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