 independent samples. Furthermore, the effect of the
presence of mutations on the probability of tumor progression, taking
into account age and sex, was evaluated through a multivariate logistic
regression model (Tab2)