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

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


I d c i
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 0 of all lung cancers With only 1 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 201 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 There are three main classes of biomarkers Predictive biomarkers interact with speci c treatments to a ect the outcome prognostic biomarkers are 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

C mm Me h d ili ed f ci c la i g cleic acid a e me
Several primary technologies have been used to assess circulating nucleic acid biomarkers Fl e ce ce i i h b idi a i 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 Imm hi chemi 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 be used as a tool for measuring protein and nucleic acid expression Schildhaus et al 2020 P l me a e chai eac i 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 Ne ge e a i e e ci g NGS NGS refers to the parallel sequencing of genomic segments There are three main kinds of NGS a Ill mi a Se e ci g In illumina sequencing the DNA bases are identi ed by their unique uorescent signals b R che Roche 454 sequencing relies on the release of visible uorescent pyrophosphate that occurs upon nucleotide addition to a new strand of DNA c I T e Ion torrent sequencing measures the release of protons due to the incorporation of nucleotide bases in the DNA Delmonico

CDKN A
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 201 Delmonico et al 201 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 from NGS assessment of 1200 Chinese NSCLC patients and The Cancer Genome Atlas TCGA data for American and European cohorts Wen et al 201 Liu et al also assessed data from TCGA to pinpoint important genes implicated in lung cancer tumor biology related to CDKN2A A54 and H322 cell lines were 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 A54 and 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 1 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 Mao et al found that plasma samples of RET ctDNA from patients with histologically con rmed lung cancer demonstrated 6 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 1 0 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 201 Thompson et al 2016

ERBB HER
Erb b2 receptor tyrosine kinase 2 ERBB2 human epidermal growth factor receptor 2 HER2 has become widely acknowledged as a potential 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 NCT01 53 26 Chuang et al 2017 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 201 Another study in 2 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 6 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 201

BRCA
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 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 clinicopathological features of BRCA mutated patient subgroups Reported BRCA prevalence without respect to mutation type was 5 3 20 out of 37 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 BRCA may 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  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

RRM
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 201 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 1 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

Wi
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 years of which 7 were male and 30 were female In normal lungs Wip1 is not expressed the results of this study showed that Wip1 was expressed in 6 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 0 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 4 patients comprising 46 males and 3 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 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 0 for Wip1 negative patients P 0 00 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

MiR
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 201 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 2 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 201 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 201 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 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 201

MiR b
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 patients 3 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 1 3 patients with varying stages of NSCLC following surgery and therapy Patients with miR 125 expression levels lower than 2 7 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 MiR 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 201 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 1 1 miR 2 3p miR 145 miR 32 and miR 1 a Through the use of liquid biopsy and PCR these miRNA levels were measured a high expression level was de ned as greater than 0 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 4 3 year death rate as opposed to low miR 145 expression which had an 7 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 201

MiR
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 1 0 total NSCLC patients and 0 control patients miR 155 and miR 21 expression levels were compared between one group of 6 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 1 6 and a 1 month median survival time in the recurrence group compared to a 57 35 mortality rate and a 2 month median survival time in the newly diagnosed group P 0 05 Xu et al 201 In combination of a three miRNA signature panel of high level miR 155 5p high level miR 223 3p and low level miR 1 a 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 4 months whereas the median survival rate for those with low  4 6 in combination with miR 30d miR 1 and miR 4 on a 4 signature miRNA signature panel high serum levels of miR 4 6 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 4 6 levels in plasma after surgical removal of tumors the recurrence free survival RFS was higher than in those that had stagnant levels of miR 4 6 the un reduced group that did not have reduced miR 4 6 levels or miR 4 6 levels did not change The group with down regulated miR 4 6 had a median unreached survival while the un reduced group had a median survival of 1 months P 0 056 Li et al 2015 In studies that focused on clinical phenotype of metastasis it was shown that miR 4 6 5p suppressed migration and invasion abilities of NSCLC cells preventing cancer progression and metastasis In one study miR 4 6 5p downregulated NSCLC through ARHGAP a protumorigenic gene miR 4 6 5p had a statistically signi cant inverse relationship with ARHGAP 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 4 6 5p and the P13R1 gene had regulatory e ects In A54 and H12 miR 4 6 5p transfected NSCLC cell lines inhibited A54 and H12 s abilities of migration and invasion after 4 hours p 0 05 showed higher apoptosis rates after 72 hours p 0 05 and inhibited cell proliferation through miR 4 6 5p suppression of P13R1 p 0 001 Tian et al 201 Furthermore it was found that miR 4 6 5p was an e ective therapeutic agent for cisplatin resistant NSCLC treatment miR 4 6 5p improved the susceptibility to cisplatin through miR 4 6 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 i o investigation of cisplatin resistance showed that miR 4 6 5p expression correlated with reduced tumor size in nude mice P 0 001 Jin et al 201

C cl i
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 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 6 2 concordance rate between liquid and tissue biopsies for RET and ROS1 and an 1 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 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