




































Circulating Nucleic Acids as Promising Biomarkers- A New Frontier of Personalized Medicine


Berkeley
Pharma Tech
Journal of Medicine

Correspondence 
CarolynSkotz@gmail.com

Keywords
Biomarker
Circulating DNA
Liquid Biopsy
MicroRNA
NSCLC
Personalized Medicine Prognosis

Submitted June 29, 2021 
Accepted July 6, 2021
Published December 17, 2021

Full Open Access

Creative Commons Attribution 
License 4.0

Abstract
Biomarkers are a range of biological signals that measure the presence and severity of the 
disease. This literature review assesses circulating DNA (ctDNA) and microRNA 
(miRNA) biomarkers detected in liquid and tissue biopsies and their importance in the 
prognosis, outcomes, and treatments for non-small cell lung cancer (NSCLC). 
These biomarkers have the potential for clinical use; however, further studies with 
requisite data and sufficiently large trials are required to refine our understanding of 
their applicability. The prognostic significance of ctDNA and miRNA biomarkers in 
NSCLC care has demonstrated that liquid biopsy and molecular diagnostic testing 
may provide a feasible and noninvasive method for tailoring treatment plans to the specific 
mutational landscape of diverse NSCLC patients. However, further testing must be 
conducted to analyze the significance and benefit of ctDNA and miRNA 
biomarkers in larger cohorts and substantiate the standardization of liquid biopsy in 
clinical practice.

Circulating Nucleic Acids as 
Promising Biomarkers: A New 
Frontier of Personalized Medicine
By: Carolyn Skotz, Samrat Thapa, Mary Nowak, Hoc Lan Phung, Akshitha 
Mamidi, Hetu Patel and Mabel Ho

Circulating DNA and microRNA are useful biomarkers in non-small cell lung cancer prognosis and treatment.                
Image Credits: N/C



Introduction

Despite representing 13% of all cancer diagnoses, lung cancer accounts for
24% of all cancer deaths in the United States each year. Among the two
subsets of lung cancer (non-small cell and small cell), NSCLC accounts for
80% of all lung cancers. With only 19% of the diagnosed patients surpassing
the ve-year survival rate, there is an urgent need for early detection,
diagnosis, and prognosis.

Recently, studies have shown that these predictions for NSCLC can be
assessed via biomarkers (Howlader et al., 2020). A biomarker is broadly
de ned as an indicator of the presence and severity of a disease state. It can
refer to a range of biological signals, including pulse, blood pressure, blood
tests, and tissue tests. Hundreds of novel biomarker-related articles are
published each year, but only a few biomarkers are currently used in
practice (Rinaldi et al., 2011). Notably, the current approach relies
primarily on the use of tissue biopsy-based biomarker testing. Tissue biopsy
refers to an invasive procedure in which solid matter from the body, usually
coming directly from the tumor or bone marrow, is sampled and assessed
for the presence of clinically actionable biomarkers. Although this method
is deemed the gold standard for NSCLC testing, it is time-intensive,
invasive, and often fails to capture tumor heterogeneity (Rijavec et al.,
2019).

Liquid biopsy is an emerging, minimally invasive process that can address
this demand for early disease monitoring. Via this method, clinicians can
quantify biological components circulating in bodily liquids, such as tumor
cells and nucleic acids, including DNA and miRNA. Circulating DNA
(ctDNA) is derived from DNA and released by cancerous cells and tumors
into the bloodstream. As a tumor grows, newer cells replace older cells. The
dead cells are broken down into their main components, releasing DNA
into the bloodstream (Mader et al., 2017). MicroRNA (miRNA) are
single-stranded, non-coding RNAs in the blood and function as antisense
RNA to regulate the target genes. Multiple miRNAs can target the same
gene (Wang et al., 2018). The pathways of ctDNA and miRNA are
depicted in Figure 2 and Figure 3, respectively.

There are three main classes of biomarkers. Predictive biomarkers interact
with speci c treatments to a ect the outcome, prognostic biomarkers are

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associated with particular outcomes regardless of the course of treatment,
and treatment-focused biomarkers are any biomarkers that in uence the
selection of a speci c course of treatment (Stein, n.d.). This paper outlines
several ctDNA and miRNA biomarkers detected from liquid biopsies and
uses previous data to assess their value in delineating NSCLC prognosis,
outcome prediction, or treatment strategies. Studies regarding these
biomarkers were reviewed according to sample size, procedure, and clinical
relevance. The speci c ctDNAs and miRNAs addressed in this paper are
summarized in Figure 1.

Figure 1: List of NSCLC ctDNA and miRNA biomarkers. This list is not comprehensive;
It serves as a brief summary of the ctDNA and miRNA biomarkers discussed in this paper.

Common Methods utilized for circulating nucleic acid
assessment:
Several primary technologies have been used to assess circulating nucleic
acid biomarkers.
1. Fluorescence in situ hybridization (FISH):
FISH utilizes a DNA sequence probe with a uorescent dye attachment to
locate and label a speci c complementary DNA strand on a chromosome.
This uorescent marker can be visualized through microscopy. This method
is applicable to blood, cytology smears, and bone marrow to detect
duplications, deletions, and total chromosomal loss or gain (Green).
2. Immunohistochemistry (IHC):
During IHC, xed tissue is exposed to antibodies, which enter and adhere
to antigenic determinants. Once this antibody-antigen substrate is created,
it catalyzes an oxidation reaction to form a visible colorized marker. This can

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be used as a tool for measuring protein and nucleic acid expression
(Schildhaus et al., 2020).
3. Polymerase chain reaction (PCR):
PCR allows for the rapid ampli cation of small DNA fragments. First, the
sample DNA is denatured at a high temperature to form two
single-stranded DNA fragments. Second, Taq polymerase, a thermostable
enzyme, attaches complementary DNA strands (known as primers) to each
exposed strand. This process results in the duplication of the original DNA
strand once elongation has been completed. Millions of copies of a speci c
DNA strand can be produced after several cycles of this process (Waters &
Shapter, 2014).
4. Next generation sequencing (NGS): NGS refers to the parallel
sequencing of genomic segments. There are three main kinds of NGS:
a. Illumina Sequencing: In illumina sequencing, the DNA bases are
identi ed by their unique uorescent signals.
b. Roche 454: Roche 454 sequencing relies on the release of visible
uorescent pyrophosphate that occurs upon nucleotide addition to a new

strand of DNA.
c. Ion Torrent: Ion torrent sequencing measures the release of protons due
to the incorporation of nucleotide bases in the DNA.

Circulating DNA Biomarkers

Figure 2: An illustration of the ctDNA pathway. At the top, the tissue cells are shedding into
the bloodstream. An immune response occurs in order to eliminate the tumor cells entering the
bloodstream and to eliminate any CTCs. As part of this elimination, many tumor cells are

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phagocytized, thus increasing the concentration of ctDNA in the bloodstream, which can be
analyzed through liquid biopsy (Delmonico et al., 2020, Bettegowda et al., 2014).

ERCC1
Excision repair cross-complementation group 1 (ERCC1) is a part of
the nucleotide excision repair (NER) pathway that acts as a mechanism
for DNA repair, and also removes the cytotoxic elements from
genomic DNA. For example, platinum is a cell-damaging agent that
causes apoptosis, therefore regulating cell growth and death in the
body. Overexpression of ERCC1 can cause lower platinum absorption,
resulting in decreased cell death and subsequent increased cell growth
and carcinogenesis. Underexpression of ERCC1 can cause
carcinogenic factors to enter genomic DNA and hinder DNA repair
mechanisms because ERCC1 typically repairs any DNA damage done
by platinum (Li et al., 2016). Hence, ERCC1 expression level directly
correlates to platinum sensitivity (Hamilton et al., 2018).

Programmed death receptor-1 is an immune-regulatory protein with
two ligands, PD-L1 and PD-L2, that is expressed by immune cells and
tumor cells. In a study done by Buderath et al., PD-L1, PD-L2, and
ERCC1 levels were measured in 83 patients with epithelial ovarian
cancer through liquid biopsy. Results showed that higher ERCC1
expression was associated with lower PD-L2 levels. Lower SP-L2 levels
were found to be associated with platinum resistance (p < 0.0001), which
supports the results from previous studies (Li et al., 2016; Hamilton et al.,
2018). Higher PD-L2 levels were associated with lower ERCC1 expression
and reduced progression free survival (PFS) and overall survival (OS), while
lower PD-L2 levels were associated with platinum resistance and higher
ERCC1 expression (Buderath et al., 2019). This demonstrates that ERCC1
expression levels detected through liquid biopsies can be used to predict the
outcomes of multiple cancers, including NSCLC and epithelial ovarian
cancer.

MET
MET is a proto-oncogene involved in cell growth, wound healing, and
post-physical injury response. It encodes the receptor tyrosine kinase
c-MET, for hepatocyte growth factor, which causes c-MET dimerization
and autophosphorylation. This results in the activation of
mitogen-activated protein kinases (MAPK), phosphatidyl-inositol-3’
phosphate kinases (PI3K), viral oncogene homologs, and signal transducers

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and activators of transcriptional signaling pathways. Mutated MET
initiates the growth of cancer cells. Unusual MET expression has been
shown to correspond with NSCLC, as well as gastrointestinal cancer and
hepatocellular carcinoma. If the MET receptor is overexpressed via
genomic ampli cation, mutation, or alternative splicing, cellular
degradation of MET can occur (Mo et al., 2017).

c-MET receptor activation is associated with EGFR-TKI resistance and has
poor prognosis. A trial consisting of 167 patients examined the prognosis,
determined by PFS, after treatment with MET inhibitors, erlotinib and
tivantinib. 84 patients were randomly assigned to receive oral erlotinib and
tivantinib (ET group), and 83 patients were randomly assigned to receive
erlotinib plus placebo (EP group). The average PFS was 3.8 months for ET
and 2.3 months for EP, which is statistically signi cant (P =.24) (Sequist et
al., 2011). Deng et al. also studied NSCLC patients with MET
ampli cation and EGFR mutation. For one patient, a combination of
crizotinib and osimertinib was administered, which greatly improved his
overall health. Results showed a 20-40% partial response rate to dual
therapy with and without prior treatment with TKIs. Unfortunately, MET
mutations have occasionally been shown to cause resistance to crizotinib
treatment (Deng et al., 2018).

In a study done by Bardelli et al., 7 patients with metastatic colorectal
tumors who had responded to panitumumab or cetuximab-based
treatment and later relapsed were analyzed. MET expression was higher in
post-relapse patients compared to rst-occurence patients. In relapse
patients with KRAS mutations, MET expression was low or undetectable.
MET ampli cation was seen in the blood before relapse, indicating that
MET monitoring could be used to predict relapse. MET ampli cation is
detected in 5-20% of EGFR-mutated lung cancers (Bardelli et al., 2013). In
metastatic colorectal tumors and NSCLC, MET can be used for predictive
outcomes and may help direct the course of treatment.

NTRK
Neurotrophic tyrosine kinase receptor (NTRK) mutations are found in
many solid malignancies, including NSCLC. Trk receptors include three
transmembrane proteins: TrkA, TrkB, and TrkC, which are encoded by
NTRK1, NTRK2, and NTRK3, respectively. In carcinogenesis, TrkA,
TrkB, and TrkC undergo changes that lead to activation of signaling

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pathways involved in cell growth and proliferation. Mutations to NTRK1,
NTRK2, and NTRK3 have also been observed in 2-3% of NSCLC
patients. For example, overexpression, in-frame deletions, and alternative
splicing of NTRK1 have been demonstrated to be potential oncogenic
mechanisms. Several NTRK inhibitors have been developed, but only
LOXO-101 and entrectinib are under clinical evaluation (Ricciuti et al.,
2017). Of 1,378 patients with locally advanced or metastatic solid tumor
malignancies with NTRK1, NTRK2, NTRK3, ROS1, or ALKmutations,
two showed NTRK1 gene rearrangements on anchored multiplex PCR.
One patient, a 45-year-old male with a history of smoking, was treated with
400 mg/m2 oral entrectinib daily. CT scans of his lungs 26 days after
beginning the treatment showed no tumor growth. After 155 days, CT
scans showed a 77% overall tumor reduction (Farago et al., 2015).

CDKN2A
The CDKN2A mutation is uniquely present in NSCLC. Studies have
suggested that the CDKN2A mutation is associated with the increased
expression of the extracellular matrix and metabolic gene sets in NSCLC
cell lines (Kim et al., 2015). This heightened expression has been suggested
as an indicator of increased mortality in lung, breast, and gastric cancers
(Gilkes, Semenza, and Wirtz, 2014). As a result, early detection of
CDKN2A mutations is critical. The e cacy of liquid biopsy for mutation
detection has been suggested in breast cancer studies, but further testing is
needed to determine whether the detection of CDKN2A alterations via
liquid biopsy would yield signi cant clinical bene t for NSCLC patients
possessing alterations in this gene (Veldore et al., 2018; Delmonico et al.,
2019).

CDKN2A alterations in NSCLC can be categorized into several discrete
classes, including copy number changes, hyperphosphorylation, and
deletion (Liu et al., 2020). A study by Wen et al. suggests that the relative
frequency of CDKN2A alterations exhibits signi cant variability among
di erent racial groups, with prevalence ranging from 5.1 to 21.5 percent in
lung adenocarcinomas and 23.2 to 43.6 percent in squamous cell lung
carcinomas. Percentages in this study were drawn fromNGS assessment of
1200 Chinese NSCLC patients and The Cancer Genome Atlas (TCGA)
data for American and European cohorts (Wen et al., 2019). Liu et al., also
assessed data from TCGA to pinpoint important genes implicated in lung
cancer tumor biology related to CDKN2A. A549 and H322 cell lines were

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cultured and evaluated through real-time PCR, Western blot analysis,
2,5-diphenyl tetrazolium bromide (MTT) assay, and cell counting,
invasion, wound healing, and migration assays. The researchers observed a
marked decrease in survival among patients with CDKN2A depletions and
found a direct correlation between CDKN2A knockdown and increased
cell invasion, migration, and proliferation in experiments in A549 and
H322 cell lines. A TCGA Provisional and Pan-Cancer Atlas survival
analysis and further studies on other diseases have suggested decreased
survival rates and other adverse e ects in patients with CDKN2A
depletion. (Reis et al., 2015; Zeng et al., 2018; Dacic et al., 2008). While
CDKN2A loss is suggested to have a negative in uence on prognosis and
survival outcomes, more research is necessary to understand CDKN2A’s
speci c implications in lung cancer (Liu et al., 2020).

With CDKN2A’s potential to be a clinically actionable biomarker, testing
and genomic panels with this gene have been bene cial and often included
in biomarker testing. Recent advances in assay techniques have established
ctDNA as a viable biomarker for detection. One study suggests that
CDKN2A ctDNA, along with the ctDNA of several other cell-cycle related
genes, exhibit only an 81.6% concordance rate to their corresponding solid
tumor biopsies (Mao et al., 2017). Studies analyzing the concordance of
CDKN2A mutation in both liquid and tissue biopsies from breast cancer
patients also revealed di erences in mutations detected through cell-free
DNA analysis and tissue biopsy, although overall mutational prevalence
was quite similar to frequencies observed in tissue samples (Delmonico et
al., 2020). Hence, the impetus for further exploration of ctDNA’s clinical
value is essential to develop increasingly accurate, noninvasive, and timely
methods of detection to ensure that CDKN2A mutations are subject to
earlier interventions, which may ultimately lead to a better prognosis for
NSCLC patients.

RET
Rearranged during Transfection (RET) activating fusion mutations are
found to occur in approximately 1% to 2% of NSCLC patients and have
been suggested to show heightened prevalence in patients who never/lightly
smoked and who are younger (Kohno et al., 2013; Kato et al., 2017). RET
codes for a receptor tyrosine kinase that recognizes growth factors for the
neurotropic factor family, which is derived from glial cell lines. Fusion of
the RET proto oncogene can activate the receptor tyrosine kinase in the

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absence of its ligand. Such an activation can have downstream e ects on
other pathways such as those including PI3K and MAPK (Kato et al.,
2017). The existence of FDA-approved therapies for RET-altered cancers
such as vandetanib and cabozantinib underscores the value of early RET
detection to improve prognosis for patients with NSCLC presenting with
RET-altered cancers. Clinical trials with vandetanib and everolimus have
demonstrated the potential of these drugs to serve viable treatments for
RET-altered cancers that have metastasized to the brain (Subbiah et al.,
2015). In addition, Kodama et al. assessed the e cacy of alectinib in RET
fusion-positive CCDC6-RET and   KIF5B-RET genes. The researchers
determined that alectinib can preclude phosphorylation of RET, thus
providing substantial inhibitory control. Furthermore, alectinib
demonstrated substantial e cacy in treating V804L and V804M mutated
cancers compared to cabozantinib and vandetanib. The suggested basis of
this di erence is supported by alectinib’s structure, which is not in uenced
by the steric e ects of the V804L and V804M gatekeeper mutations
(Kodama et al., 2014).

Mao et al. found that plasma samples of RET ctDNA from patients with
histologically con rmed lung cancer demonstrated 96.2% concurrence with
solid tumor tissue DNA samples. Concordance rates in this study were
determined by an analysis of 40 tissue and plasma samples collected from
participants aged 18-80 who did not qualify for rst-line surgical treatment
(Mao et al., 2017). Similar studies have also demonstrated high concurrence
rates between tissue and liquid biopsies, although the concurrence
percentage was shown to drop signi cantly in one study when analysis was
limited to speci c subtypes of genomic alterations, such as copy number
variations, which demonstrated concordance rates as low as 3.5%; however,
sample size in this cohort was limited to 45 patients (Chae et al., 2017). The
variability in concordance rates between ctDNA and tissue biopsy shows
that further analysis is required to determine whether ctDNA is a viable
standalone method of detection for RET-altered cancers, given that RET
mutations are included in the National Comprehensive Cancer Network’s
recommendation for genomic targets in NSCLC (Yang et al., 2018;
Thompson et al., 2016).

ERBB2 (HER2)
Erb-b2 receptor tyrosine kinase 2 (ERBB2), human epidermal growth
factor receptor 2 (HER2), has become widely acknowledged as a potential

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target for therapy (Kirs et al., 2015; Chuang et al., 2017). While alterations
in ERBB2/HER2 have primarily been studied in breast cancer, recent
research has assessed the potential for targeted therapies in treating
ERBB2/HER2- positive lung cancers (de Melo Gagliato et al., 2016;
Chuang et al., 2017; Veatch et al., 2019). Some studies have shown that
HER2 commonly presents in female patients with adenocarcinoma
histology with no history of smoking (Garrido-Castro and Enriqueta Felip,
2004). ERBB2/HER2 receptors can also be phosphorylated to form
heterodimers with other epidermal growth factor receptors (EGFR), such as
HER1, and play a critical role in signaling pathways that regulate
transcription, cell proliferation, and prevention of apoptosis (de Melo
Gagliato et al., 2016). Approximately
1-5% of lung cancers were found to harbor ERBB2/ HER2 aberrations, but
some studies have demonstrated that the percentage for the NSCLC
subtype likely lies between 1-2% (Kris et al., 2015; Chuang et al., 2017).
Regarding detection methods, Mao et al. demonstrated that ERBB2, along
with other driver genes, exhibits a 96.2% concordance between tissue and
liquid biopsy methods (2017).

Chuang et al. performed a retrospective assessment with nine patients,
seven of whom presented with ERBB2 mutations and advanced NSCLC
between 2013-2016 and two of whom met similar criteria from a
2014-2015 cohort. All patients were treated with a regimen of paclitaxel,
vinorelbine, and trastuzumab. To pinpoint ERBB2 mutations, NGS-based
methods Solid Tumor Actionable Mutation Panel, PCR-based ERBB2
sizing assay, Geneseq assay, or NGS-based FoundationOne assay were
executed on tissue samples, while NGS-based pro ling using deep
sequencing was performed on ctDNA samples. Therapeutic response was
assessed according to Response Evaluation Criteria in Solid Tumors
(RECIST) version 1.1. The results of this study demonstrated that 44% of
the patients exhibited response to targeted therapies. Although the sample
size was small, clinical indications and availability of targeted therapies for
ERBB2/ HER2 may warrant the use of these treatments. The authors
caution that genotypic di erences in ERBB2 mutations may result in
di erential e cacy of targeted therapies. Trastuzumab in conjunction with
chemotherapy has demonstrated therapeutic promise in clinical settings,
although it has not been shown to penetrate the blood-brain barrier,
rendering it less useful for metastases to the brain and spinal cord. Other
potential therapies include dacomitinib and neratinib (NCT01953926)
(Chuang et al., 2017).

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In conjunction with targeted therapies, detection remains a critical factor in
prognosis. Early detection is optimal, but tissue sampling can be di cult at
certain periods of cancer development. As such, ctDNA analysis may
provide a reliable baseline for determining tumor mutational landscape. In
other cancer types, such as cholangiosarcoma, ctDNA analysis resulted in
the successful detection of ERBB2 (HER2) mutations. Liquid biopsy was
selected in one case study in lieu of tissue biopsy due to challenges in tissue
acquisition. As a result of ERBB2 (HER2) mutation detection via liquid
biopsy, the patient was able to begin dual anti-HER2
pertuzumab/trastuzumab therapy as an o -label treatment regimen
(Yarlagadda et al., 2019).

Another study in 29 colon cancer patients demonstrated that proper
clinically veri ed circulating free tumor DNA analysis may provide a
method of detection for HER2 copy number changes (concordance with
tissue equaled 96.6%), allowing clinicians to identify patients who may
bene t from anti-HER2 therapies. In addition to high concurrence rates,
ctDNA has also been suggested to surmount the issue of tumor
heterogeneity. Nevertheless, more studies are required to determine
whether the observed concordance rates are a ected by di erential release
of tumor DNA into circulation (Siravegna et al., 2019).

BRCA1/2
Accurate DNA replication is integral to genomic integrity and protection
against deleterious mutations. The BRCA1/2 genes work sequentially to
ensure that this integrity is maintained, speci cally by assisting in DNA
lesion repair. Despite deriving its name from its frequent association with
breast cancer, BRCA1/2 genes have also been implicated in NSCLC (Hu et
al., 2019; Remon et al., 2020; Ji et al., 2020). With regards to targetability,
germline BRCA-mutated (gBRCAm) cancers have demonstrated
promising responses in poly-(ADP ribose) polymerase (PARP) clinical trials
and other studies (Hu et al., 2019; Ji et al., 2020). In a study by Remon et
al., analysis was performed on advanced NSCLC patient specimens
negative for ALK rearrangements and positive for activating EGFR
mutations. NGS and single nucleotide polymorphism (SNP) arrays were
performed on the samples in order to assess genomic features in patients
new to targeted therapy. Using molecular data, the authors determined the
prevalence of pathogenic mutations in BRCA genes, RECIST-assessed
response rate to platinum-based chemotherapy, and characteristic

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clinicopathological features of BRCA mutated patient subgroups.
Reported BRCA prevalence without respect to mutation type was 5.3%
(20 out of 379 patients), although pathogenic mutations in BRCA1/2
constituted only 2.1% of advanced NSCLC patients. The results of Ramon
et al.’s study demonstrated that there may not be a signi cant link between
BRCA mutation in NSCLC and improved response to platinum based
chemotherapy; a similar inconclusiveness on BRCA’s role in predicting
treatment outcomes was noted in another study on tumor samples that
exhibited positive IHC staining for BRCA1 (Ramon et al., 2020; Watchers
et al., 2005). Based on study data, it appeared that BRCA variants of
unknown signi cance (VUS) exhibit longer OS (p=0.07), whereas tumor
protein p53 (TP53) mutation status in BRCA-positive subgroups did not
yield a statistically signi cant di erence in prognosis (p=0.3). Although the
authors found that biallelic mutations in BRCAmay warrant further study
to determine their prognostic signi cance, they noted that data gathered in
their patient cohort did not yield su ciently robust evidence to support
implementation of speci c targeted therapies in monoallelic BRCA1/2
patients (Remon et al., 2020). In contrast to the data presented by Ramon
et al., Ji et al. also analyzed BRCA1/2 in determining e cacy of targeted
olaparib (PARP inhibitor) therapy. Assessment of data demonstrated that
BRCA1 and BRCA2 depleted cells are more susceptible to olaparib.
Similar to its role in breast cancer cells, olaparib was found to exhibit a
similar apoptosis-inducing function in BRCA-mutated NSCLC cells (Ji et
al., 2020). Moreover, other studies have demonstrated that heightened
BRCA1 expression correlates with lower overall survival rates (Joerger et al.,
2011).

While a common concern with the use of ctDNA assays is their ability to
provide an accurate indication of a patient’s mutational landscape, recent
studies have assessed the utility of a BRCA1 selective polyethylene glycol,
gold nanoparticle-infused biosensor. This biosensor showed substantial
selectivity and a BRCA1 lower limit of detection of 1.72 femtomolar
(Wang et al., 2015). These results suggest that biosensor-based detection of
speci c biomarker sequences may serve as a viable method of detection to
address the issue of sensitivity in liquid biopsies (Wang et al., 2015; Yang et
al., 2016).

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PTEN
Phosphatase and tensin homolog (PTEN) serves as a phosphatase for

phosphatidylinositol-(3,4,5)-triphosphate (PIP3). PTEN decreases PIP3
concentration, which in turn serves to downregulate phosphorylated AKT
(P-AKT). In healthy cells, PTEN serves as an indirect downregulator of
P-MAPK and P-AKT, resulting in lower rates of cell division and   increases
in apoptosis (Teresi et al., 2006). Patients carrying EGFR mutations have
reaped signi cant bene t from EGFR-TKIs; however, resistance to these
TKIs poses a signi cant obstacle in treating patients. PTEN loss represents
one mechanism of resistance to TKIs such as ge tinib and erlotinib, and
continuing studies have set out to discover methods to circumvent the
e ects of this resistance mechanism (To et al., 2018).

Peroxisome proliferator-activated receptor agonists (PPARs) are implicated
in the development and di erentiation of cells, tumorigenesis, and
metabolism. Studies have demonstrated that PTEN loss is correlated with
resistance to ge tinib (To, Wu, and Loong, 2018; Lee et al., 2006). Increases
in apoptosis were observed in cell lines treated concurrently with PPARγ
agonists and ge tinib (P<0.017) (To et al., 2018). Hence, PPARγ agonists
in conjunction with EGFR-TKIs may serve as a viable treatment strategy to
circumvent EGFR-TKI resistance in patient subsets that exhibit PTEN loss
(To, Wu, and Loong, 2018; Ni et al., 2017; Lee et al., 2006). Although
future studies require further analysis of PTEN’s ctDNA and solid tumor
concordance rates in NSCLC, comparative analysis of liquid and tissue
biopsies in prostate cancer patients revealed an 88.9% agreement between
individual copy number calls of PTEN (Wyatt et al., 2017).

Despite the higher concordance rate observed in some studies, others
caution that PTEN copy number alterations, when present in low
circulating concentrations, can go undetected (Vandekerkhove et al., 2019).
Thus, a predominant factor in the standardization of ctDNA assessment is
the development of su ciently sensitive assays that have lower limits of
detection, which may preclude the aforementioned issue.

BRAF (V600E and non-V600E)
BRAF (v-RAF murine sarcoma viral oncogene homolog B) is a
serine/threonine kinase involved in the RAS/RAF/MAPK pathway.
BRAF mutations can be categorized in three classes. BRAF V600/E/R/K

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mutations are class I mutations that result in strong activation of BRAF’s
kinase activity and MAPK pathway. The mechanism of V600E mutations
is that it can form salt bridges with K507 and the c-terminal of the alpha-C
helix. They signal as monomers but mimic the dimerization process and
respond to BRAF inhibitors (Dankner et al., 2018).
Class II and Class III mutations are non-V600E mutations. Class II
mutations have high or intermediate kinase activity and serve as RAS-
independent mutations, which signal as dimers and can respond to MEK
and Pan-RAF inhibitors. Class III mutations show low kinase activity and
are also called RAS-dependent mutant BRAF-wild-type. Class II and class
III non-V600 BRAF mutants dimerize with BRAF or CRAF (Dankner et
al., 2018).

A study by Lin et al. was performed to determine the frequency of BRAF
mutations in Chinese individuals. The study determined that 1.2% to 4.2%
of NSCLC patients in the Chinese population had the BRAF mutations.
Authors also determined that only 30% of patients with BRAF V600E
mutation bene ted from BRAF inhibitors. Furthermore, Lin et al. also
reported that the Class I mutation is predominantly found in females,
while class II and III mutations co-occur with the KRAS mutation. This
study’s impact is limited because of its analysis of the BRAF mutation in
speci c races and ethnicities, and thus the ndings cannot be readily
extrapolated to other groups (Lin et al., 2019). In the clinical setting,
detection of BRAF mutation via liquid or tissue biopsy appears equally
reliable, as one study demonstrated that assessment of BRAF alteration
yields a 96.2% concordance rate between liquid and tissue biopsies (Mao et
al., 2017).

NF1
NF1 is a tumor suppressor gene that shuts down activity of RAS protein
via a protein called neuro bromin, which under normal physiological
conditions stimulates GTPase activity of RAS and converts active
RAS-GTP complex to its inactive RAS-GDP complex. This inactivation
can lead to the shutdown of RAS associated GTPases (i.e. KRAS, NRAS,
MRAS, HRAS, RRAS, and RRAS2). In patients with the NF1 mutation,
there is a loss of functional neuro bromin, which can lead to accumulation
of active RAS GTPases. This results in excessive cellular proliferation and
tumor formation which can lead to the development of additional
mutations, apart from those a ecting RAS activity. Beau-champ et al.

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found that loss of NF1 has shown response to dasatinib in
dasatinib-resistant lung cancer (2014). Furthermore, low levels of NF1 with
MAP-ERK expression can be sensitive to erlotinib and reverse the erlotinib
resistance (Tao et al., 2020). TKI resistant NF1 mutations develop by the
following mechanisms:

1. Activation of heat shock factor 1
2. Inhibition of tumor cell apoptosis
3. Promotion of epithelial-mesenchymal transformation
4. Promotion of sustained angiogenesis

Chian et al. performed a case-control study in the blood samples of 187
patients with NSCLC and 310 non-cancer patients. Authors used
quantitative PCR to identify age and gender-based biomarkers (CPEB4,
DUSP6, EIF2S3, GRB2, MCM4, MDM2, NF1, POLDIP2, RNF4,
STAT2, and WEE1). The majority of the population was comprised of
males 66 years and older, as well as smokers. Participants who never smoked
expressed NF1 at lower levels. NF1 also had protective e ects compared to
MDM2 gene. NF1 was shown to be an age-dependent marker (OR: 0.16,
CI: 0.14 to 0.88, P-value: 0.0255).

PIK3CA
Hyperactivation of the PI3K/AKT pathway is common in NSCLC
patients. This pathway is involved in interfering with various cellular
mechanisms such as proliferation, migration, invasion, and resistance to
therapy. Mutations in AKT, the catalytic subunit of PIK3CA, or PTEN
downregulation can activate the AKT pathway. Sawa et al. determined a
relationship between PIK3CA mutation and lung cancer in patients with
COPD. The researchers extracted DNA from the surgical specimens of 197
patients and analyzed using deep sequencing techniques. The frequency of
PIK3CAmutation was signi cantly higher in the COPD group than in the
non-COPD group (10.4% vs. 1.7%, p = 0.015).

The PIK3CA gene mutation can a ect the prognosis of NSCLC through
several mechanisms. A systematic review and meta-analysis performed by
Wang et al. showed that PIK3CA expression status was not an independent
risk factor for OS of NSCLC patients (HR = 0:80; 95% CI:
0.58-1.12; P = 0:193), which is consistent with the results of 3 included
studies.

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ROS1
ROS1 is a receptor tyrosine kinase similar to ALK. It is a single-pass
transmembrane protein with an intracellular C-terminal tyrosine kinase
domain and an extracellular N-terminal domain. ROS1’s extracellular
domain has similar amino acid sequences to that of cell adhesion molecules
and the extracellular matrix. Typically, ROS1 is expressed more in the
kidneys than in the lungs. The mechanism of ROS1 mutations is currently
unknown. In general, ROS1 rearrangement occurs in the absence of other
known oncogenic drivers (EGFR mutations, KRAS mutations, ALK
rearrangements). There are some exceptions as well, which exclude it to be
useful in screening. ROS1 can be identi ed via the FISH and RT-PCR
techniques, but RT-PCR sometimes yields false negatives. IHC is another
technique used to detect mutations of ROS1 (Luk et al., 2018).
Concordance rates for detecting ROS1 alteration is 96.2% between liquid
and tissue biopsies, according to analysis by Mao et al. (2017).

A multicenter prospective cohort study by Mezquita et al. was conducted
on 128 patients to determine liquid biopsy’s clinical usefulness in detection
of ALK/ROS1 fusion and mutation to extrapolate with e cacy of tyrosine
kinase inhibitors. Patients aged 18 years and older with ALK and
ROS1-fusion-positive were enrolled during October 2015 and August
2018. Samples were collected at diagnosis and during radiation therapy.
Blood samples were collected, and ct-DNA analysis as well as fusion load
were evaluated. Absent mutation showed better OS. The median OS was
58.5 months in patients with ALKmutations (95% CI, 26.9 to not reached
[NR]) while 44.1 months in patients with non-ALK mutations (95% CI,
21.7 to NR) and it was found to be 105 months in patients who were
detected negative for ctDNA (P = .001). Also, patients who had complex
ALK mutations showed poor OS (median: 26.9 months; 95% CI, 13.9
months to NR) compared to patients with single ALKmutations (median:
NR; 95% CI, 57.0 months to NR; P = .003). The median PFS was 20.7
months (95% CI, 6.3 to NR) in the negative ctDNA group while it was
found to be 2.8 months (95% CI, 1.2 to NR) in the patients with one or
more ALK mutations (P = .03).

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ALK
ALK is most common in NSCLC patients, presenting itself in about 3-5%
of cases per year. ALK alteration is not hereditary and is most commonly
present in younger patients (55 years old or below) or patients who have
never smoked. IHC or FISH remain the most common strategies to
identify this biomarker. ALK normally encodes for a protein that plays a
role in neural development. Alterations in this gene are caused by
chromosomal translocations and are almost always mutually exclusive to
EGFR and KRAS. ALKmutations are most common among patients with
a never/light smoking history, an adenocarcinoma histology, of a younger
age, of the female gender, and in tumours with the wild type for EGFR and
KRAS. The most common rearrangement of ALK in NSCLC is
EML4-ALK (echinoderm microtubule-associated protein-like 4 (EML4)-
ALK). This mutation leads to uncontrolled proliferation, migration, and
tumorigenesis. Increasing research in fusion genes such as ELM4-ALK
show that alongside coding for proteins, they generate non-coding RNAs
that contribute to tumor progression (Camidge et al., 2010).

In 2011, the FDA approved the rst TKI for ALK mutations, crizotinib.
Since then, researchers began studying ALK as a predictive biomarker. For
instance, Vincent et al. outlined biomarkers involved in clinical practice in
2012, indicating the importance and extensive research that has been done
on ALK over the past couple years. Currently, IHC combined with
crizotinib is considered a gold standard for the detection and treatment of
ALK-positive NSCLC (Shaw et al., 2011). EML4-ALK fusion variant 3
(V3) serves as a new potentially targetable biomarker for higher-risk cases.
Evidence shows that the presence of the V3 protein in ELM4-ALK leads to
acceleration of disease, failure at early stages of treatment, and a poorer OS.
ELM4-ALK V3 is also associated with shorter PFS, even after non-TKI
treatments and chemotherapy (Christopoulos et al., 2018). This suggests
the di erences between the V3 variant and other more common variants,
such as EML4-ALK V2, are clinically distinguishable due to V3’s stark
di erences (P<0.001) in PFS regardless of previous treatment to other ALK
tumor types. V3 variants progress faster in response to TKI treatments,
which is likely due to the developments of TKI resistant mutations. A
study conducted by Madsen et al., analyzed ctDNA of NSCLC patients
with the ALK gene. The results showed the presence of ctDNA before
treatment initiation is associated with inferior PFS as well as the presence of
ctDNA shortly after the start of ALK TKI treatment. This study

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emphasizes the value of genomic pro ling through ctDNA during all stages
of the treatment. Although there are multiple targeted therapies for ALK,
fusion variants of ALK that show poorer PFS and OS still need to be
studied (Madsen et al., 2020).

KRAS
Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most frequent
oncogenic mutation in western countries for NSCLC (Liu et al., 2020). It
is involved in the RAS/MAPK pathway and codes for the K-ras protein
(GTPase), the most commonly mutated gene in its pathway.
Mutations in the KRAS gene impair the GTPase activity, resulting in a
constantly active GTPase, which leads to cell survival, proliferation, and
di erentiation. The most common nucleotide mutation in KRAS is the
G12C mutation, which has been directly linked to smoking. Further
research is necessary to establish a relationship between variant KRAS types
and prognosis. Cox regression and multinomial logistic regression were
used to di erentiate the e ect of KRAS mutation subtype on OS. G12C
subtype was associated with poor OS (P = 0.021), compared to G12D
(Aredo et al., 2019). Data was extracted from patients in the Thoracic
Malignancies Cohort (TMC) to compare clinical features and OS of the
wild type KRAS to KRAS G12C, amongst other mutated types. 100% of
the patients with the KRAS G12C subtype were active smokers. Results
showed that the treatment and survival of KRAS mutant subtypes were
similar (Cui et al., 2020). However, KRAS G12C in particular proved to be
an important predictive biomarker compared to the other KRAS mutant
types, such as wildtype KRAS and mutated KRAS (P = 0.74) (Cui et al.,
2020). Although there are no current KRAS inhibitors on the market, it
has been marked as a clinically applicable biomarker in a paper by Vincent
et al. in 2012. Subsequently, research conducted since then corroborates the
potential for KRAS to serve as a potential predictive biomarker in NSCLC.
Data collected from plasma based liquid biopsies by NGS showed that 36
(18.6%) out of the 194 liquid biopsies were a KRAS G12C variant. Since
tissue biopsies are often rejected (17%) due to insu cient cellularity, KRAS
detection in ctDNA may bene t from clinical implementation.

TSC1/2
The TSC1-TSC2 complex is a key regulator of the mTORC1 (mammalian
target of rapamycin complex 1), which controls cell growth. Hamartin
(TSC1) and tuberin (TSC2) are the mutations of genes found in the

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tuberous sclerosis complex (TSC). Inhibition of TSC1/2 results in inactive
mTORC1, leading to uncontrolled cell growth. Fuchs et al., determined
the inverse correlation between hamartin and p-mTOR (the catalytic
subunit of two distinct protein complexes which controls cell proliferation)
expression in NSCLC and SCLC samples. FISH and western blot analysis
were performed on 166 NSCLC and SCLC cell lines. Furthermore,
cytoplasmic hamartin expression was expressed in greater than 50% of
NSCLC cell lines (Fuchs et al., 2014). Fuchs et al., concluded that
activation of lung cancer cell lines is caused by the inhibition of the
mTORC by the TSC. Unlike most other NSCLCmutations, TSC1/2 may
be caused by EGFR expression, but is not dependent on it. Wang et al.
utilized 144 NSCLC patients to study the characteristics of TSC1 and
TSC2 in NSCLC. The researchers found 27 of the 144 NSCLC patients
exhibited TSC1 mutation and 40 exhibited TSC2 mutation. It must also be
noted that most patients with TSC mutations expressed other oncogenic
gene alterations as well. The expression of TSC1/2 was slightly more
common in NSCLC compared to SCLC (53.7% vs. 43.6%) (Wang et al.,
2020). Furthermore, the TSC1 mutation had a median OS of 14.1 months,
whereas patients with TSC2 mutation had a median OS 110.6 months;
however, the di erence was not statistically signi cant (P = 0.201). Wang et
al. concluded that TSC1/2 de nes a unique NSCLC population and often
coincides with other mutations such as TP53. In an experiment conducted
by Rulli et al., liquid biopsies were used to measure the number of
circulating tumor cells (CTCs), the quantity of cell free tumor DNA
(cftDNA), and the mutational pro le of DNA from CTCs (ctcDNA) and
cftDNA in early stage breast cancer patients. NGS of ctcDNA and
cftDNA showed that 52% of the patients expressed mutations in multiple
genes including TSC1 (Rulli et al., 2020). In addition to the signi cance of
TSC in NSCLC prognosis and treatment, initial research shows strong
promise for it to be implemented via liquid biopsies. Currently, there are no
targeted therapies on the market for TSC1/2, although PD-1 and/or
CTLA-4 inhibitors show success in clinical trials for TSC1/2 associated
tumors (Liu et al., 2018).

FGFR1/2
Fibroblast growth factor receptor 1 and broblast growth factor receptor 2
are members of the broblast growth factor receptor family. FGFR1 and
FGFR2 span the cell membrane and interact with broblast growth factors
outside the cell. This results in a cascade of downstream signaling within

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the PI3K and MAPK pathways, which control embryonic development,
cell proliferation, di erentiation, and migration (Theelen et al., 2016). In a
study conducted by Theelen et al., 653 early stage NSCLC samples were
assessed. The levels of expression in FGFR1 and FGFR2 directly correlated
to clinicopathological features. FGFR1 expression was associated with light
smoking (p = 0.02), FGFR2 correlated negatively with age for the whole
cohort (p = 0.27), and there was a negative correlation between tumour
stage and FGFR2 expression (p = 0.002). FGFR1 was expressed in 10.6%
and FGFR2 in 12.9% of all NSCLC tumor samples (Theelen et al., 2016).
Protein expression of FGFR is related to worse OS. Moreover, while
FGFR1 is associated with light smoking, FGFR2 is more common in
females and younger age patients (age 31 to 53 years). The study by Theelen
et al. demonstrated an abundance of FGFR mutations present in NSCLC,
suggesting that they play a role in growth and malignant progression of
NSCLC. A study by Santiago-Walker et al. demonstrated a 63%
concordance rate for detecting FGFR mutations in temporally unmatched
blood and tissue, which supports the potential for patient selection with
blood-based testing. Clinical di erences were observed between
ctDNA-FGFR positive and negative patients, but they were not statistically
signi cant (Santiago-Walker et al., 2019). Furthermore, FGFR1’s indication
of a worse OS suggests that it may serve as a prognostic biomarker (Theelen
et al., 2016).

EGFR
Epidermal growth factor receptor (EGFR) mutations are observed in
10-35% of all patients with adenocarcinomas. EGFR-targeting TKIs
(EGFR-TKIs) have shown higher objective response rates and longer PFS.
EGFR-TKIs are considered more successful than chemotherapy for EGFR
mutated patients. Unfortunately, about 30% of patients develop resistance
to these EGFR TKIs. A study done with 502 EGFR-mutated NSCLC
patients was conducted between 2003 and 2014 to examine the relationship
between EGFR mutations and TP53 mutations. TP53 is a tumor
suppressor that is commonly mutated in many cancers including NSCLC.
TP53 mutations are found in 35-55% of NSCLC patients. Of the 502, 43
had both TP53 and EGFR mutations. Those with mutant TP53 had
relapse-free survival (RFS) for a median of 42.2 months whereas those with
wildtype had RFS for a median of 37.7 months (P=0.59). 60 patients
received EGFR-TKIs as treatment. Of these 60 patients, 24 had mutant
TP53 and 36 had wildtype TP53. The results of the treatment were not

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statistically di erent (Labbe et al., 2017). EGFR can be used to direct the
course of treatment.

RRM1
Ribonucleotide reductase catalytic subunit M1 (RRM1) is a biomarker
involved in tumor proliferation, invasiveness, and metastasis. In a study
done by Mlak et al., 60 NSCLC patients’ RRM1 expression was measured
through use of liquid biopsy. All of the patients had not been treated for
NSCLC yet. High RRM1 expression was associated with higher risk of oral
mucositis, but it was not signi cantly associated with disease-free survival
(DFS) or OS shortening (Mlak et al., 2018). In another study done by
Zhang et al., RRM1 and ERCC1 levels were measured through liquid and
tissue biopsy in NSCLC patients using PCR. Patients with lower level
RRM1 expression, as seen in the liquid biopsy, had longer median OS.
Their OS was 18.5 months, as opposed to patients who had higher level
expression whose OS was 13.0 months (P = 0.043).
Higher level RRM1 was also associated with prolonged PFS. Patients with
low expression level had a median PFS of 6.0 months whereas patients who
had lower expression level had a median PFS of 4.0 months (P = 0.044)
(Zhang et al., 2012). RRM1 levels, measured through use of liquid and
tissue biopsy, can be used to predict outcomes such as OS and PFS.

Wip1
Overexpression of wildtype p53-induced phosphatase 1 (Wip1) is
commonly found in many types of tumors and is associated with poor
prognosis. In a study by Zhao et al., 117 NSCLC patients were examined
and Wip1 expression was determined through IHC. The patient cohort
had a mean age of 56.9 years, of which 87 were male and 30 were female. In
normal lungs, Wip1 is not expressed; the results of this study showed that
Wip1 was expressed in 69.3% of the NSCLC patients. Wip1 overexpression
was observed more in lung adenocarcinomas than other subtypes of
NSCLC. Wip1 negative patients survived for a longer time than Wip1
positive patients. After 80 months, the survival of Wip1 positive patients
was less than 20% but the survival of Wip1 negative patients was over 40%
(P=0.014) (Zhao et al., 2016). In another study, 84 patients, comprising 46
males and 38 females, were examined and treated. All of them had lung
adenocarcinomas. Wip1 levels were examined through IHC. Patients were
considered Wip1 positive if 10% or more cancer cells within the tumor were
strongly stained for Wip1 after IHC. Wip1 expression was positive in

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64.3% of the patients. The survival of Wip1 negative patients was
signi cantly greater than Wip1 positive patients. The OS rate was around
50% for Wip1 positive patients 100 months after surgery whereas the OS
rate was around 90% for Wip1 negative patients (P = 0.0099). These results
demonstrate that Wip1 expression correlates with negative prognosis
(Satoh et al., 2011). Wip1 can be used as a predictive outcome biomarker in
order to foresee the prognosis of the patients.

miRNA Based Biomarkers

Figure 3. This figure depicts the miRNA pathway. 3A: miRNA genes are transcribed to
primary miRNA (Pri-miRNA). 3B, 3C: Pri-miRNA is processed to precursor miRNA
(Pre-miRNA). 3D: Pre-miRNA is exported from the nucleus to the cytoplasm, maturing into
miRNA duplex. 3E: miRNA duplex is cleaved to produce mature miRNA. 3F: Mature
miRNA participates in the regulation of cellular pathways (Kwak et al., 2010).

Let-7
The miR let-7 family regulates components of cellular development and
di erentiation. Although typically a tumor suppressor, some cases have
been recorded in which let-7 acts as an oncogene (Chirshev et al., 2019).
Xie et al. examined the role of let-7 in detecting time of acceleration
re-proliferation, a key contributor to radiotherapy failure. The study
enrolled 19 eligible patients and via qRT-PCR and statistical analyses found
a high level of serum let-7 was associated with a better OS rate (P=0.024),
suggesting that serum let-7 could re ect the proliferation of tumor tissue
(Xie et al., 2016). Another study tested let-7 prognostic capability as part of
a miR panel consisting of let-7e, 125a-5p, miR-30a, miR-30e, and

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miR-30-3p. The study found NSCLC dedi erentiation was associated with
reduced expression of miR-125a-5p, let-7e and miR-30e (P=0.038); the loss
of expression of let-7e and miR-125a-5p was associated with shorter
postoperative survival in NSCLC patients (P=0.007) (Zhu et al., 2014).
Dedi erentiation is a process required for tumorigenesis, occurring when
specialized cells become less specialized and acquire tumor cell plasticity or
self-renewal ability (Friedmann-Morvinski & Verma, 2014, p. 245). A more
recent study performed qRT-PCR analysis on 120 NSCLC patients and
360 healthy controls and found plasma let-7c and miR-152 expressions
were lower in NSCLC patients. Furthermore, receiver operating
characteristic curves displayed an association between low let-7c level and
poor di erentiation status (P<0.001), cancer metastasis (P=0.021), and
advanced stage classi cation (P=0.013). Lastly, comparing postoperative
and preoperative plasma from 96 NSCLC cases showed an increase in let-7c
expression level post-operation: suggesting the potential of let-7c as a
prognosis biomarker for NSCLC (Dou et al., 2015).

MiR-21
miR-21 is an extensively studied oncogenic miRNA due to its association
with tumor suppressor genes that regulate proliferation, apoptosis, and
invasion (Feng et al., 2016) More recently, researchers have sought to utilize
the presence of miR-21 as a predictive marker of NSCLC. Wang et al.
utilized serum samples of 88 NSCLC patients and 17 healthy controls
obtained from Jiangsu Province People Hospital. The researchers found
that the 3-year OS in NSCLC patients with high serummiR-21 expression
was lower (39.8%) compared to 58.2% in patients with low serummiR-21
(P<.001) (Wang et al., 2011). Liu et al. reported consistent results with the
prior study regarding OS, but noted that the miR-21 levels in serum and
tumor miR-21 levels had no signi cant correlation (2012).

miR-21 levels have also been used to predict NSCLC tumor recurrence in
patients post surgical resection. Munagala et al. studied cancer recurrence
in mouse models and found that miR-21 was upregulated in recurrent
tumors compared to primary tumor tissue, and more notably, serum
miR-21 also mirrored the tumor pro le tumor miRNA results (Munagala
et al., 2011). Dejima et al. con rmed this observation by examining
miR-21 levels in 195 NSCLC patients and 30 healthy patients; nding that
exosomal miR-21 levels showed signi cant increase in NSCLC patients
with recurrent tumors (P<0.01) (Dejima et al., 2017). In the clinical

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setting, Han-Bo Le et al. found that miR-21 levels in post-operative
patients’ serum samples was lower than pre-operative levels, which further
suggests miR-21’s role as predictive biomarker for tumor recurrence.

The use of miR-21 to evaluate treatment options has also been investigated.
Wei J et al. followed up with 53 patients who received platinum-based
chemotherapy to treat NSCLC. They found that patients with partial
response to the therapy had miR-21 plasma levels several fold lower than
patients with stable orprogressive disease (Wei et al., 2011). Gao et al. noted
similar results via tumor miRNA analysis of 58 patients, with high levels of
miR-21 expression correlating with increased platinum resistance (Gao et
al., 2012).

MiR-30
The miR-30 family serves in a regulatory capacity during tissue and organ
development. The miR-30 family possesses tumor suppressor abilities,
which include its roles in the pathogenesis of cancers, including breast
cancer, thyroid cancer, colon cancer, and lung cancer (Mao et al., 2018).

A study investigated the prognostic value of plasma miR-30b and miR-30c
from EGFR-mutated lung cancer patients undergoing erlotinib treatment.
Blood qPCR analyses from 29 erlotinib-treated, EGFR-mutated lung
cancer patients demonstrated associations between low plasma levels of
miR-30b and miR-30c and increased e cacy of erlotinib in
EGFR-mutated NSCLC patients as indicated by PFS (P<0.05 for both
miRNAs). The study concluded that miR-30b and miR-30c may serve as
potential biomarkers to predict erlotinib e cacy in EGFR-mutated
NSCLC patients (Hojbjerg et al., 2019).

Another study on 104 lung cancer cases with benign lesions and 20 healthy
controls from West China Hospital investigated the prognosis value of
miR-30a-5p. Through qRT-PCR, high plasma miR-30a-5p levels
correlated with increased tumor size (P=0.02), advanced tumor
di erentiation (P=0.03), and advanced tumor node metastasis (TNM)
stage (P=0.0001). The plasma level of miR-30a-5p was also found to
signi cantly decrease post-surgery (P<0.0001) (Liang et al., 2019). Through
Kaplan–Meier survival analysis, low miR-30a-5p expression was associated
with longer survival compared to high miR-30a-5p expression (P=0.0001).
Kaplan– Meier survival analysis is an approach to measure the portion of

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samples surviving post-treatment after a certain time period (Kishore et al.,
2010). Finally, Cox multivariate regression analysis con rmed the
correlation between miR-30a-5p, advanced TNM stage, and OS, signifying
the prognostic value of miR-30a-5p in NSCLC (Liang et al., 2019).

MiR-125b
The dysregulation of miR-125b is a common feature across many cancers
that a ects tumor cell proliferation, di erentiation, invasion, migration,
drug resistance, and tumor immunity. Due to its tendency to act as both
tumor-suppressor and oncogene, depending on the cancer type and
molecular contexts, miR-125b has not been utilized for clinical purposes.
Recently, however, several studies have noted miR-125b’s potential as a
prognostic biomarker for advanced NSCLC patients. Cui et al. compared
serum miRNA-125b across 260 inoperable advanced NSCLC patients and
260 healthy patients. Utilizing qRT-PCR to measure circulating miR-125b,
and evaluating e cacy of chemotherapy in accordance with the Radiologic
RECIST, the study found that 99 patients (38%) responded to
chemotherapy with partial or complete response; 161 (62%) patients were
not responsive, but instead exhibited stabilization or disease progression.
miR-125b was signi cantly associated with chemotherapeutic response,
with nonresponsive patients exhibiting signi cantly higher expression levels
than responsive patients (P=0.003). The authors postulate that miR-125b
may act as a viable biomarker to predict chemotherapy resistance.
Con rming this observation, Shi et al. measured the relative expression of
miR-125b in 74 patients with advanced NSCLC pre- and post
chemotherapy using RT-qPCR and noted that sensitivity to chemotherapy
in NSCLC patients with high expression of miR-125b was lower than
those with low expression of miR-125b (p<.05) (2020).

The utility of miR-125 to predict survival outcomes has also been studied.
Yuxia et al. compared miR-125b serum levels in 193 patients with varying
stages of NSCLC, following surgery and therapy. Patients with miR-125
expression levels lower than 2.79 were put into the low-expression group,
and those above, in the high expression group. Kaplan-Meier survival
curves revealed that high expression signi cantly correlated with poor
survival (p<. 00001).

Multivariate Cox hazard analysis also showed miR-125b to be an
independent prognostic marker on NSCLC (Yuxia et al., 2012).

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MiR-145
miR-145 has been reported to have decreased levels in many di erent
cancers, such as pancreatic cancer, prostate cancer, breast cancer, and
colorectal cancer (Liu et al., 2018). miR-145’s tumor suppressive abilities
and its prevalence in many types of cancer makes it a potential biomarker
for prognosis. A study on immune responses and cancer progression
assessed 345 NSCLC patients and focused on a 5 miRNA panel, which
consisted of miR-191, miR-28-3p, miR-145, miR-328 and miR-18a.
Through the use of liquid biopsy and PCR, these miRNA levels were
measured; a high expression level was de ned as greater than 80%. The
median survival time for those with high expression of all 5 miRNAs was
3.6 to 5.7 months shorter than those with low expression. Out of the
miRNAs investigated in the study, miR-145 demonstrated higher
expression in normal patients than in NSCLC patients. Nevertheless,
higher levels of miR-145 in NSCLC patients is associated with poorer
survival. High miR-145 expression had an 88.4% 3 year death rate as
opposed to low miR-145 expression which had an 79.2% 3 year death rate
(P = 5.21E−03). miR-145 is associated with Ras, mitogen-activated protein
kinase, ATM and estrogen receptor signaling pathways, which are closely
associated with resistance to chemoradiotherapy and/or targeted therapy in
advanced NSCLC (Zhang et al., 2019).

MiR-155
MiR-155 is overexpressed in many diseases and is signi cant in
carcinogenesis. It is most commonly overexpressed in solid tumors and
hematopoietic malignancies. Its overexpression in NSCLC has
foreshadowed its potential as a biomarker for prognosis and predictive
purposes. MiR-155 has exhibited high expression in NSCLC patients and
has been seen to induce proliferation of NSCLC cancer cells. In a study
with 180 total NSCLC patients and 80 control patients, miR-155 and
miR-21 expression levels were compared between one group of 68 newly
diagnosed patients and 112 patients with recurrent or metastasized
NSCLC. The expression of miR-155 and miR-21 was higher in those with
NSCLC when compared to the control group (P < 0.01), while expression
of both miR-155 (P < 0.05) and miR-21 (P < 0.01) was higher in those in
the recurrence group than those in the newly diagnosed group. Higher
levels of these miRNAs were found to a ect the prognosis negatively, with
a mortality rate of 91.96% and a 19 month median survival time in the

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recurrence group compared to a 57.35% mortality rate and a 28 month
median survival time in the newly diagnosed group (P < 0.05) (Xu et al.,
2019). In combination of a three-miRNA signature panel of high level
miR-155-5p, high level miR-223-3p, and low level miR-199a-5p, there was
a mean DFS of 46 months in 52 resectable NSCLC patients (San orenzo et
al., 2013). In three independent cohorts of NSCLC patients in Maryland,
Norway, and Japan, miR-155 was concluded in addition with miR-17 and
miR-21 in association with mortality rates and DFS (Saito et al., 2011).

The prognosis impact of miR-155 expression is in uenced by other factors
as well. In another study, 335 NSCLC patients were examined, and it was
discovered that squamous cell carcinoma expressed higher levels of
miR-155 than adenocarcinomas. Higher miR-155 expression has a negative
role in disease speci c survival (DSS) prognosis in patients with
adenocarcinomas, but a positive role in DSS in patients with squamous cell
carcinomas. The median survival for those with high miR-155 expression
was 84 months whereas the median survival rate for those with low
miR-155 expression was 190 months (P=0.43). Thus the prognostic
impact of miR-155 di ers by histological subtype (Donnem et al., 2011).
Hana et al. also found that in 52 patient serum samples high miR-155
expression level correlated with poor prognosis, and is correlated with a
median survival (MS) of 69 days (P=0.034) in adenocarcinomas and a MS
of 58 days (P=0.023) in patients with positive EGFR gene mutations
(Hana  et al., 2020).

MiR-486
While miR-486 is widely controversial, with many studies reporting
con icting results in miR-486’s role as an oncogene or tumor suppressor, it
has been proven to be a signi cant, non-invasive biomarker for prognosis.
Sromek et al. measured miR-486 levels in patients that underwent tumor
resection and found increased miR-486 levels expressed in their plasma
samples (n=14) one year after surgery (Sromek et al., 2017).

A meta-analysis conducted by Jiang et al. among 7 studies demonstrated
that there was no increased risk of poor outcome with lower expression of
miR-486 (Jiang et al., 2018). Poor outcome was de ned using 3 factors:
OS, PFS, and RFS. Conversely, in a study by Gao et al. high levels of
miR-486 in 140 NSCLC patients was directly correlated with shorter OS,
speci cally in Stage I NSCLC cancer (Gao et al., 2020). When using

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miR-486 in combination with miR-30d, miR-1, and miR-499 on a 4
signature miRNA signature panel, high serum levels of miR-486 also
showed similar results of unfavorable survival. More speci cally, it was
shown that within the 60 patient cohort, those that carried two or more of
the high-risk miRNA would have signi cantly shorter survival (P all <
0.001) compared to those who had none or one high-risk miRNA (Hu et
al., 2010). In patients that showed down-regulated miR-486 levels in
plasma after surgical removal of tumors, the recurrence free survival (RFS)
was higher than in those that had stagnant levels of miR-486, the
un-reduced group, that did not have reduced miR-486 levels or miR-486
levels did not change. The group with down-regulated miR-486 had a
median unreached survival, while the un-reduced group had a median
survival of 19 months (P=0.056) (Li et al., 2015).

In studies that focused on clinical phenotype of metastasis, it was shown
that miR-486-5p suppressed migration and invasion abilities of NSCLC
cells, preventing cancer progression and metastasis. In one study,
miR-486-5p downregulated NSCLC through ARHGAP5, a
protumorigenic gene. miR-486-5p had a statistically signi cant inverse
relationship with ARHGAP5 (P=0.0156), which correlated with further
cancer progression (n=76, frozen NSCLC samples) (Wang et al., 2014).
Moreover, in correlation with the P13K-Akt signaling pathway,
miR-486-5p and the P13R1 gene had regulatory e ects. In A549 and
H1299 miR-486-5p transfected NSCLC cell lines, inhibited A549 and
H1299’s abilities of migration and invasion after 48 hours (p < 0.05),
showed higher apoptosis rates after 72 hours (p < 0.05), and inhibited cell
proliferation through miR-486-5p suppression of P13R1 (p < 0.001) (Tian
et al., 2019). Furthermore, it was found that miR-486-5p was an e ective
therapeutic agent for cisplatin resistant NSCLC treatment. miR-486-5p
improved the susceptibility to cisplatin through miR-486-5p’s suppression
of TWF1, twin lin actin binding protein 1, a gene that plays a role in
tumor invasion and chemotherapy resistance. An in vivo investigation of
cisplatin resistance showed that miR-486-5p expression correlated with
reduced tumor size in nude mice (P < 0.001) (Jin et al., 2019).

Conclusion
In the literature about miR, we found several articles that indicate
signi cant promise for their utility as a biomarker for prognosis, outcome
prediction, or treatment strategies. First, recent studies regarding miR

Berkeley Pharma Tech Journal of Medicine | 143



biomarkers have begun to diversify from general metrics such as OS. More
speci c metrics now include predicting tumor recurrence, resistance to
treatments, medication performance, and much more. Second, the articles
we reviewed have indicated miR testing to be feasible in most clinical
settings. We found the measurement of miR typically involves some
variation of PCR procedure performed on plasma, serum, and saliva
samples.
Lastly, while we failed to observe standardization in the studies we reviewed,
each independent study demonstrated signi cant promise for miR utility as
a biomarker for prognosis, outcome prediction, or treatment strategies.
These studies consisted of large sample sizes with little error indicated in the
observed results. If these studies are veri ed, they present great potential for
miR biomarkers that would assist clinicians in determining treatment
strategies.

Similar trends were found in the ctDNA literature. Our review paper noted
that many ctDNA biomarkers indicate predictive outcomes including PFS
and OS. Many ctDNAs are used for directing the course of treatment,
including MET, BRCA, PTEN, and EGFR. This is often because
abnormal expression of these biomarkers can lead to resistance, which
requires the use of alternate therapies. Furthermore, while comparing
ctDNA from liquid biopsies with tissue biopsies, we found high
concordance rates between liquid biopsies and tissue biopsies. There is a
96.2% concordance rate between liquid and tissue biopsies for RET and
ROS1 and an 81.6% concordance rate for CDK2NA. High concordance
rates between tissue and liquid biopsies furthers the case for clinical
implementation of liquid biopsy as a means to detect genetic alterations in
ctDNA. Unlike standard tissue biopsy options, ctDNA and miRNA are
obtained through less invasive methods (usually via a simple blood test).
However, the question remains whether ctDNA or miRNA will serve as
viable standalone methods in the detection of actionable or prognostic
biomarkers. Based on current literature, circulating nucleic acid biomarkers
appear to hold promise in indicating targeted treatment plans for NSCLC
patients presenting with speci c genomic pro les. With the increasing
number of emerging biomarkers, it is critical to analyze the clinical utility of
these biomarkers in light of their prognostic value and clinical indications.
Clinical actionability remains a signi cant factor in determining biomarker
value.

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Ease of implementation and invasiveness of testing procedures are
important considerations in developing assay methods that ease patient
discomfort without diminishing speci city and accuracy of detection. The
impetus to ensure reliable and streamlined testing measures is paramount, as
the current literature suggests that early detection of speci c biomarkers can
provide a remarkable clinical bene t. Moreover, in an e ort to ensure equal
access to advanced cancer care, the nancial burden of testing should be
assessed. Ultimately, the prognostic signi cance of ctDNA and miRNA
biomarkers in NSCLC care has demonstrated that liquid biopsy and
molecular diagnostic testing hold promise for the future of personalized
cancer care. Further testing must be conducted to analyze the signi cance of
ctDNA and miRNA biomarkers in larger cohorts and to determine the
precise clinical bene t of their standardization in clinical practice.

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