EGFR itself (Singh, C., et al., 2014; Larsen, J.,

EGFR gene feeds directions for producing a small chemical, the epidermal growth factor receptor, which are generated by a cell and
bind to other protein (ligand) on the same cell or outside the cell – like
fitting of a key into locks. The attachment of the ligand to the epidermal
growth receptor will create a chemical reaction inside the cell. This will
trigger activation of signal pathways promoting controlled cell growth and
proliferation. In cancer, the mutated gene “believes” the growth factor is bind
though it is actually not attached. As a result, the tumor cell proliferates
uncontrollably. F (Kobayashi, K., et al., 2013). Alteration usually happen in
exon 18-21, but 90% of deletion and point alteration take place in exon 19 and
L858R, respectively (Zappa, C., et al 2016).

P53 regulates cell cycle, initiates apoptosis and preserve genome integrity,
also works as a master growth regulatory switch (Singh, C., et al., 2014). Alterations mainly suggest G to T substitution caused by bulky DNA adducts
(Massion, P., et al., 2003). P53 gene will affect tumor-suppression functioning resulting to uncontrolled cellular
proliferation. P53 was the
frequently mutated protein in lung cancer in both SCLCs and NSCLCs, approximately 100 % and 90 %, respectively. P53 genes has the capacity to
transform by binding with normal P53 and then inactivate itself (Singh, C., et
al., 2014; Larsen, J., et al., 2011).

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Anaplastic Lymphoma Kinase (ALK) is a growth factor receptor that can be seen in cancer cells.
Generally, it can be found in adult’s brain tissue and researched has
established that it is vital receptor in fetal development. In the course of
cell mutation, intron 10 will merge with intron 13 and “turn it on” resulting
in cancer multiplication (Roh, M. 2014; Eldridge, L. 2017).

BRAF
is a serine/ threonine kinase molecular pathway that is responsible for the
regulation of cell multiplication. It can be found on the upper part of the MEK
and ERK signalling cascade. BRAF mutation will stimulate MEK and ERK
resulting to cancer cell activation. (Guanghui, C., et al.,).

FGFR1 is one of the commonest amplified genetic
factor in human malignancies. No recognized targeted therapeutic agents for
SQCs. Research suggest that FGFR
inhibitors will result to considerable tumor reduction in size, therefore, it could possibly be an advantageous
therapeutic option in FGFR1 amplification (Okimoto, R., et al., 2014).

Molecular testing of lung cancer

Molecular biomarkers were characterized
into three groups (Mutation, gene rearrangement and amplification)

Mutations
including EGFR, KRAS, BRAF, and HER2

 

Methodology

Direct sequencing, real-time
polymerase chain reaction (PCR), and commercial kits are some of the various
techniques employed to identify mutations.
The analytical sensitivity, benefits, drawbacks turnaround time of each
technique must be taken into consideration prior to the utilization of the
system (Hyo, S., et al., 2017). The
gold standard for cell mutation analysis is direct sequencing. However, percentage
of inaccuracy level is high if tumor tissue is insufficient, thus, it
necessitates more than 50 percent (tumor purity) of the total tumor tissue to
normal tissue for accurate results (Davis, M., et al., 2014).
On the other hand, polymerase chain reaction (PCR) techniques only requires small amount of
cell (1 percent) for analysis. PCR technique is high in sensitivity and is a
quick test. Nonetheless, PCR method can only identify certain cell mutation.
Direct sequencing and real-time PCR are utilized for the analysis EGFR, KRAS,
BRAF, and HER2 mutations. EGFR mutations is generally researched
and analyzed in the study of lung cancer. T790M is a test high in sensitivity,
is also beneficial in detecting EGFR (Hyo, S., et al., 2017;  Lindeman, N. et al., 2013).

Sample types

Specimen for analysis
can be collected from a various type of technique: lung biopsy and needle
aspiration (transbronchial), ultrasound-guided (endobronchial), and CT–guided
biopsy. Transthoracic biopsy is invasive, time-consuming and not suitable for unresectable growths (Davies,
M., et al., 2014). In addition to that, tissue
biopsies are frequently inadequate for molecular analysis. Because of this, new test is being introduced, liquid
biopsy, identification of malignant growth in a person’s bodily fluid which comprises
of circulating malignant cell or malignant DNA. Implementation of plasma analysis will prevent patient from undergoing
repeat biopsy procedures, and lessen sequencing artifact caused by formalin
fixation of samples. However, negative results necessitate biopsy because of
high frequency of false-negative result (Sholl, L., 2017; lung., anon).

Gene
rearrangements including ALK, FGFR

 

Methodology

Fluorescence in situ hybridization
(FISH), immunohistochemistry (IHC), reverse-transcriptase polymerase chain
reaction (RT-PCR), and NGS are the existing procedures to evaluate gene
rearrangement.

Presently, FISH is the
standard technique in detecting gene rearrangements while IHC is being utilized
as screening technique. This test is high in sensitivity and specificity in
detecting gene rearrangement despite of fusion partners. It is also generally used in testing alteration
in gene copy number (Hubers, A., et al., 2013; Naidoo, J., et al., anon).

Limitations
encountered in using FISH includes technical complexity in performing and
interpreting the test, and preparation and storage of tissues. Contrary to
Rt-PCR, FISH can detect fusion with different partners (Sholl, L., 2017).

With regards to mRNA expression, RT-PCR is
beneficial in finding fusion transcript with unique sequences. RT-PCR is
considered to be a highly sensitive test. However, only recognized variants are
being detected, thus, primer design necessitates specific details about fusion
partners and breakpoints. Moreover, in comparison to fresh frozen tissue, RNAs
may severely damage prompting the use of FFPE samples in most molecular testing (Roberts, S., 2016; Liao, B., et
al., 2015).

The
next generation sequencing (NGS), are revolutionary tool that can concurrently identify gene fusions by
means of targeted DNA or RNA sequencing (Sholl, L., 2017; Roberts, S., 2016). Due
to its sensitivity, a small number to several hundred nanograms of DNA sample
can provide reliable results. However, sensitivity is often reduced when tumor
content is relatively low. Similar method (FISH) should be used to confirm or invalidate
low quality results (Sholl, L., 2017).

Immunohistochemistry
(IHC) has been integrated by
various recognized institution for a speedy
diagnosis of EGFR mutations, as in occasions wherein the result is needed
immediately. Moreover, it has been recognized that it is suitable for testing
when limited tissue prevents sequencing. Such situations are small biopsy
specimen and specimen with removed calcium deposits. Despite the fact that is
is usefeul, IHC are incapable of detecting the detailed amount of base pair
deletions (15 base pairs) and the range of mutation in 9 or 12 base pairs (Naidoo, J., et al.,
anon).

Sample type

Sample
obtained thru biopsy, cytology specimen and smear slides are suitable for gene
rearrangement analysis. Cell block are primarily utilized when sufficient tumor
cells are available. Tumor purity is not a requirement for FISH test, however, a
well-conserved 50-100 cancer cells are needed. Nevertheless, RT-PCR and NGS
technologies’, cancer percentage is crucial in gene rearrangement analysis. If
needed, tumor dissection may be performed to enhance growth content

Amplifications including FGFR1, EGFR, 
and HER2

Methodology

The
standard technique in detecting gene amplification is FISH using locus-specific
intensifier (LSI) protein and chromosome-specific centromere (CEP) probe. FISH
assay has the benefit of measuring gene amplification by selecting specific
malignant cells.

Sample type

Biopsy
samples that normally comprise of FFPE tissues may be utilized to test for
molecular amplification.

Molecular Treatment of lung cancer

Targeted Treatments

An interesting evolution in treatment of lung
cancer is the development of targeted treatments. They are so called “targeted therapies” since they
are intended to target specific cancer cell by interfering a particular protein. Comparing to
chemotherapy drugs, which cannot identify the normal from cancer cells,
targeted therapies are analytically designed and generated to attack and block the progression specific
malignant target cells. Chemotherapy drugs are cytotoxic, whereas targeted
therapies are cytostatic – they block proliferation of malignant cell. People
suffering from lung cancer may be treated with only targeted drug or with
combination of chemotherapy.

One method to detect a possible target is by comparing protein in tumor
and normal cell. Proteins that exist numerously in tumor cells would be a
possible target especially if they affect cell multiplication or survival. A
different method is identifying tumor cells which generates abnormal proteins
that initiates tumor development. A good example is the cell growth signaling
protien – BRAF. Abnormal
structure of BRAF is known as BRAF V600E which are present in melanomas. Vemurafenib (Zelboraf® is used to target this altered protein (Chen, D., et al.,2014; Okimoto, R., et al.,
2014). Some of the targeted therapies are approved
to target specific biomarkers for the treatment of lung cancer. Some of the
prominent biomarker includes Epidermal growth factor receptor (EGFR),
Anaplastic lymphoma kinase (ALK) and K-ras mutations (KRAS 1) (Pendharkar , D., et al.,
2013).

Some of the recommended drugs
to treat alteration in EGFR: 1) Erlotinib (Tarceva) this drug is used to treat people with NSCLC by blocking the receptor –
epidermal growth factor receptor (EGFR) which can be found on the surface of
the cell. 2)Afatinib (Gilotrif) drug
used for patients with metastatic NSCLC and with alteration or deletion in
EGRF. 3)Gefitinib (Iressa)
approved by the FDA as the first-line management of patients with NSCLC. Crizotinib (Xalkori) and Ceritinib (Zykadia)
are used for the treatment of patient with ALK protein alteration. The former’s
mechanism of action is blocking and stopping the multiplication of cancer cell,
while the latter is used by patient who do not tolerate and respond to
Crizotinib (Pirker, R., et al., 2016).

Types
of targeted therapy

Immunotherapy

Immunotherapy is a
well-tolerated cancer treatment that aids immune system
to fight malignant cells. It is used for
metastatic squamous NSCLC which was ineffectively cured with chemotherapy. Overall, immunotherapy utilizes
individual’s own immune system to treat lung cancer effectively.

Main categories of immunotherapy include monoclonal antibodies, adoptive T-cell transfer
and checkpoint inhibitors. Monoclonal antibodies are drugs
that target specific tumor antigen. Some monoclonal antibodies “mark” malignant
cells, hence, antibodies can easily locate and spoil them (Patel, J., 2016).

Adoptive T-cell transfer is a treatment in which it enhances T-cells
capacity to compete with cancer. In this technique, T-cells are extracted from
the tumor, the genes are then modified in the lab to strengthen anticancer
response. T-cells are white blood cells and
plays a vital role in the immune system (Lung., anon).