Editorial

Liquid Biopsy as a Follow-Up Tool: Comment on Longitudinal

Monitoring of Somatic Genetic Alterations in Circulating
Cell-Free DNA During Treatment With Epidermal Growth
Factor Receptor–Tyrosine Kinase Inhibitors
1 2
Umberto Malapelle, PhD ; and Christian Rolfo, MD, PhD, MBA ; on behalf of the International Society of Liquid
Biopsy (ISLB)

A major issue in the management of non–small cell lung cancer (NSCLC) is the relative lack of availability of these
samples (ie, small histological biopsies and cytological specimens) for morphological, immunohistochemistry, and molecular
analysis.1,2 In addition to the limitations of these paucicellular specimens, up to 30% of NSCLC patients do not have
tissue availability due to poor general conditions or technical and medical limitations.2 In this complex clinical scenario,
an increasingly valid option is molecular assessment with a minimally invasive method that utilizes cell-free nucleic acids
(mainly DNA and/or RNA as a new approach), commonly known as “liquid biopsy.”3 Traditionally, indications for liquid
biopsy in NSCLC were limited to assess the mutational status of the epidermal growth factor receptor (EGFR) in order to
evaluate patients’ eligibility for the administration of tyrosine kinase inhibitors (TKIs) in an upfront setting (in patients
naïve to any treatment) when tissue is unavailable or inadequate or after progression from a first- or second-generation
EGFR-TKI using a single target determination.4 In the new paradigm, the development of more sensitive detection
methods, such as next-generation sequencing (NGS), allows simultaneous evaluation of multiple somatic mutations; fur-
thermore, the addition of other techniques, such as hybrid capture, makes it possible to determine the presence of other
genetic alterations, such as fusions. This approach demonstrated a marked increase in the detection of therapeutically
targetable mutations and improved delivery of molecularly guided therapy compared with tissue genotyping,5,6 with a
shorter turnaround time.6 These results support a hypothetical change in the upfront management of NSCLC patients
from “tissue first” to “blood first.”
Recently, the therapeutic landscape of EGFR-mutated NSCLCs has been revolutionized by the advent of the
third-generation EGFR-TKI osimertinib in a first-line setting,7 replacing first- and second-generation inhibitors and
changing the scenario of mechanisms of acquired resistance, including druggable alterations such as MET amplification,
EGFR C797S mutation, PIK3CA mutation, BRAF mutation, and HER2 amplification/mutation, among others.8 In this
issue of Cancer, Iwama et al9 evaluated the role of a longitudinal monitoring of somatic genetic alterations in cell-free
DNA (cfDNA) of EGFR-mutated NSCLC patients throughout EGFR-TKI treatment. Using digital droplet polymerase
chain reaction (ddPCR) and NGS, the authors evaluated the presence of somatic mutations and alterations at baseline
and every 12 weeks until progression in 100 NSCLC patients who harbored sensitive EGFR mutations. This population
included 87 EGFR-TKI–naïve patients (group A) and 13 EGFR-TKI and chemotherapy-pretreated patients (group B).
Bone and adrenal glands metastases were more frequent among patients with detectable EGFR-activating mutations
in cfDNA at baseline than among those without (bone, 56.5% vs 21.6%, P = .001; adrenal glands, 13.0% 6.0 vs 0.0%,
P = .02).9 These results are in line with a recent pooled analysis of 10 studies suggesting that metastatic site location
influences the diagnostic accuracy of circulating tumor DNA (ctDNA) EGFR mutation testing in NSCLC with a higher
sensitivity in patients with extrathoracic disease (M1b) versus pulmonary contralateral metastases or pleural/pericardial
effusion (M1a) (odds ratio, 5.09; 95% CI, 2.93-8.84).10

Corresponding author: Christian Rolfo, MD, PhD, MBA, Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine,
22 S. Greene Street, Room N9E08, Baltimore, MD 21201; [email protected]
1
Department of Public Health, University Federico II of Naples, Italy; 2 Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of
Medicine, Baltimore, Maryland
We thank Pasquale Pisapia, Alessandro Russo, and Ranee Mehra for assistance with the manuscript.
See referenced original article on pages 219-27, this issue.
DOI: 10.1002/cncr.32482, Received: July 7, 2019; Revised: July 12, 2019; Accepted: July 26, 2019, Published online September 10, 2019 in Wiley Online Library
­(wileyonlinelibrary.com)

22 Cancer  January 1, 2020
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Liquid Biopsy as a Follow-up Tool/Malapelle and Rolfo

TABLE 1. Studies Longitudinally Evaluating the Mutational Status of Clinical Relevant Genes in Advanced
NSCLC Patients

First Author No. of Patients Target Blood Collection Time Techniques

9
Iwama et al 100 EGFR Baseline and every 12 weeks ddPCR, NGS
Lee et al11 81 EGFR Baseline and every 8 weeks ddPCR
Oxnard et al16 13 EGFR Variable number of times during routine care ddPCR
Tseng et al17 72 EGFR Baseline, 10 weeks after EGFR-TKI treatment, at time of disease Peptide nucleic acid–zip nucleic
progression acid clamp PCR
Iwama et al18 35 EGFR Baseline, 4 and 24 weeks after EGFR-TKI treatment, at time of Nanofluidic dPCR, NGS
disease progression

Abbreviations: ddPCR, digital droplet polymerase chain reaction; EGFR, epidermal growth factor receptor; NSCLC, non–small cell lung cancer; NGS,
next-generation sequencing; PCR, polymerase chain reaction, TKI, tyrosine kinase inhibitor.

Interestingly, median progression-free survival (PFS) expression of this mutation (>20 copies/mL) than first-
was significantly longer in patients with undetectable or second-generation TKIs.9 To overcome the limited ref-
EGFR mutations at baseline (19.0 months vs 7.9 months), erence range of ddPCR, with the aim to identify other
suggesting a prognostic role for cfDNA quantity.9 Other resistance mechanisms than the traditional one (EGFR
investigators have evaluated the role of cfDNA quantity as exon 20 p.T790M) in the NSCLC patients population
a prognostic factor, in addition to the detected mutation considered in the study by Iwama et al, the authors per-
variants.11,12 In particular, a prespecified secondary objec- formed an NGS-based analysis of collected ctDNA sam-
tive of the EURTAC trial used a very sensitive TaqMan ples, showing that the number of mutations identified in
assay and considered patients with available baseline ctDNA was significantly higher in patients experienced a
serum or plasma samples. In this study, 78% (76/97) disease progression than at baseline (P = .04).9
of patients were found to have an EGFR mutation in Collectively, and considering the data provided
cfDNA; the median OS was shorter in patients with the from other studies in the same research setting9,11,16-18
L858R mutation in cfDNA than in those with the exon (Table 1), the study by Iwama et al represents an import-
19 deletion (13.7 months [95% CI, 7.1-17.7 months] vs ant step forward to implement liquid biopsy, and in par-
30.0 months [95% CI, 19.3-37.7 months]; P < .001).12 ticular the evaluation of mutational status on cfDNA, as
This pivotal analysis underlined the role not only of the a follow-up tool in patients with an oncogene addiction,
quantity of cfDNA, but also of the quality (in this case not only to predict the resistance disease, but also to plan
the type) of mutation detected. the specific second-line treatment basing on the presence
In the study by Iwama et al, this point was not ad- of a specific mutation detected. Of note, in this study a
dressed in the group with the evidence of EGFR mutations low percentage of patients received osimertinib as upfront
at baseline, and the PFS was longer in patients without treatment, which is the new standard of care in the United
evidence of these alterations in 12 or 24 weeks (11.4 months States and in most European countries. Although ddPCR
vs 4.6 months and 16.1 months vs 7.1 months, respec- has a high sensitivity for the detection of known EGFR
tively).9 Most of the patients enrolled in the present study mutations, the impossibility to simultaneously evaluate
were treated with first- or second-generation EGFR- multiple genetic alterations as well as other genetic ab-
TKIs (96.6% in group A and 61.5% in group B), and errations (eg, amplifications and fusions) compromise its
only 1 patient was treated with the new first-line agent use as a real-time monitoring tool in EGFR-mutated pa-
osimertinib. Early plasma clearance of EGFR mutations tients, missing clinically relevant mechanisms of acquired
has been reported to be a prognostic factor for improved resistance compared with NGS. Multiplex panels using
outcome with osimertinib, both in first-line13 and EGFR- NGS platforms are reliable and should be preferred as
TKI–pretreated patients.14,15 they detect beyond the common mutations, indels, copy
Another relevant point raised by the authors was the number changes, and translocations, with acceptable lev-
high rate of progression in patients with an increase of els of sensitivity, as suggested in the IASLC liquid biopsy
EGFR mutation detection during TKI treatment (PFS, statement4 and confirmed in the present study.9
9.1 months vs 19.0 months; hazard ratio, 4.72).9 In ad- The essential contribution of this study to the
dition, a crucial point was represented by the detection of liquid biopsy application is the confirmatory data
EGFR exon 20 p.T790M. The authors confirmed a high coming from real-time monitoring with NGS at serial
efficacy of osimertinib in patients with an elevated basal time points during treatment with an EGFR-TKI. The

Cancer  January 1, 2020 23
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Editorial

Figure 1. Example of real-time monitoring in clinical practice in a 47-year-old patient with non–small cell lung cancer (NSCLC) who
was treated with first-line osimertinib using a 73-gene plasma NGS test. After 3 months of treatment, the patient reported a mixed
response, with a partial response in the lung and substantial stability of the liver metastasis. A liquid biopsy revealed a marked drop
of variant allele fraction, with a reduction of cell-free DNA (cfDNA) percentage of the EGFR exon 21 L858R mutation (from 9.8% to
0.2%), exon 18 EGFR R108K (from 5.4% to 0.1%), and PIK3CA E545K (from 8.6% to 0.3%). After 7 months, the liver lesion showed
progression, whereas there was a response in the lung. The liquid biopsy results revealed a marked increase of all 3 mutations (EGFR
L858R 18.9%, EGFR R108K 12.8%, and PIK3CA E545K 24.1%) and the acquisition of a novel EGFR mutation (C797S) (4.5% cfDNA).
Treatment with osimertinib was discontinued and treatment with afatinib was started. ALK indicates anaplastic lymphoma kinase;
CT, computed tomography; EGFR, epidermal growth factor receptor; PD, progressive disease.

application of this approach in clinical practice (Fig. 1) patients and could represent a new standard of care
can lead to a capitalization of precision medicine arma- in NSCLC patients if confirmed in larger prospective
mentarium to improve the overall survival of our cancer studies in the future.

24 Cancer  January 1, 2020
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Liquid Biopsy as a Follow-up Tool/Malapelle and Rolfo

FUNDING SUPPORT 8. Ramalingam SS, Cheng Y, Zhou C, et al. LBA50Mechanisms of ac-

No specific funding was disclosed. quired resistance to first-line osimertinib: preliminary data from the
phase III FLAURA study. Ann Oncol. 2018;29(suppl 8).
9. Iwama E, Sakai K, Hidaka N, et al. Longitudinal monitoring of somatic
genetic alterations in circulating cell-free DNA during treatment with
CONFLICT OF INTEREST DISCLOSURES epidermal growth factor receptor–tyrosine kinase inhibitors. Cancer.
Umberto Malapelle reports a consulting or advisory role for Boehringer 2020;126:219-227.
Ingelheim, MSD, AstraZeneca, and Roche. Christian Rolfo reports a speak- 10. Passiglia F, Rizzo S, Rolfo C, et al. Metastatic site location influences the
er’s bureau role for MSD and GuardantHealth, a scientific advisory role diagnostic accuracy of ctDNA EGFR-mutation testing in NSCLC pa-
for Mylan, receipt of an institutional grant from Biomark, nonremunerated tients: a pooled analysis. Curr Cancer Drug Targets. 2018;18:697-705.
collaboration with OncoDNA, and a steering scientific committee role for 11. Lee JY, Qing X, Xiumin W, et al. Longitudinal monitoring of EGFR
Oncopass. mutations in plasma predicts outcomes of NSCLC patients treated
with EGFR TKIs: Korean Lung Cancer Consortium (KLCC-12-02).
Oncotarget. 2016;7:6984-6993.
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