Keywords
EGRF - EML4-ALK - lung cancer - NGS - oncogenic driver mutations
Introduction
According to GLOBOCAN 2020 statistics, lung cancer is the most common cancer in men
(14.3%) and third most common cancer in female (8.4%). Overall lung cancer is the
second most common cancer in both sexes (11.4%) after female breast cancer (11.7%)
but accounts for overall highest cancer-related mortality (18%).[1] In India, lung cancer is fourth most common cancer both in incidence and cancer-related
death.[2] Histologically, lung cancer is divided into non-small cell (NSCLC) and small cell
lung cancer (SCLC). NSCLC is the most common type accounting approximately 80 to 85%
cases and includes adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma.
Adenocarcinoma subtype accounts for more than half and is further defined according
to different molecular subtypes by the identification of oncogenic drivers.
Over the last few decades, conventional platinum-based chemotherapy remains the mainstay
of treatment in metastatic NSCLC, but it has produced only a modest increase in patient's
overall survival, reaching a plateau, with response rates around 35% and median survival
time of 10 to 12 months.[3] With recent advances in the knowledge of NSCLC biology, various oncogenic driver
mutations are identified that cause aberrant activation of intracellular signaling
pathways associated with the sustained growth of lung cancer cells. This leads to
the development of molecularly directed targeted therapies with significant improvement
in patient's outcomes. Updated recommendations from the College of American Pathologists,
International Association for the Study of Lung Cancer, Association for Molecular
Pathology, and European Society of Medical Oncology have strengthened the 2013 guidelines
and suggest genomic testing for endothelial growth factor receptor (EGFR), ALK, and
ROS1 for all advanced NSCLC, regardless of patient's characteristics which was revised
in 2018.[4] The minimal conventional genomic study generally includes, sequentially or in parallel,
at least, the EGFR, ALK, and ROS 1 mutational analysis. Sequential testing requires
a substantial DNA amount, is time-consuming, caused sample exhaustion, and often leads
to under genotyping and treatment delay.[5]
[6] Therefore “National Comprehensive Cancer Network (NCCN)” strongly advises to perform
broad panel-based testing like next-generation sequencing (NGS) in appropriate patients
with stage IV NSCLC, because it is more efficient and cost-effective than testing
for one biomarker at a time for detection of rare mutations to which targeted therapies
are available.[7] NGS allows the sequencing of several genomic regions in a single test, in a single
platform and even in samples with low DNA content. An NGS-based approach potentially
provides a more sensitive and comprehensive genetic characterization of lung cancer,
which may impact the therapeutic options and patient's prognosis.
Aims and Objective
The aim of this study was to evaluate the mutational profile by NGS method in patients
with metastatic NSCLC, diagnosed in a Tertiary Hospital in North East India.
Materials and Methods
This study is a hospital-based prospective observational study performed on 88 patients
evaluated and treated under the Department of Medical Oncology, State Cancer Institute,
Guwahati during a period of 1 year from June 1, 2020 to May 31, 2021. All patients
aged 18 years or above, who were diagnosed as metastatic NSCLC with Eastern Cooperative
Oncology Group performance status (ECOG PS) 0 to 2 and gave consent for the study
and underwent mutational study by NGS were included in the study. Patients with histology
large cell lung cancer, SCLC, or ECOG PS 3 or more were excluded from the study.
Ethical clearance was obtained from the ethical committee of the institution prior
to the study vide letter no. SCI/ECR/2020/15 held on April 29, 2020 at Seminar Hall
State Cancer Institute, Guwahati
The data for the study was collected in a predesigned proforma. The patients were
fully informed about the study and their informed consent was taken prior to their
participation in the study. A detailed clinical history, physical examination, and
relevant investigations were done. Nonsmokers are those patients who never had a smoking
history or smoked less than 100 cigarettes in lifetime. Ex-smokers are those who smoked
more than 100 cigarettes in his lifetime but quit smoking 1 year back and current
smokers are those who continue to smoke.
NGS was performed in fresh frozen paraffin embedded tissue block. DNA from samples
was extracted and subjected to NGS using the ion S5 system. To achieve optimal sensitivity,
this assay requires a minimum of 10 to 100 ng DNA. High-quality nucleic acids that
passed quality control checked were subjected to library preparation and analyzed
for relevant genomic alternations. In this study, we have included five genes, for
example, EGFR, EML4-ALK, BRAF, MET, and ROS1 that are under current recommendation
from NCCN guideline for broad-based panel testing for metastatic NSCLC and they have
specific actionable targeted therapy recommended against them. Sequencing was performed
to achieve a minimum 500x depth of coverage. Reporting was done only for variant with
a minimum coverage of more than500x. The output sequences were aligned to the human
reference genome hg19 (GRCh37). High-quality sequencing data was then analyzed using
the optimized ION (a software used in NGS data analysis) torrent suite and the ION
Reporter software to accurately detect rare somatic variants. The hotspots, indels,
and fusions were analyzed with the help of the ION reporter software and variants
were annotated according to the American College of Medical Genetics and American
Medical Oncologist (AMO) guidelines.
NGS panel study for mutational analysis was not available in study the center, so
it was done using a sponsored coupon form Novartis Healthcare under their patient
support program.
Results
This study is a hospital-based prospective observational study performed on 88 patients
in department of Medical Oncology, State Cancer Institute, Guwahati, during the study
period of 1 year from June 1, 2020 to May 31, 2021. After considering the inclusion
and exclusion criteria, a total of 88 cases were studied. The results and observations
of this study are described in the following text.
Majority of cases (n = 30) of mNSCLC cases were in the age group of 41 to 50 years (34.1%). The next majority
of patients (n = 21) were in the age group of 51 to 60 years (23.9%). Highest age was 75 years and
lowest was 26 years in this study. Average age at presentation in the study was 53.74
years. Majority of the patients (n = 47) were male (53.4%). Females comprised of 46.6% of cases (n = 41). The male: female ratio in the study was 1.14:1. Out of 88 patients, 70 patients
(79.5%) were from rural areas and 18 patients (20.5% cases) were from urban areas.
Most patients in the study were nonsmoker in the study (n = 47, 53.4%), while 21.6% (n = 19) were ex-smoker and 25% (n = 25) were current smoker. In this study, adenocarcinoma (n = 75, 85.2%) was the most common histological type followed by squamous cell carcinoma
(n = 10, 11.4%) and NSCLC, not otherwise specified (n = 3, 3.34%) as shown in [Fig. 1].
Fig. 1 Histopathological types of metastatic non-small cell lung carcinoma (NSCLC). NOS,
not otherwise specified.
mNSCLC cases with adenocarcinoma histology had the overall highest mutational status
(n = 55, 62.5%). Squamous cell carcinoma and NSCLC, not otherwise specified type had
very rare mutational status (n = 1 each) as shown in [Table 1] and [Fig. 2]. Total 31 patients (35.3%) had no detectable mutation in the study.
Table 1
Mutational pattern among mNSCLC cases
|
Histology
|
Mutation detected
|
Mutation not detected
|
|
Number (n)
|
Percentage (%)
|
Number (n)
|
Percentage (%)
|
|
Adenocarcinoma
|
55
|
62.5
|
19
|
21.6
|
|
Squamous cell carcinoma
|
1
|
1.1
|
10
|
11.4
|
|
NSCLC, NOS
|
1
|
1.1
|
2
|
2.3
|
|
Total
|
57
|
64.77
|
31
|
35.3
|
Abbreviations: mNSCLC, metastatic non-small cell lung cancer; NOS, not otherwise specified.
Fig. 2 Mutational landscape in metastatic non-small cell lung carcinoma (NSCLC). NOS, not
otherwise specified.
As shown in [Table 1] and [Fig. 2], EGFR (n = 32, 56.14%) is the most common mutation detected in mNSCLC followed by EML4-ALK
(n = 19, 33.33%). ROS1 mutation was detected in four patients, while MET and BRAF V600E
mutations were detected in one patient each. Among the mNSCLC cases where driver mutations
were detected (n = 57, 64.77%), adenocarcinoma is the most common histology (n = 55, 96.49%). Again, among metastatic adenocarcinoma of lung (n = 55), EGFR is the most common mutation (n = 31, 56.33%) followed by EML4-ALK (n = 19, 33.33%). ROS1, BRAF V600E, and MET were the rare mutations detected in 5.45%
(n = 3), 1.81% (n = 1), and 1.81% (n = 1) each.
Most common variant in EGFR mutation was Exon 19 (n = 17, 53.12%) followed by Exon 20 (n = 8, 25%) and Exon 21 (n = 6, 18.75%) as shown in [Fig. 3]. EGFR mutation in Exon 18 was the rarest mutation (n = 1, 3.13%). Two patients with EGFR Exon 20 have upfront T790M mutation. p.Glu746_Ala750
was the most common variant in EGFR Exon 19 mutation, while L858R was the commonest
variant in EGFR Exon 21 mutation.
Fig. 3 Subtypes of epithelial growth factor receptor mutations.
Among patients with mNSCLC with detectable driver mutation, skeleton is the overall
most common site of metastasis (n = 39) followed by pleural effusion (n = 13), brain (n = 12), and opposite lung (n = 9). Also, skeleton is the most common site of metastasis across all subtypes of
driver mutations, while brain is the most common site in the EML4-ALK mutated patients.
Discussion
This study is a prospective hospital-based study of mutational profile of metastatic
NSCLC patients. NSCLC is the most common histological type of lung carcinoma that
can be further divided into adenocarcinoma, squamous cell carcinoma, and large cell
carcinoma. In this era of precision medicine, NSCLC is further divided based on driver
mutation like EGFR, EML4-ALK, and ROS1. These targets are important not only for classification
but also for therapeutic purpose and for prognostication.
Chun et al[8] and Zhang et al[9] in their study found that the median age of mNSCLC was 63 years (range, 34–83) and
63 years (range, 33–87), respectively, and in our study, we found that majority of
cases (n = 30) were in the age group of 41 to 50 years (34.1%) followed by 51 to 60 years
(n = 21, 23.9%) age group. Average age at presentation in the study was 53.74 years.
There was male preponderance of mNSCLC cases in this study like other studies from
China[9] and Portugal.[11] Smoking plays an important role in oncogenesis of lung cancer, but most mNSCLC cases
in this study were nonsmoker like in other studies. In compared to the study by Zhang
et al[9] and Reis et al,[12] our study also found that adenocarcinoma was the most common histology for mNSCLC.
In this study, we have found that EGFR (n = 32, 56.14%) is the most common mutation detected in mNSCLC followed by EML4-ALK
(n = 19, 33.33%). ROS1 mutation was detected in four patients, while MET and BRAF V600E
mutations were detected in one patient each. As shown in [Fig. 4], in most studies[8]
[9]
[11]
[12] EGFR was found to be the most common driver mutation in NSCLC.
Fig. 4 Mutational landscape in metastatic non-small cell lung carcinoma (mNSCLC). EGFR,
epithelial growth factor receptor.
The incidence of EGFR mutations is much higher in the Indian population than in whites
and is closer to the incidence observed in East Asian countries.[13] The incidence of EGFR mutation differs by ethnicity, with 10 to 23% of adenocarcinomas
in whites being driven by activating EGFR mutations, compared with 40 to 50% of adenocarcinomas
in Asians as shown in [Fig. 5].[14]
[15]
[16]
Fig. 5 Mutational pattern in studies from India. EGFR, epithelial growth factor receptor.
Conclusion
In this study, an approach was made to know the mutational profile and clinical status
of metastatic NSCLC patients attending Medical Oncology Department State Cancer Institute,
Guwahati.
At the end of this study analysis, it was seen that mNSCLC here is common in younger
age group compared with other studies, common in nonsmoker male, and adenocarcinoma
is the most common histology. Most patients were residing in rural areas of the state.
As most patients in stage IV disease presented with ECOG PS 2 (60.23%), palliative
chemotherapy in them may further deteriorate the quality of life and ECOG PS, so searching
for a targetable driver mutation in them is the current standard of care which in
this study was done with NGS technique. We have found that EGFR is the most common
driver mutation in them followed by EML4-ALK. Overall skeleton is the most common
site of metastasis, while presence of brain metastasis may also indirectly suggest
EML4-ALK derived mNSCLC.
There is very limited data from North East region till now regarding mutational study
in mNSCLC. Most current recommendation is based on western data. However, geographical
and demographic differences of this part of world may have impact on the mutational
profile in lung cancer patients from this region. About 64.77% of our mNSCLC patients
have driver mutation detected by NGS method as per current recommendation of which
56.14% had EGFR and 33.33% had EML4-ALK mutation. Although this study comprises relatively
a small sample size for a short period of time, it opens up wide range of possibilities
for further studies. Larger prospective study with a large sample size targeting multiple
mutations for a long duration is required to give us more comprehensive understanding
of the mutational landscape of mNSCLC in this part of world in the current era of
precision medicine.
Limitation of the Study
A limitation in our study is that it was conducted on a small sample size of 88 patients.
Since the duration and study population were less, further studies with a large number
of patients over a larger duration of period are required.
