Non-invasive these patients harboring EGFR mutations acquire resistance to

Non-invasive approaches, including
ctDNA material which are usually based on serum or plasma samples, are showing
great potential to monitor EGFR-TKI treatment in NSCLC. ctDNA has a high degree
of specificity to detect EGFR mutations. Moreover, ctDNA is capable of
monitoring disease progression during EGFR-TKI treatment since it reflects the
tumor burden.  Effectively, these liquid specimens can complement with
tumor tissues and help to guide EGFR-TKI therapy in NSCLC especially in those
where tissue biopsy is hard to obtain regularly. Thus, liquid samples obtained
by non-invasive approaches possess great potential to be valuable materials
applied for guiding individual treatment (Sun et al., 2015).

Nowadays, EGFR gene
mutations are the standard predictive biomarkers for selecting the treatment of
NSCLC patients: to receive EGFR-TKI treatment. Multiple treatment options (such
as small-molecule inhibitors directed at these molecular targets) have been
developed, and some of them are migrating from bench to bedside; however,
despite these rapid progresses, tyrosine kinase inhibitors of epidermal growth
factor receptor (EGFR-TKIs) are still the most successful example of targeted therapy
in NSCLC. Compared with conventional treatment options for cancer patients
especially chemotherapy, EGFR-TKIs are able to achieve prolonged
progression-free survival (PFS) with reduced side effects in NSCLC patients
harboring activating EGFR mutation (Sun et al., 2015).  

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Somatic mutations in the
epidermal growth factor receptor (EGFR) gene have been identified in patients with
radiographic responses to EGFR-tyrosine kinase inhibitors (TKIs). Currently,
the response to EGFR-TKIs depends on patients harboring EGFR-sensitive
mutations, and the median progression-free survival (PFS) is approximately 12
months. Despite an initial response, most of these patients harboring EGFR mutations
acquire resistance to EGFR-TKIs (Hata et al., 2013). Effectively, the
resistance to treatment will lead to tumor progresses. For this reason,
identification of the molecular mechanisms of EGFR-TKI resistance is necessary.
This acquired resistance has many identified mechanisms including the second
T790M mutation of EGFR, amplification of MET or HER2, and mutations of PIK3CA
or BRAF; but the secondary EGFR mutation, a point
mutation in exon 20 (T790M) accounts for approximately half of acquired
resistances to EGFR-TKIAs and monitoring T790M mutation is useful for
estimating EGFR-TKI resistance. The point mutation in exon 21 (L858R) or
deletion in exon 19 predicts good response to EGFR-TKIs, while the point
mutation (T790M) in exon 20 implies resistance to EGFR-TKIs. Taniguchi et al.
performed a study to quantitatively detect the T790M-resistant mutations in
ctDNA. In 43.5 % (10/23) of patients who had progressive disease after
EGFR-TKI treatment, the T790M mutation in ctDNA was detected (Sun et
al., 2015).  

The introduction of
first-generation EGFR inhibitors (erlotinib, gefitinib) and subsequently
afatinib for non–small cell lung carcinoma (NSCLC) patients with activating
somatic mutations in EGFR has led to improved
tolerability and efficacy compared with first-line chemotherapy, as mentioned
above. But due to the emergence of resistance during treatment, a novel, oral,
irreversible tyrosine kinase inhibitor was created for the treatment of
patients with mutant EGFR NSCLC: Rociletinib
(CO-1686). It has demonstrated efficacy against the activating mutations (L858R
and del19) and the acquired primary resistance mutation (T790M), while sparing
wild-type EGFR. An ongoing phase I/II trial of rociletinib has demonstrated a
promised clinical activity with a 59% ORR in T790M mutation-positive patient
population (Karlovich et al., 2016).

In a study done by Thress et al., ctDNA was
shown to be more precise and informative than tissue as the blood mirrors the
entire tumor burden. In addition, the rate of clinical response to AZD9291 (osimertinib, which is an orally administered EGFR-TKIis highly
potent against EGFR-TKI-sensitizing mutations and the T790M resistance
mutation, but with a margin of selectivity against wild-type EGFR activity) was
almost identical in patients positive for the T790M mutation in plasma and in
tissue, indicating that plasma detection may be a suitable alternative to
tissue genotyping in patients with NSCLC (Thress et al., 2015).

On the other
hand, in a retrospective analysis done by Oxnard et al., the sensitivity of
plasma genotyping for detection of T790M was 70%; patients positive for T790M
in plasma have outcomes with osimertinib that are equivalent to patients
positive by a tissue-based assay. This confirms that obtaining plasma samples
could avoid a tumore biopsy for T790M testing (Oxnard et al., 2016).

To confirm more the importance of T790M
detection by liquid biopsy, Crowley et al. showed that third-generation
inhibitors have shown activity in the presence of this mutation (such as
WZ4002114) and novel drug combinations have shown promising preclinical
activity. In addition, they emphasized on the utility of plasma samples for
T790M detection by reporting that this mutation was first observed in relapsed
patients and later confirmed through the non-invasive analysis of plasma
samples, proving that resistance to targeted therapies can be monitored
in the blood (Crowley et al., 2013).

Some evidence indicates that
T790M doesn’t only appear after TKI therapy but it may be present at low levels
in EGFR mutated AdenoCAs prior to EGFR drug therapy. Wang et al. used
dPCR and ARMS to analyze T790M cfDNA mutations in 135 TKI-treated lung cancer patients
who had progression-free survival on the TKI therapy for over six months. T790M
was identified in 31.5% using dPCR and 5.5% using ARMS of these patients prior
to TKI therapy, demonstrating that dPCR was a more sensitive test. Post TKI
therapy, T790M was identified in 43.0% of patients by dPCR, and in 25.2% by ARM.
Patients with high pre-TKI therapy T790M levels had a poorer progression-free
and overall survival, demonstrating the importance and prognostic value of T790M detection (Ansari
et al., 2016).

More studies were done to confirm the
accuracy of plasma for mutation detection. The study done by Reckamp et al., using
Qiagen therascreen EGFR Rotor-Gene Q PCR Kit for mutation analysis, reported
the concordance data for matched tissue/cytologic and plasma samples tested
were: concordance 95% 131 of 138, sensitivity 73% 16 of 22 and specificity
99% 115 of 116. A comparable sensitivity of EGFR mutation detection was
observed in plasma: 93% (38 of 41 specimens) for T790M, 100% (17 of 17) for L858R,
and 87% (34 of 39) for exon 19 deletions. Moreover,
the study showed EGFR mutation frequency that was
9% for evaluable plasma samples. In addition, the frequency of T790M was 3%,
57% for exon 19 deletions, 32% for L858R, 0% for exon 19 deletions and T790M
occurring together and 2% for L858R and T790M together (Reckamp et al.,

Another study reported the
frequency of EGFR mutations: three subgroups of EGFR mutation
positive patients were identified, including 19Dels or L858R alone, 19Dels or L858R
plus T790M, and T790M alone. The patients with EGFR 19Dels or
L858R plus T790M accounted for the majority of the EGFR mutation
positive patients. Furthermore, EGFR19Dels or L858R alone was found
in 6.5% to 14.3% of the patients in plasma throughout the study. T790M alone
was also detected in 3 to 6.5% of the patients. Effectively, the study showed
that the majority of T790M positive patients carried simultaneously 19Dels or
L858R in plasma throughout the course of their disease (Zheng et al., 2016).

The mutation status concordance observed
between 1162 matched tissue/cytologic and plasma samples (89%) suggests that
ctDNA is a feasible sample for real-world EGFR mutation analysis if
robust/sensitive DNA extraction and mutation analysis methodologies are used (Reckamp
et al., 2016).

The 2011 publication by Liu et
al. reported the successful EGFR mutation analysis of 86
matched plasma-derived ctDNA and formalin-fixed, paraffin-embedded samples
using a Scorpion-amplification refractory mutation system (ARMS). 27 EGFR mutation-positive
were identified in plasma out of the 40 tumor samples, showing a sensitivity of
67.5% (Douillard et al., 2014).

In another study done by Wang et al., EGFR
mutations were detected in the plasma samples from 17 NSCLC patients (12.7%). With
consistency with results from the paired tumor tissues of two NSCLC patients, 11
plasma samples had an Exon 19 deletion, 4 samples had a L858R mutation, and 2
samples had a L861Q mutation. Finally, sensitivity and specificity for EGFR
mutation detected in plasma were 22.06% (15/68) and 96.97% (64/66) compared
with tissue sample as a reference (Wang et al., 2014).

Finally, in a study done by Kuang et al., using
the SARMS assay they detected 12 patients with EGFR del 19 (E746_A750), 7
patients with L858R, and 8 patients with EGFR T790M mutations. The
overall concordance of tumor EGFR mutation with
plasma EGFR mutation was 74% (32 of
43), and this concordance as a function of the specific type of mutation (exon
19 deletion versus L858R). 85% (17 of 20) concordance with the plasma EGFR mutation
was detected in the patients with a known tumor exon 19 deletion mutation whereas
in those with a tumor L858R mutation the concordance rate was only 29% (2 of 7)
with the plasma EGFR mutation. Moreover, the
relationship with prior clinical response to gefitinib or erlotinib was
evaluated in patients in whom EGFR T790M was detected in
plasma DNA. There was no evidence for the presence of EGFR T790M
(0 of 8; 0%) in patients with progressive disease to gefitinib or erlotinib or
in patients who had never been treated with these agents (0 of 4; 0%). Thus, the EGFR T790M
mutation detected in plasma DNA is associated strongly with a prior clinical
response to gefitinib or erlotinib. Moreover, using the SARMS
technique, only 39% of EGFR mutations were detected
in plasma (7 out of 18 known EGFR-mutant patients); EGFR T790M
was identified in 71% (5 of 7) in plasma of patients whose tumors were positive
for EGFR T790M
mutation (Kuang et al., 2009).

Another study performed by
Sorensen et al. confirms the point
that prior TKI treatment is a must for the development of T790M. EGFR mutations
were examined in plasma samples collected during erlotinib treatment from 23
lung cancer patients, where EGFR mutations were identified in their blood
sample taken before the initiation of treatment. 9 patients had T790M mutation
combined with the sensitizing EGFR mutation, 6 patients had the sensitizing
mutation without the T790M mutation, and 8 patients had neither the sensitizing
nor T790M mutations, with the appearance of T790M always occurring together
with an increase in the amount of the sensitizing EGFR mutation. These results demonstrated
that the T790M mutation was not present in the blood before treatment with
erlotinib in any of the 23 patients with presence of the sensitizing EGFR
mutations in the pretreatment blood sample. In the end, the study reported
that the T790M mutation continued to increase in all 9 patients until disease
progression while there was no trend toward an increase in the original
sensitizing EGFR mutation noted in patients who did not develop T790M
mutations (Sorensen et al., 2014).