Plasma-based next generation sequencing in advanced non-small cell lung cancer (NSCLC): significance in diagnosis and treatment in Asian patients

Abstract

NGS is recommended by NCCN and ESMO in evaluation of advanced NSCLC prior to treatment as being eligible for target therapy has important treatment implications. However, up to 20% of tissue biopsies are insufficient to run molecular testing for all nine FDA approved biomarkers. Currently, major guidelines recommend in the metastatic treatment naïve setting to pursue first line plasma-based NGS only if there is insufficient tissue. In our retrospective cohort, it was found that in 43 patients who underwent first line plasma-based and tissue NGS, 22 were Asians. Eleven of the twenty-two (50%) patients had an actionable mutation. The overwhelming majority of those mutations were EGFR actionable mutation (81.8%). All EGFR actionable mutations found on tissue was seen on plasma-based NGS, but 2 EGFR actionable mutation were seen on plasma-based NGS only. 22.2% of EGFR actionable mutations would have been missed if tissue-based NGS was pursued alone in the first line. In addition, in both plasma-based and tissue NGS, there is a statistically significant higher chance of detecting an EGFR actionable mutation if you were Asian compared to White (P = 0.004) or a nonsmoker compared to a smoker (P = 0.017). A small number of patients were found to have an EGFR actionable mutation on plasma-based NGS but not tissue NGS despite sufficient tissue to run NGS. They derived varying degrees of benefit from targeted therapy. Based on these findings patients who are Asian or nonsmoker plasma-based NGS should be considered in the diagnostic setting even if there is sufficient tissue for NGS.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-025-15295-2.

Introduction

The emergence of actionable biomarkers and targeted therapy changed the treatment paradigm in advanced non-small cell lung cancer (NSCLC) [1, 2]. Epidermal growth factor receptor (EGFR) is present in about a third of all patients with advanced non-squamous NSCLC, with higher prevalence in Asians, non-smokers, and females [3, 4]. Tyrosine kinase inhibitors targeting EGFR mutation showed improved progression free survival and overall survival compared to previous standard-of-care chemotherapy [5–7]. Similarly, drugs have been developed targeting ERBB2 mutation, ALK rearrangement, ROS1 rearrangement, BRAF V600E, MET Exon 14 skipping mutation, NTRK fusion, and KRAS G12C [8–15]. As many as 50% of patients with advanced NSCLC have an actionable oncogenic driver, with the majority being EGFR mutations [16–18].

Next generation sequencing (NGS) is recommended by National Comprehensive Cancer Network (NCCN) and European Society of Medical Oncology (ESMO) in evaluating advanced NSCLC prior to treatment [19, 20]. The gold standard for molecular analyses remain tissue molecular testing. However, this can be limited by insufficient tissue or technically difficult-to-access biopsy site. As high as 20% of all cases in clinical practice have insufficient tissue to run all guideline recommended biomarker testing [21–23]. It is in this setting that plasma NGS or liquid biopsy has gained interest as it is a minimally invasive procedure that can overcome these limitations.

Plasma-based NGS is a powerful ancillary tool. Tumors can shed cancer cells and nucleic acids into the peripheral blood [24, 25]. Through assessing various substances secreted by tumor such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), cell-free nucleic acid, exosomes, or proteins plasma-based NGS presents the potential to find actionable biomarkers [26]. Various plasma-based NGS such as Foundation one and Guardant 360 CDx have been approved by the FDA as a comprehensive genomic assay for assessment of mutations of potential clinical significance in NSCLC [27].

The optimal application of plasma-based NGS in the treatment of advanced NSCLC has not been clearly defined. Plasma-based NGS has high concordance with tissue biopsy (>98%), as well as the potential to detect actionable tumor mutations in patients who tested negative on tissue or had insufficient tissue sample. Plasma-based NGS can increase the detection of targetable mutations by up to 48% when used concurrently with tissue biopsy [28]. It’s been shown that patients with advanced NSCLC who had treatment determined based on plasma NGS alone derived response to targeted therapy [29]. However, to date, concurrent plasma NGS with tissue NGS has not shown improved progression free survival or overall survival [30]. At present in the metastatic treatment naïve setting, plasma-based NGS is recommended as an ancillary tool when there is insufficient tissue to run tissue based NGS. In the second line setting plasma NGS represent a non-invasive way of monitoring for persistence of an actionable biomarker, as well as potentially discovering acquired resistance mechanism. In patients with targetable EGFR mutation, plasma NGS monitoring can be helpful in progression as the most common mechanism by TKI resistance is through the development of EGFR T790M or C797S mutation [31]. Similarly, plasma NGS can detect resistance mutations in patients who progress on an ALK TKI [32].

To date, real-world studies describing how plasma-based NGS is used in clinical practice for advanced NSCLC are scarce. We conducted a retrospective study to see how plasma-based NGS was used at our academic institution in Massachusetts. Based on prior studies, EGFR actionable mutations are more likely to be detected on tissue molecular testing in Asians, females, and nonsmokers [4, 33–36]. Given high concordance of tissue and plasma-based NGS, we expect a higher frequency of EGFR actionable mutation to be detected on plasma-based NGS in Asians, females, and non-smokers. In addition, we hypothesized that tissue and plasma-based NGS would be highly concordant.

Methods

A retrospective study of all adult patients > 18 years old who received plasma-based NGS at Tufts Medical Center was conducted between January 1, 2015 and December 31 st, 2023. January 1 st 2015 was chosen as our starting date as that was when our institution began utilizing Guardant 360 assay for all patients with NSCLC. At our institution, Guardant360 CDx was our preferred plasma-based NGS using ctDNA to assess for actionable mutations. Through the Guardant portal, we identified all patients who had plasma-based NGS done at our institution. Patients were included if they had lung cancer listed as the diagnostic reason for ordering plasma-based NGS. Chart review was continued until 01/30/2024 to give at least one month for follow-up. Patients were eligible if they had a diagnosis of metastatic lung adenocarcinoma, and were not on active treatment at the time of diagnosis of metastatic disease. Patients were excluded if plasma-based NGS was done in the setting of localized or regional disease, or they declined diagnostic work-up for complete staging.

Patient characteristics that were recorded included age, gender, race, smoking status, Eastern Cooperative Oncology Group (ECOG) performing status, tissue histology, staging, and status of central nervous system (CNS) metastasis at the time of diagnosis. If a patient received a tissue biopsy, molecular testing was done according to physician’s preference which consisted primarily of Esoterix EGFR mutation test or Onkosight NGS. Both tissue and plasma-based NGS used at our insitution covered clinically important EGFR mutations including atypical mutations such as S768I, L861Q, and G719X. It is thought that atypical EGFR mutations such as G719X or Exon 20 typically have a less durable response to targeted therapy [37, 38]. Results of tissue biopsy were abstracted from our electronic medical record. Information collected includes the adequacy of tissue for molecular testing, nine guideline driven biomarkers, non-FDA approved molecular mutations, PD-L1 status, and circumstance of testing whether at diagnosis or progression. Turnaround time was calculated from the time the test was ordered and when it resulted.

If a patient received plasma-based NGS, it was recorded whether this was done in the setting of diagnosis of metastatic disease or on progression. We recorded if any of nine FDA-approved molecular mutation was detected, as well as non-FDA approved molecular mutation and variant allele frequency. Turnaround time was calculated based on when physician ordered the test and when test resulted.

For patients that received both tissue and plasma-based NGS in the first line, sensitivity, specificity, positive predictive value, negative predictive value, and concordance were calculated. Chi-square/Fisher’s exact tests were used to compare the difference in actionable mutation seen on plasma-based NGS by race, smoking status, and gender. P values were adjusted for multiple comparisons using the Bonferroni method, as needed. P values < 0.05 was considered as statistically significant. All the analyses were conducted via SPSS (version 25.0).

Results

One hundred and fifty-four patients met the inclusion criteria of NSCLC and received plasma-based NGS. Twenty-two patients were excluded as the test was done in an earlier stage of the disease, and 41 patients were excluded as they were non-adenocarcinoma. Of the 91 patients, 18 had more than one plasma-based NGS. Sixteen patients had plasma-based NGS alone in the treatment-naive diagnostic setting. Seventy-five patients had tissue biopsy in the treatment-naïve diagnostic setting, of which 55 patients had concurrent tissue and plasma-based NGS. Twelve patients did not have sufficient tissue for testing, leaving 43 patients with concurrent plasma-based NGS and tissue molecular testing in the diagnostic setting. Twenty of the forty-three (46.5%) patients were found to have an actionable mutation, of which 11 (55.0%) were Asians (Fig. 1).

Fig. 1.

Consort diagram

The median age of our patient population was 68.5 years old (range 29.0–98.0). Of the 91 patients, 45 were male (49%). By race, 34 patients were White (37%), 49 Asian (54%), 4 African American (4.5%), and 4 with other/unknown races (4.5%). Seventeen (19%) were current smokers, 38 were former smokers (42%), 3 patients had exposure to secondhand smoke (3%), and 33 patients were never smokers (36%). In terms of ECOG status, 54% of our patients were ECOG 0 or 1. Thirty four of our ninety-one (37%) patients had CNS metastasis at the time of diagnosis (Table 1).

Table 1.

Baseline patient characteristics of patients who received plasma-based NGS in the first- or second-line setting

Patient characteristics (N = 91)
Age (years, mean (range))	68.5 (29.0–98.0)
Gender
Male	45 (49%)
Female	46 (51%)
Race
White	34 (37%)
Asian	49 (54%)
African American	4 (4.5%)
Other/Unknown	4 (4.5%)
Smoking status
Current smoker	17 (19%)
Former smoker	38 (42%)
Exposed to secondhand smoke	3 (3%)
Never smoker	33 (36%)
ECOG status
0	9 (10%)
1	40 (44%)
2	17 (19%)
3	5 (5%)
4	2 (2%)
Unknown	18 (20%)
CNS metastasis at time of diagnosis	34/91 (37%)

Of the 43 patients who had sufficient tissue to undergo both tissue and plasma-based NGS in the treatment naïve diagnostic setting, results of NGS for the nine FDA approved actionable mutations were evaluated. There was high concordance in all 9 mutations with > 90% between tissue and plasma-based NGS. Calculation was made assuming tissue-based NGS as the gold standard and reference value. Specifically, for EGFR actionable mutation, sensitivity was 85.7% [6/(6 + 1)], with specificity of 92.9% [33/(3 + 33)]. Positive predictive value for EGFR actionable mutation was 66.6% [6/(6 + 3)] and negative predictive value was 97.1% [33/(33 + 1)]. This translates to a concordance of 90.7% [(33 + 6)/(33 + 6 + 1 + 3)] for EGFR actionable mutation (Table 2). There were several mutations for which no mutation was detected on tissue or plasma-based NGS (NTRK fusion, BRAF V600, ROS1 rearrangement, or RET rearrangement (Supplementary Table 1).

Table 2.

Concordance rates of positive findings from 9 FDA approved actionable mutations in lung cancer amongst the 43 patients who underwent concurrent tissue and plasma-based NGS in the treatment-naïve metastatic setting

	EGFR mutation	ALK rearrangement	KRAS G12C	HER2 mutation
(+)on both tissue and plasma	6	0	1	1
(+) on tissue alone	1	1	1	0
(+) on plasma alone	3	1	2	1
(-) on both tissue and plasma	33	41	39	39
Sensitivity	85.7%	0%	50%	100%
Specificity	91.6%	97.6%	95.1%	92.9%
Positive predictive value	66.6%	N/A	33.3%	25.0%
Negative predictive value	97.1%	97.6%	97.5%	100%
Concordance	92.9%	95.3%	93.0%	93.0%

Of the 43 patients who underwent concurrent plasma and tissue based NGS 22 were Asians (51.3%), whereas 17 (39.5%) were white. The remaining 4 were unidentifiable race or Black. Full breakdown of the results of tissue and concurrent plasma-based NGS in the 43 patients stratified by race can be found in supplementary Table 8. Twenty of the forty-three (46.5%) patients were found to have an actionable mutation on either plasma-based NGS or tissue biopsy, of which 11 were Asian (55.0%), and 6 were White (30.0%).

It was noted that 9 of the 11 (81.8%) actionable mutations found in Asians were EGFR actionable mutations. In Table 3, a breakdown was made of specific EGFR actionable mutations in Asians found in the treatment naïve metastatic setting on either tissue NGS, plasma-based NGS, or both. All mutations seen on tissue NGS was found on plasma-based NGS. The majority of the mutations detected were EGFR Exon 19 deletion of EGFR Exon 21 mutation (77.8%). In addition, plasma-based NGS picked up two additional actionable EGFR mutations that was not seen on tissue NGS. This translates to a concordance of 77.8%. The other two actionable mutations seen in Asian on concurrent tissue and plasma-based NGS in the treatment naïve metastatic setting can be found in supplementary Table 9.

Table 3.

EGFR actionable mutations in Asians who underwent concurrent tissue and plasma-based NGS in the treatment naïve metastatic setting

EGFR actionable mutation detected in Asians (N = 9)	Tissue	Plasma
EGFR Exon 19 deletion	4	5
EGFR Exon 21 mutation	2	2
EGFR Exon G719A mutation	0	1
EGFR Exon 20 mutation	1	1
Concordance	7/9 (77.8%)

As the majority of our patients were White or Asian, we stratified the FDA actionable mutation profile by the two predominant races. In addition, we stratified by gender and smoking status. In Table 4, we compared all patients who received tissue molecular testing on diagnosis of metastatic disease. As our eligibility criteria required plasma-based NGS, this included patients who had concurrent plasma-based and tissue molecular testing, and also patients who received plasma-based NGS on progression. The only clinically significant difference found of the 9 FDA marker was in EGFR and is presented in Table 4. Asians had significantly higher percentage of actionable EGFR mutations compared to Whites (53.8% vs. 12.5%, P = 0.001). Similarly, a higher percentage of nonsmokers had an actionable EGFR mutation compared to those who had a history of smoking exposure (53.6% vs. 25.6%, P = 0.017). This however did not reach statistical significance after Bonferroni correction. There was no significant difference in EGFR actionable mutation when stratified by gender. All other actionable mutations were not statistically significant when stratified by race, smoking status, or gender.

Table 4.

Tissue molecular testing: EGFR actionable mutations stratified by race, smoking status, and gender in patients who underwent tissue-based NGS in the metastatic setting

EGFR actionable mutation stratified by:	Number of patients	P-Value
Race
Asian	21/39 (53.8%)
White	3/24 (12.5%)	P = 0.001
Smoking status
Nonsmoker	15/28 (53.6%)
Smoker	11/43 (25.6%)	P = 0.017*
Gender
Female	16/37 (43.2%)
Male	10/35 (28.6%)	P = 0.195

*Not significant after correction by Bonferroni correction

Similar analysis was done for patients who received plasma-based NGS on the diagnosis of metastatic disease (Table 5). This included patients who had concurrent plasma-based and tissue molecular testing, and those who were too frail to receive a tissue biopsy. Asians had a significantly higher percentage of detected actionable EGFR mutations detected compared to Whites (35.7% vs. 3.8%, P = 0.004). Nonsmokers were also found to have a significant higher proportion of detected actionable EGFR mutation compared to those who had a history of smoking exposure (38.9% vs. 7.5%, P = 0.009). Furthermore, patients who were smokers had a significant higher proportion of TP53 mutation vs. non-smokers (57.5% vs. 16.7%). This was statistically significant at 0.004 (Supplementary Table 6). We found no difference in actionable mutations on plasma-based NGS when stratified by gender.

Table 5.

Plasma-based NGS: EGFR actionable mutations stratified by race, smoking status, and gender in patients who received plasma-based NGS in the treatment- naïve metastatic setting

EGFR actionable mutation stratified by:	Number of patients	P-Value
Race
Asian	10/28 (35.7%)
White	1/26 (3.8%)	P = 0.004
Smoking status
Nonsmoker	7/18 (38.9%)
Smoker	3/40 (7.5%)	P = 0.009
Gender
Female	7/29 (24.1%)
Male	4/30 (13.3%)	P = 0.287

Looking at turnaround time, on average, it took 9.7 days for plasma-based NGS to return, whereas on average it took 12.2 days for tissue molecular testing results to return.

Finally, we discovered that in a subset of Asian patients, an actionable mutation was seen on plasma-based NGS alone. This occurred in a few scenarios. One was when the patient was too frail to undergo a tissue-based NGS. An alternative reason was there was insufficient tissue to run molecular NGS. In 4 instances, there was sufficient tissue to run molecular testing on tissue pathology but only a mutation was detected on plasma-based NGS. Here, we show that in some of these patients, they still derived a benefit from targeted therapy (Table 6). The first patient was a 98-year-old Asian female with an ECOG of 3. She was too frail to undergo tissue molecular testing, but an EGFR exon 19 mutation was detected on plasma-based NGS. She ultimately was started on Erlotinib and had an OS of 1295 days. Patient 2 was a 69-year-old Asian male with an ECOG of 1. He had an actionable EGFR G719A mutation detected on plasma-based NGS, and no mutation detected on tissue biopsy despite sufficient tissue to run testing. He was started on Osimertinib, and had a PFS of 175 days, but an overall survival of 1046 days.

Table 6.

Outcomes in Asian patients who were treated based on results of plasma-based NGS alone

Age	Race	Sex	ECOG	Actionable mutation detected on plasma-based NGS alone	Was tissue biopsy done	Did tissue biopsy detect an actionable mutation	1 st line treatment	PFS	OS
98	Asian	F	3	EGFR (Exon 19 deletion)	No	N/A	Erltoinib	N/A	1295
69	Asian	M	1	EGFR (G719A)	Yes	No	Osimertinib	175	1046
87	Asian	F	1	(EGFR E746_A750del)	Yes	No	Osimertinib	195	378
76	Asian	F	1	EGFR E746_T751delinsV	Yes	QNS	Osimertinib	50	NR

NR Not reached

N/A not applicable

QNS quantity not sufficient

Discussion

Our retrospective study is unique from other real-world studies given our high proportion of Asian patients, reflecting the patient population that we serve. In addition, as it was a single institution study, we had insight into each patient’s detailed treatment plan. In terms of age and gender, it was similar to prior studies of advanced NSCLC^5,36,37. At our institution, the majority of plasma-based NGS was used in the metastatic setting (81%), although a fair number were used in the localized setting. We were surprised at the number of patients who received concurrent tissue and plasma-based NGS in the metastatic treatment naïve setting. The high representation of Asians in our study likely reflects our institution’s practice of ordering concurrent plasma-based and tissue molecular testing if clinicians had a high suspicion of the patient having an underlying actionable mutation.

To the best of the authors’ knowledge, this is the first study looking at the utility of plasma-based NGS and actionable mutations stratified by race. As prior studies on molecular testing in tissue biopsy have shown, patients who are Asian or nonsmokers have a higher probability of having a detectable EGFR mutation on tissue NGS [4, 33–36]. Our study showed similar findings in Asians compared to Whites on tissue molecular testing although surprisingly this was not seen in nonsmokers vs. smokers. However, it is worth noting the P-value to reach significance was 0.0167 after Bonferroni’s correction and the P-value for likelihood of detecting EGFR mutation in nonsmokers vs. smokers on tissue-based NGS was 0.017. This suggest the result may be in part due to our modest sample size as the P value was close to statistical significance, and that race may be a more important factor than smoking status in having an EGFR mutated metastatic NSCLC.

What is novel to our study is that we verified that there was a higher rate of EGFR actionable mutation being detected on plasma-based NGS with Asians compared to Whites. Similarly, we found nonsmokers had a higher rate of EGFR actionable mutation on plasma-based NGS compared to smokers. Taken together, this suggests that patients who are nonsmokers or Asian are more likely to have a detectable EGFR mutation on plasma-based NGS. This also suggests plasma-based NGS has similar sensitivity and specificity to tissue NGS.

Given our small sample size, we were able to collect detailed information about a patient’s exact treatment course. This led to some key and important findings. At our institution 11 of 22 (50%) of Asian patients in the first line underwent concurrent tissue and plasma-based NGS were discovered to have an actionable mutation, and the overwhelming majority of those mutations were EGFR actionable mutation (81.8%). All EGFR actionable mutations found on tissue-based NGS was seen on plasma-based NGS, but 2 EGFR actionable mutation were seen on plasma-based NGS only. This meant 22.2% of EGFR actionable mutations would have been missed if tissue-based NGS was pursued alone in the first line. The clinical significance of this cannot be understated as it would change first line management for these patients.

The natural question that follows is do patients in the first line derive benefit from actionable mutations seen on plasma-based NGS alone. We focused on the Asian population in our study given the high proportion of actionable mutation detected relative to other races, a small cohort of Asians patients went onto treatment based on actionable mutation detected only on plasma-based NGS in the treatment naïve metastatic setting. This occurred in three scenarios; patients who were too frail to undergo tissue biopsy in the diagnostic setting, insufficient tissue to run molecular NGS, or there was sufficient tissue for molecular NGS but the mutation was detected only on plasma-based NGS. In Table 6, we showed the clinical outcomes of those patients with some who derived meaningful clinical benefit from targeted therapy.

How do we reconcile these actionable mutations detected on plasma-based NGS but not tissue-based NGS even with sufficient tissue to run molecular testing? One explanation is that there’s marked intra and inter-tumor heterogeneity even within the tissue itself, and the area biopsied may not contain the driver mutation. Another explanation is that tumors evolve over time, and in certain patients the original pathology specimen may have been done in the setting of localized disease prior to the development of relapse. Lastly, an interesting area worth exploring is the significance of lower variant allele fraction (VAF) and lower limit of detection plasma-based NGS has compared to tissue NGS. Guardant 360 can detect variant allele fraction as low as 0.1%, with data showing even at this low VAF therapy continues to retain efficacy [39, 40]. Currently, it is unknown if higher VAF conveys better response to therapy and OS [41, 42]. There is no guideline from the FDA or prospective studies about the impact of VAF on treatment outcomes for patients who have an actionable mutation detected on tissue or plasma-based NGS. We postulate some of these mutations being detected on plasma-based NGS could be due to the low percentage of tumor harboring this mutation and could explain variability in treatment response.

In prior studies, sensitivity, specificity, positive and negative predictive value were calculated under the assumption tissue biopsy is the gold standard [30]. In a setting where a plasma-based NGS detected an actionable mutation but a tissue biopsy did not, the finding of a plasma-based NGS was felt to be a false positive. Under this assumption, we can see that similar to other studies, although tissue and plasma-based molecular testing is highly concordant, the concordance is driven largely by the high specificity of the study. However as described above patients who received first-line treatment based on actionable mutations detected only on plasma-based NGS can derive clinically meaningful benefit. In one patient, overall survival (OS) approached that seen in the FLAURA study [43]. Mutations identified exclusively through plasma-based NGS may not be false positives, highlighting an area that warrants further exploration.

To date concurrent tissue and plasma-based NGS has not been shown to improve progression free survival (PFS) or OS [30]. However, we believe what that data reflects is not all treatment-naive metastatic NSCLC patients should receive concurrent plasma-based and tissue NGS given the substantial financial toxicity to the patient and our healthcare system. Based on the practice of our institution and the data we have shown, we believe if plasma-based NGS is used judiciously in the treatment-naïve metastatic setting, it can translate to clinically meaningful and significant benefit.

Our study should prompt reconsideration of how we approach the diagnostic evaluation of newly diagnosed metastatic lung adenocarcinoma. Based on the current guideline, if there is sufficient tissue to run molecular genotyping and no actionable mutation is found in the first line, patients would be treated with chemotherapy, immunotherapy, or chemoimmunotherapy. However, our study shows that if a patient was Asian, or a nonsmoker, they have a high likelihood of having an actionable EGFR mutation detected on plasma-based NGS. Furthermore, a small subset of Asian patients derived clinically meaningful benefit from actionable mutation found on plasma-based NGS only even with sufficient tissue for NGS. This suggests that these actionable mutations seen on plasma-based NGS only may not be false positives, and should warrant consideration of targeted therapy or re-biopsy. This important finding has the potential to change how we approach the evaluation of newly diagnosed metastatic NSCLC. Therefore, we suggest if a patient is Asian, or a nonsmoker, upfront plasma-based NGS in the treatment naïve metastatic setting should be considered even if tissue molecular testing returns negative. Below we propose a modified diagnostic algorithm based on the International Association for the Study of Lung Cancer (IASLC) guidelines (Fig. 2) [44].

Fig. 2.

Proposed algorithm for evaluation of new metastatic NSCLC

Limitations of our study include being limited to a single institution, retrospective nature, and a modest sample size. Our study only included patients who received plasma-based NGS for advanced NSCLC. There was a lack of a control arm. In this study, we also did not have sufficient representations of other races such as blacks.

Notably our study did not find any RET rearrangements or NTRK fusions. Despite NGS being recommended by ESMO for evaluation of fusions, ctDNA-based NGS may not be the optimal platform. ctDNA based NGS requires optimization of the panel for evaluation of the intronic regions to detect the fusion mutation given their lengths. As RET rearrangements can have unusual breakpoints or an unidentified fusion partner, FISH may have higher sensitivity. There can be substantial discordance in results between fluorescent in situ hybridization (FISH) and ctDNA based NGS [45]. However in clinical practice availability of tissue to do molecular testing must also be considered and the advantage of NGS lies in its ability to assess for multiple mutations at once [46].

Similarly, due to the length of the intronic regions of NTRK fusions the majority of fusions detected through ctDNA based NGS is via the fusion partner ETV6. FISH has lower sensitivities for evaluation of NTRK mutations due to the nature of fusions being formed in a intrachromosomal manner [47]. One way to bypass these limitations is through RNA-based NGS where introns are already spliced. However, RNA tends to fragment and degrade rapidly which can lead to failure of sequencing [48]. Recently, it was shown the addition of circulating tumor RNA (ctRNA) to ctDNA can increase diagnostic yield of tissue-NGS confirmed gene rearrangement by 28.6%. The combined modality was non-inferior to Guardant 360 ctDNA platform, detected 8.8% more actionable mutation than Guardant 360 ctDNA, although did not meet its endpoint of non-inferiority relative to tissue-based NGS [49]. This suggests using ctDNA and ctRNA may increase sensitivity and specificity of plasma-based NGS assays.

Future directions include having a prospective study with a larger sample size, as it would be important to accrue a sufficient sample size to have adequate power to answer if patients do derive clinical benefits based on the actionable mutations seen on plasma-based NGS alone in the treatment-naïve metastatic setting. As shown in our case series, clinical responses were variable if treated based on results of plasma-based NGS alone.

It also remains to be determined if patients with actionable mutations detected on plasma-based NGS only derive the same benefit from targeted therapy as tissue detected actionable mutation. If so, this carries important implications about the use of plasma-based NGS in the adjuvant setting given approval of targeted therapy based on ADAURA and more recent LAURA trial [50, 51]. An area that needs further exploration related to this would be the significance of VAF and whether it patients with higher VAF derive a more robust response to therapy.

Clinical Practice Points:

• Asians, females, and nonsmokers are more likely to have an EGFR mutation on NGS.

• To date, the addition of plasma based NGS in the first line with tissue NGS has not shown to improve PFS or OS even though more actionable mutations are detected when used together.

• Plasma based NGS does also have a higher rate of detecting actionable mutation if a patient is Asian or nonsmoker. In addition, patients who went onto treatment based on mutations detected only on plasma-based NGS but not tissue-based NGS can derive meaningful benefit.

• Based on these findings, we suggest there may be a role for plasma-based NGS in the first line in patients with metastatic lung cancer who is Asian or a nonsmoker.

Authors’ contributions

Dr. Cherng-Horng Wu was the primary author and responsible for the conceptualization, design, definition of methodology, collection, analysis of data, and writing of the manuscript. Dr. Lori Pai was the senior investigator and mentor for Dr. Cherng-Horng Wu. She provided guidance and supervised the project throughout the duration of the study. She also assisted in defining the hypothesis, methodology, and was instrumental in revision of the manuscript. Dr. Xia Wu helped with design, collection, and analysis of data, as well as editing of the manuscript. Dr. Xiao Hu helped with design, collection of data, and editing of manuscript. Alice Kennedy helped with design and collection of data.

Funding

There was no funding received in this research project.

Data availability

The data that support the findings of this study are available from the authors but restrictions apply to the availability of these data which were obtained with the permission of our IRB at our institution and so are not publicly available. Data are, however, available from the authors upon reasonable request and with permission from our institution’s IRB. Please reach out to Dr. Cherng-Horng Wu at cwu1@uw.edu to obtain this dataset.

Declarations

Ethics approval and consent to participate

The authors have complied with the applicable privacy laws and obtained approval from the Tufts Social, Behavioral, and Educational Research IRB to conduct this original research. An exemption 7 was obtained from the IRB for the purposes of this research. The Tufts Social, Behavioral, and Educational Research IRB waived requirement for informed consent from patients as many of them have deceased prior to the start of this study. This study adhered to the Declaration of Helsinki.

Consent for publication

An exemption 7 was obtained from the IRB for the purposes of this research.

Competing interests

The authors declare no competing interests.