<figure><img src="image_1.png" /></figure>

# Genomic testing among patients with newly diagnosed advanced non-small cell lung cancer in the United States: A contemporary clinical practice patterns study

Luis Paz-Ares<sup>a, *</sup>, Adam Gondos<sup>b</sup>, Diego Saldana<sup>b, 1</sup>, Marlene Thomas<sup>b</sup>, Celine Mascaux<sup>c, d, e, f</sup>, Lukas Bubendorf<sup>g</sup>, Fabrice Barlesi<sup>c, d, h</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

**Keywords:**

- Non-small cell lung cancer
- Genetic information databases
- Gene expression profiling
- Next-generation sequencing
- Targeted molecular therapy
- Tumor biomarker

## ABSTRACT

**Objectives:** According to 2018 United States and international lung cancer and pathology guidelines, testing of EGFR, ALK, ROS1 and BRAF genes is a minimum requirement to identify targeted therapy options in patients with advanced non-small cell lung cancer (aNSCLC). We describe real-world use and clinical features of next-generation sequencing (NGS) and other non-NGS testing technologies in these patients.

**Materials and methods:** Patients were from the Flatiron Health electronic health record-derived de-identified database and were newly diagnosed with non-squamous aNSCLC between 1 January 2018 and 30 June 2019. We describe occurrence and patterns of NGS- (including comprehensive genomic profiling [CGP]) and non-NGS-based genomic testing before the start of first-line therapy, unsuccessful genotyping (<4 genes tested) and incidence of potentially missed targeted therapy options (<4 genes tested with no positive results).

**Results:** Among 3050 patients, 2356 received any type of genomic testing (NGS: 1406 [59.7%]). Unsuccessful genotyping occurred in 13.2% and 52.2% of NGS- and non-NGS-tested patients, respectively. Among NGS-tested patients, 10.0% had a potentially missed targeted therapy option (CGP: 2.9%), compared with 40.2% in the non-NGS tested group. While all four guideline-recommended genes were tested in ≥ 92% of patients who had NGS testing, when only non-NGS testing was used, although EGFR and ALK had similarly high testing proportions, BRAF and ROS1 (56.1% and 83.7%, respectively) were examined less often.

**Conclusions:** Our findings suggest that in aNSCLC clinical practice, NGS testing may help to avoid potentially missed targeted therapy options and improve testing uptake for recently approved biomarkers. Results therefore support the use of guideline-recommended broad-panel NGS testing in clinical practice.

## 1. Introduction

Recent years have witnessed an unprecedented expansion in the

availability of molecularly guided therapies for patients with advanced non-small cell lung cancer (aNSCLC) [1–9]. For many of these therapies, treatment decisions rely on upfront verification of the presence of

**Abbreviations:** aNSCLC, advanced non-small cell lung cancer; CGP, comprehensive genomic profiling; FDA, Food and Drug Administration; FMI, Foundation Medicine, Inc.; NGS, next-generation sequencing; PD-L1, programmed death-ligand 1; US, United States.

* Corresponding author at: Hospital Universitario, 12 de Octubre, Av. De Córdoba, s/n, 28041 Madrid, Spain.

E-mail address: lpazaresr@seom.org (L. Paz-Ares).

¹ Present address: Novartis Institutes for BioMedical Research (NIBR), Quantitative Sciences & Innovation, Translational Data Science, Wallstrasse 25, 4051 Basel, Switzerland.

<footnote><sup>a</sup> Hospital Universitario 12 de Octubre, CNIO-H120 Lung Cancer Unit, Complutense University and Ciberonc, Madrid, Spain</footnote>

<footnote><sup>b</sup> F. Hoffmann-La Roche Ltd, Pharmaceutical Division, Personalised Healthcare Centre of Excellence, Basel, Switzerland</footnote>

<footnote><sup>c</sup> INSERM UMR 1068, CNRS UMR 725, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix-Marseille Université, Marseille, France</footnote>

<footnote><sup>d</sup> Department of Multidisciplinary Oncology and Innovative Therapeutics, Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France</footnote>

<footnote><sup>e</sup> Department of Pulmonology, Strasbourg University Hospital, Strasbourg, France</footnote>

<footnote><sup>f</sup> INSERM UMR S1113 Inserm, IRFAC, Laboratory Streinth (STress Response and INnovative Therapy Against Cancer), ITI InnoVec, Université de Strasbourg, Strasbourg, France</footnote>

<footnote><sup>g</sup> Pathology, Institute of Medical Genetics and Pathology, University Hospital Basel, University of Basel, Switzerland</footnote>

<footnote><sup>h</sup> Gustave Roussy Cancer Campus, Villejuif, France</footnote>
genomic alterations in the tumour by molecular testing. From 2018, according to guidelines from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology [10], the testing of EGFR, ALK, ROS1 and BRAF genes have been considered to be a minimum requirement [11], given that these genes have all had targeted therapy options in newly diagnosed patients with non-squamous aNSCLC approved by the United States (US) Food and Drug Administration (FDA) [9,10]. Along with NTRK and programmed death-ligand 1 (PD-L1), such biomarker testing recommendations have also been seen in the latest European Society for Medical Oncology Clinical Practice Guidelines for metastatic NSCLC [12].

NSCLC guidelines discuss the option of broad next-generation sequencing (NGS) [10], but little is known about the contemporary patterns of use and clinical features of NGS and other non-NGS testing technologies in the real world [13]. In this study, we examine contemporary use of genomic testing among patients with newly diagnosed aNSCLC who were overwhelmingly cared for in the US community oncology setting.

Both clinically, and from the point of view of the patient, the desired outcome of genomic testing for targetable oncogenic drivers is the identification of a targeted therapy option or the clarification that no option for targeted therapy exists, within either standard of care or clinical development programmes. Accordingly, biomarker testing may be considered suboptimal if a patient does not have a positive test result that opens the way for targeted treatment options and targetable genes remain untested.

## 2. Materials and methods

### 2.1. Data and patients

The Flatiron Health electronic health record-derived database is a longitudinal, demographically and geographically diverse de-identified database. It is sourced from over 280 cancer clinics (~800 sites of care) representing more than 2.4 million US patients with cancer and available data for analysis. Disease-specific data are curated from patient-

level structured and unstructured electronic health records via technology-enabled abstraction.

Patients included in this study were newly diagnosed with non-squamous aNSCLC between 1 January 2018 and 30 June 2019 and had commenced first-line therapy ≤ 90 days after diagnosis. Exclusion criteria included missing biomarker testing date information (Supplementary Fig. 1 and Supplementary Table S1), a biomarker test date > 30 days before the date of advanced disease diagnosis, an unknown type of biomarker test or an unknown test result and histology other than non-squamous cell carcinoma (Fig. 1).

Patients were grouped according to the type of biomarker test(s) received between 30 days before the date of advanced diagnosis and the start of systemic first-line therapy. Testing groups were NGS, including a subgroup of patients who received comprehensive genomic profiling from Foundation Medicine Inc. (FMI CGP; Cambridge, MA, US), non-NGS (all methods that are used in practice and are not NGS-based) and no genomic testing. FMI CGP is based on massively parallel DNA sequencing of routine formalin-fixed and paraffin-embedded clinical specimens, to characterise mutations and other types of genomic changes across > 300 cancer-related genes [14,15]. A blood-based test that used a smaller number of genes was also available during the study [16]. Both types of tests covered all four genes specifically examined in this study (ALK and ROS1 rearrangements, and EGFR and BRAF mutations [any and BRAF<sup>V600E</sup>, respectively]). Grouping of tests was performed hierarchically, whereby patients who received any NGS test were assigned to the NGS group regardless of whether they received additional non-NGS testing (Supplementary Fig. 2). Similarly, patients with an FMI CGP test were assigned to that group regardless of whether they received other NGS or non-NGS tests (Supplementary Fig. 2).

### 2.2. Analysis

We defined successful biomarker testing as at least one successful test for each of the four examined genes, ALK, BRAF, EGFR and ROS1 (Supplementary Fig. 3). We describe the following aspects of genomic testing: occurrence of testing, patterns of use of testing technologies (including side-by-side use), occurrence of successful biomarker testing,

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Patient attrition chart (N = 3050 remaining patients). aNSCLC: advanced non-small cell lung cancer.</figcaption></figure>
unsuccessful genotyping (defined as at least one of four genes with missing test order and/or a test failure and no result) and percentage of patients with a potentially missed targeted therapy option. The latter measure was defined as patients without a positive biomarker test result who did not receive successful tests for all four of the specified genes. All analyses were descriptive.

For individual biomarkers, all rearrangements in ALK and ROS1, BRAF<sup>V600E</sup> mutations, as well as all EGFR mutations, were assumed to be treatable with targeted therapy.

## 3. Results

### 3.1. Patient demographics and use of genomic testing

Overall, 3050 patients with newly diagnosed, non-squamous aNSCLC were included in the study. The mean patient age at advanced disease diagnosis was 68 years old and the sex ratio was balanced (male: 50.2%; female: 49.8%); the majority of patients had a history of smoking (n = 2589/3050; 84.9%), were diagnosed with stage IV disease (n = 2389/3050 [78.3%]); of the rest, the majority were stage III [396/3050; 13.0%]) and were cared for in a community oncology setting (n = 2822/3050; 92.5%) (Table 1). Among the 3050 patients included in the analysis, 694 (22.8%; of whom 436/694 [62.8%] were stage IV) had no genomic testing performed on their tumour for any of the four guideline-recommended genes prior to first-line treatment. Among the other 2356 (77.2%) patients whose tumour had a genomic test, 1406 (59.7%) were tested with an NGS test (FMI CGP: n = 443 [18.8%]) and 950 (40.3%) with a non-NGS test only (Table 1). Non-NGS techniques used included polymerase chain reaction, immunohistochemistry, fluorescent in situ hybridisation, mass spectrometry, amongst others. The proportion of patients with a history of smoking was higher among those not tested versus those who were tested (93.2% versus 81.8% to 83.5%) (Table 1).

Among patients tested by NGS technologies, parallel use of other testing technologies did occur, but with considerable differences (Fig. 2 and Supplementary Table S2). Among patients tested by FMI CGP (n = 443), 93 (21.0%) received an additional test using another NGS or non-NGS method; however, in the subgroup of patients tested by another NGS test (n = 963), the majority (70.3%; Fig. 2 and Supplementary Table S2) received additional testing with a non-NGS method.

### 3.2. Features of biomarker testing

Among the 1406 patients who received an NGS test prior to first-line treatment, the majority were successfully genotyped for all four examined genes (n = 1220/1406; 86.8%; Fig. 3A and Supplementary Table S2). Successful genotyping was numerically more frequent for patients who received FMI CGP compared with those who received another type of NGS test (95.3% versus 82.9%; Fig. 3B). Unsuccessful genotyping occurred in 13.2% (n = 186/1406) of patients who received an NGS test, mainly due to a missing test order (n = 128/186; 68.8%; Fig. 3A and Supplementary Table S2), although the frequency of test failures was numerically greater in those who received an NGS test other than FMI CGP (n = 42/963 [4.4%] versus n = 1/443 [0.2%]; Fig. 3B and Supplementary Table S2). Among the 950 patients who received a non-NGS test prior to first-line treatment, unsuccessful genotyping occurred in 496 patients (52.2%; Fig. 3A and Supplementary Table S2). This was mainly due to a missing order (n = 430/496; 86.7%). In addition, test failures played a minor role (n = 28/496; 5.7%; Fig. 3A and Supplementary Table S2).

### 3.3. Potentially missed targeted therapy options

Of the 1406 patients who received an NGS test prior to first-line treatment, a positive genomic finding was identified in 28.3%, whereas targeted therapy was ruled out following negative results in all

<table><caption>**Table 1**

Patient demographics and disease characteristics by testing group.</caption><thead><tr><th>Characteristic, no. (%)</th><th>All N = 3050</th><th>CGP (+other) n = 443 (18.8)</th><th>Other NGS (+non-NGS) n = 963 (40.9)</th><th>Non-NGS n = 950 (40.3)</th><th>No Test n = 694 (22.8)</th></tr></thead><tbody><tr><td><strong>Mean age, years (standard deviation)</strong></td><td>68.3 (9.9)</td><td>68.1 (10.6)</td><td>68.5 (9.5)</td><td>68.3 (10.2)</td><td>68.4 (9.3)</td></tr><tr><td><strong>Age group</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>20-34</td><td>5 (0.2)</td><td>0</td><td>2 (0.2)</td><td>2 (0.2)</td><td>1 (0.1)</td></tr><tr><td>35-44</td><td>28 (0.9)</td><td>8 (1.8)</td><td>5 (0.5)</td><td>12 (1.3)</td><td>3 (0.4)</td></tr><tr><td>45-54</td><td>219 (7.2)</td><td>38 (8.6)</td><td>68 (7.1)</td><td>67 (7.1)</td><td>46 (6.6)</td></tr><tr><td>55-64</td><td>834 (27.3)</td><td>124 (28.0)</td><td>250 (26.0)</td><td>269 (28.3)</td><td>191 (27.5)</td></tr><tr><td>65-74</td><td>1052 (34.5)</td><td>126 (28.4)</td><td>359 (37.3)</td><td>312 (32.8)</td><td>255 (36.7)</td></tr><tr><td>75-84</td><td>853 (28.0)</td><td>134 (30.2)</td><td>256 (26.6)</td><td>273 (28.7)</td><td>190 (27.4)</td></tr><tr><td>≥85</td><td>59 (1.9)</td><td>13 (2.9)</td><td>23 (2.4)</td><td>15 (1.6)</td><td>8 (1.2)</td></tr><tr><td><strong>Sex</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Female</td><td>1518 (49.8)</td><td>234 (52.8)</td><td>507 (52.6)</td><td>476 (50.1)</td><td>301 (43.4)</td></tr><tr><td>Male</td><td>1532 (50.2)</td><td>209 (47.2)</td><td>456 (47.4)</td><td>474 (49.9)</td><td>393 (56.6)</td></tr><tr><td><strong>Race</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>White</td><td>2012 (66.0)</td><td>324 (73.1)</td><td>651 (67.6)</td><td>573 (60.3)</td><td>464 (66.9)</td></tr><tr><td>Asian</td><td>106 (3.5)</td><td>10 (2.3)</td><td>40 (4.2)</td><td>48 (5.1)</td><td>8 (1.2)</td></tr><tr><td>Black or African-American</td><td>263 (8.6)</td><td>25 (5.6)</td><td>70 (7.3)</td><td>95 (10.0)</td><td>73 (10.5)</td></tr><tr><td>Hispanic or Latino</td><td>4 (0.1)</td><td>0</td><td>1 (0.1)</td><td>2 (0.2)</td><td>1 (0.1)</td></tr><tr><td>Other</td><td>284 (9.3)</td><td>38 (8.6)</td><td>82 (8.5)</td><td>104 (10.9)</td><td>60 (8.7)</td></tr><tr><td>Missing</td><td>381 (12.5)</td><td>46 (10.4)</td><td>119 (12.4)</td><td>128 (13.5)</td><td>88 (12.7)</td></tr><tr><td><strong>Ethnicity</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Hispanic or Latino</td><td>78 (2.6)</td><td>9 (2.0)</td><td>17 (1.8)</td><td>34 (3.6)</td><td>18 (2.6)</td></tr><tr><td>Missing</td><td>2972 (97.4)</td><td>434 (98.0)</td><td>946 (98.2)</td><td>916 (96.4)</td><td>676 (97.4)</td></tr><tr><td><strong>Stage at initial diagnosis</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Not reported</td><td>19 (0.6)</td><td>3 (0.7)</td><td>4 (0.4)</td><td>1 (0.1)</td><td>11 (1.6)</td></tr><tr><td>Stage I</td><td>143 (4.7)</td><td>21 (4.7)</td><td>32 (3.3)</td><td>34 (3.6)</td><td>56 (8.1)</td></tr><tr><td>Stage II</td><td>73 (2.4)</td><td>13 (2.9)</td><td>21 (2.2)</td><td>18 (1.9)</td><td>21 (3.0)</td></tr><tr><td>Stage III</td><td>396 (13.0)</td><td>44 (9.9)</td><td>99 (10.3)</td><td>94 (9.9)</td><td>159 (22.9)</td></tr><tr><td>Stage IIIC</td><td>30 (1.0)</td><td>5 (1.1)</td><td>9 (0.9)</td><td>5 (0.5)</td><td>11 (1.6)</td></tr><tr><td>Stage IV</td><td>2389 (78.3)</td><td>357 (80.6)</td><td>798 (82.9)</td><td>798 (84.0)</td><td>436 (62.8)</td></tr><tr><td><strong>Practice type</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Academic</td><td>228 (7.5)</td><td>11 (2.5)</td><td>139 (14.4)</td><td>32 (3.4)</td><td>46 (6.6)</td></tr><tr><td>Community</td><td>2822 (92.5)</td><td>432 (97.5)</td><td>824 (85.6)</td><td>918 (96.6)</td><td>648 (93.4)</td></tr><tr><td><strong>Smoking status</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>History of smoking</td><td>2589 (84.9)</td><td>370 (83.5)</td><td>788 (81.8)</td><td>784 (82.5)</td><td>647 (93.2)</td></tr><tr><td>No history of smoking</td><td>459 (15.0)</td><td>73 (16.5)</td><td>175 (18.2)</td><td>165 (17.4)</td><td>46 (6.6)</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Characteristic, no. (%)</th><th>All N = 3050</th><th>CGP (+other) n = 443 (18.8)</th><th>Other NGS (+non-NGS) n = 963 (40.9)</th><th>Non-NGS n = 950 (40.3)</th><th>No Test n = 694 (22.8)</th></tr></thead><tbody><tr><td>Unknown/not documented</td><td>2 (0.1)</td><td>0</td><td>0</td><td>1 (0.1)</td><td>1 (0.1)</td></tr><tr><td>ECOG status at advanced disease diagnosis</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>0/1</td><td>705 (23.1)</td><td>117 (26.4)</td><td>187 (19.4)</td><td>239 (25.2)</td><td>162 (23.3)</td></tr><tr><td>2</td><td>119 (3.9)</td><td>21 (4.7)</td><td>25 (2.6)</td><td>41 (4.3)</td><td>32 (4.6)</td></tr><tr><td>3/4</td><td>17 (0.6)</td><td>3 (0.7)</td><td>4 (0.4)</td><td>5 (0.5)</td><td>5 (0.7)</td></tr><tr><td>Missing</td><td>2209 (72.4)</td><td>302 (68.2)</td><td>747 (77.6)</td><td>665 (70.0)</td><td>495 (71.3)</td></tr></tbody></table>

CGP: comprehensive genomic profiling; ECOG: Eastern Cooperative Oncology Group; NGS: next-generation sequencing.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Use of testing technologies in newly diagnosed patients with aNSCLC. aNSCLC: advanced non-small cell lung cancer; CGP: comprehensive genomic profiling; NGS: next-generation sequencing. <sup>a</sup> Includes CGP only or other NGS testing methods only. <sup>b</sup> For CGP, includes other NGS and/or non-NGS testing methods; for other NGS, other non-NGS testing methods were included.</figcaption></figure>

four biomarkers in 61.7% (Table 2). Among NGS-tested patients, 10.0% were found to have a potentially missed targeted therapy option, which was numerically less pronounced in those receiving FMI CGP compared with those receiving another type of NGS test (2.9% versus 13.2%; Table 2). Among the 950 patients who received a non-NGS test only, a potential targeted therapy was identified in 20.7% and a potentially missed targeted therapy option was found in 40.2% (Table 2). When adjusting for the detection of a non-BRAF<sup>V600E</sup> mutation (as these were not treatable, they were considered negative findings), 23.5% of patients with NGS testing had a result that indicated a treatable targeted therapy option, compared with 20.4% among those who received non-NGS testing only. At the same time, in patients tested with NGS, targeted therapy was ruled out in 66.4%, compared with 39.4% among those who received non-NGS tests only.

### 3.4. Testing of individual biomarkers

Regardless of the technology used, EGFR and ALK testing was performed in ≥ 95% of patients (Table 3). However, while BRAF and ROS1 were also tested in a high proportion of patients who received an NGS test (≥92%), corresponding proportions were numerically lower when non-NGS tests only were used (56.1% and 83.7%, respectively; Table 3). Details of the BRAF and EGFR mutations detected are included in Supplementary Tables S3 and S4, respectively.

### 4. Discussion

We describe contemporary clinical patterns of genomic testing in patients with newly diagnosed non-squamous aNSCLC who were cared for within the Flatiron network, reflecting the community oncology care setting in the US. Overall, we found that not using genomic testing in aNSCLC remains a concern in recent clinical practice, with almost a quarter of patients not receiving any genomic testing before initiation of first-line therapy. Our results show that NGS tests are used in 60% of patients and that not using NGS testing may more often lead to a potentially missed targeted therapy option (compared with cases where NGS testing was used). We also found that when only non-NGS tests were used, testing for recently approved biomarkers such as ROS1 and BRAF was lower. Furthermore, we found a slightly lower probability of finding an actionable BRAF mutation with non-NGS testing. There were no differences in testing between age groups, although there was a greater proportion of non-testing among smokers versus non-smokers and a greater use of CGP in a community versus academic setting; these findings are in line with previous research [17–19]. Unsuccessful genotyping, regardless of testing technique, occurred mainly due to a missing test order, with test failures playing a minimal role.

Previous studies suggest that while genomic testing has increased in recent years [17,20], there remains room for improvement. Previous research has indicated that potential reasons for the lack of biomarker testing include waiting time for results, insufficient tissue samples from biopsies for biomarker analysis, requirement for re-biopsies that may be challenging from a risk, cost and patient perspective [20] and insufficient payer coverage of testing [21]. However, in 2018, the US Centers for Medicare and Medicaid Services approved a national coverage decision for FDA-approved NGS testing in patients with aNSCLC [22]. Combined with the past and more recent FDA approval of NGS-based platforms for genomic testing in patients with aNSCLC [15,23,24], this should, based on the reduced overall costs and enhanced molecular sensitivity of these platforms [14,25,26], help to increase the proportion of patients who undergo testing both when tissue samples are available and when only a blood sample can be analysed.

In the present study, use of NGS-based techniques in patients with non-squamous aNSCLC, particularly CGP, was found to increase the frequency of successful genotyping and reduce the likelihood of a potentially missed targeted therapy option, which is critical given the observed clinical benefit of such treatments, both in clinical trials and in the real world [6,7,27–32]. Analysis of individual biomarkers revealed that when only non-NGS techniques were used, testing was numerically lower for BRAF and ROS1 genes than for EGFR or ALK. Slower adoption of these more recently approved biomarkers (i.e., from 2018) [10,11], technical difficulties, possibly including a lack of material for testing due to prior biomarker analysis, and patients being directed to immunotherapy without genomic testing because of high PD-L1 expression (not recommended by guidelines) [11], may explain these findings. Regarding the latter, clinical implementation of both PD-L1 testing and testing of molecular biomarkers recommended by NSCLC guidelines can be challenging, due to lack of adequate tissue quantity and quality. Indeed, a real-world, retrospective cohort study demonstrated an increase in PD-L1 testing among patients diagnosed with aNSCLC since 2016, and, concomitantly, an increase in first-line cancer immunotherapy, suggesting a high level of enthusiasm for use of PD-L1 testing
A

<figure><img src="image_5.png" /></figure>

Not successfully genotyped due to no record of test only

Not successfully genotyped due to no record of test and test failure

B

<figure><img src="image_6.png" /></figure>

Not successfully genotyped due to test failure only

Successfully genotyped

<caption>**Fig. 3.** Overall results of genotyping in patients by (A) NGS and non-NGS testing methods and (B) CGP and other NGS testing methods. CGP: comprehensive genomic profiling; NGS: next-generation sequencing.</caption>

**Table 2**

Overall outcomes of genomic testing strategies: Identification or exclusion of potential targeted therapy options, including adjusted values for non-BRAF<sup>V600E</sup> mutations.

<table><thead><tr><th rowspan="2"></th><th colspan="3">NGS n = 1406</th><th rowspan="2">Non-NGS n = 950</th></tr><tr><th>CGP (+other) n = 443</th><th>Other NGS (+non-NGS) n = 963</th><th>Overall</th></tr></thead><tbody><tr><td><strong>Potential targeted therapy identified, % (adj.%)<sup>a</sup></strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Mutation positive in at least 1 marker, 4 tested</td><td>28.0 (21.4)</td><td>23.7 (19.8)</td><td>25.0 (20.3)</td><td>8.7 (8.4)</td></tr><tr><td>Mutation positive in at least 1 marker, &lt;4 tested</td><td>1.8 (1.8)</td><td>3.9 (3.8)</td><td>3.3 (3.2)</td><td>12.0 (12.0)</td></tr><tr><td>Total</td><td>29.8 (23.2)</td><td>27.6 (23.6)</td><td>28.3 (23.5)</td><td>20.7 (20.4)</td></tr><tr><td><strong>Targeted therapy ruled out, % (adj.%)<sup>a</sup></strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Mutation negative in 4 markers</td><td>67.3 (73.8)</td><td>59.2 (63.0)</td><td>61.7 (66.4)</td><td>39.1 (39.4)</td></tr><tr><td><strong>Potentially missed targeted therapy, % (adj.%)<sup>a</sup></strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Only negative tests in &lt; 4 markers and at least 1</td><td>2.9 (2.9)</td><td>13.2 (13.3)</td><td>10.0 (10.0)</td><td>40.2 (40.2)</td></tr></tbody></table>

CGP: comprehensive genomic profiling; NGS: next-generation sequencing.

<sup>a</sup> adj. %: Adjusted percentage values accounted for the finding of a non-BRAF<sup>V600E</sup> mutation; as these were not treatable, they were considered negative findings for the adjusted values. Detailed information on BRAF mutation findings in the study are provided in Supplementary Table S3.

and cancer immunotherapy that may supersede recommendations for biomarker testing [33]. Nevertheless, given the emergence and recent approval of new biomarkers (e.g., KRAS, NTRK, MET, RET, HER2) [10,11], our results support an increasing clinical need for broader NGS-based testing, which tends to be more cost- and time-effective than single-gene testing, in patients with aNSCLC [26]. This supports the application of standardised testing algorithms in pathology to ameliorate waiting for individual test results, i.e., EGFR, BRAF, ALK, ROS1 and any other approved genomic biomarker should be tested by pathologists in every patient with stage IV non-squamous NSCLC by default.

A limitation of the current study is that the reasons why patients received genomic testing were not available; it is possible that for some patients, the lack of a solid tumour sample or insufficient tumour tissue, or other technical barriers [34,35], may have prevented any or some of the testing. In patients for whom a solid tumour sample was unavailable, both in the first-line setting [36] and otherwise [37,38], NGS-based liquid biopsies may be considered, which are minimally invasive, have

a shorter turnaround time for test results and may serve to reduce overall healthcare costs [39,40]. This technique has already shown high concordance with tumour tissue genotyping for detection of genomic alterations and subsequent clinical decision-making in aNSCLC [25,37,40–42], and a number of NGS-based liquid biopsy platforms have since been granted FDA approval [43,44]. Compared with tumour tissue genotyping, NGS liquid biopsies have also been demonstrated to enable enhanced detection of therapeutically relevant genomic alterations in aNSCLC and thus stratification to targeted therapy in cases of insufficient tissue availability, failed quality control, incomplete genotyping or a negative biomarker result from tissue testing [40,45,46].

Furthermore, some patients may have tested positive for rare genomic alterations that were not documented routinely in our data, e. g., NTRK fusions; however, given the rarity of these alterations in aNSCLC (~0.1%) [47], it seems unlikely that they would have had a major influence on the results.
<table><caption>**Table 3**
Proportion of patients tested for individual biomarkers who had a successful test and mutation positivity by test approach.</caption><thead><tr><th rowspan="2"></th><th colspan="2">NGS n = 1406</th><th rowspan="2">Non NGS n = 950</th></tr><tr><th>CGP (+other) n = 443</th><th>Other NGS (+non-NGS) n = 963</th></tr></thead><tbody><tr><td><strong>ALK, %</strong></td><td></td><td></td><td></td></tr><tr><td>Tested<sup>a</sup></td><td>98.0</td><td>95.5</td><td>94.9</td></tr><tr><td>Successful<sup>b</sup></td><td>99.5</td><td>97.9</td><td>96.0</td></tr><tr><td>Positive<sup>c</sup></td><td>2.5</td><td>3.2</td><td>3.3</td></tr><tr><td>Negative<sup>c</sup></td><td>97.5</td><td>96.8</td><td>96.7</td></tr><tr><td><strong>BRAF, %</strong></td><td></td><td></td><td></td></tr><tr><td>Tested<sup>a</sup></td><td>96.4</td><td>95.3</td><td>56.1</td></tr><tr><td>Successful<sup>b</sup></td><td>99.8</td><td>97.8</td><td>97.9</td></tr><tr><td>Positive<sup>c</sup></td><td>10.3</td><td>6.7</td><td>1.5</td></tr><tr><td>Negative<sup>c</sup></td><td>89.7</td><td>93.3</td><td>98.5</td></tr><tr><td><strong>EGFR, %</strong></td><td></td><td></td><td></td></tr><tr><td>Tested<sup>a</sup></td><td>98.4</td><td>99.3</td><td>94.9</td></tr><tr><td>Successful<sup>b</sup></td><td>99.3</td><td>97.6</td><td>98.6</td></tr><tr><td>Positive<sup>c</sup></td><td>17.8</td><td>18.8</td><td>17.4</td></tr><tr><td>Negative<sup>c</sup></td><td>82.2</td><td>81.2</td><td>82.6</td></tr><tr><td><strong>ROS1, %</strong></td><td></td><td></td><td></td></tr><tr><td>Tested<sup>a</sup></td><td>98.0</td><td>92.4</td><td>83.7</td></tr><tr><td>Successful<sup>b</sup></td><td>99.8</td><td>96.2</td><td>95.6</td></tr><tr><td>Positive<sup>c</sup></td><td>1.4</td><td>0.9</td><td>1.1</td></tr><tr><td>Negative<sup>c</sup></td><td>98.6</td><td>99.1</td><td>98.9</td></tr></tbody></table>

CGP: comprehensive genomic profiling; NGS: next-generation sequencing.

a % of patients overall.

b % of patients tested.

c % of successful tests (positive = rearrangement present for ALK or ROS1, or mutation positive for EGFR or BRAF).

## 5. Conclusions

The findings of this study suggest that among US patients with newly diagnosed non-squamous aNSCLC, a lack of NGS testing may increase the probability of a potentially missed targeted therapy option. The use of NGS, particularly CGP, may also help to avoid suboptimal biomarker testing, minimise test failures, improve uptake of testing for recently approved biomarkers and enable enhanced access to clinical trials of molecularly guided therapies and individualised, targeted therapy. Our results provide support for the use of guideline-recommended broad-panel NGS testing in clinical practice, which is also warranted by the ever increasing molecularly guided treatment options for aNSCLC.

**Additional Contributions:** Research support for third-party writing assistance for this manuscript, furnished by Stephen Salem, BSc, of Health Interactions, was provided by F. Hoffmann-La Roche Ltd, Basel, Switzerland.

**Prior Presentation:** These analyses have been presented in part: Gondos A, Paz-Ares L, Saldana D, et al. Genomic testing among patients (pts) with newly diagnosed advanced non-small cell lung cancer (aNSCLC) in the US: a contemporary clinical practice patterns study. Poster presentation at the American Society of Clinical Oncology 2020 Virtual Congress, May 29–May 31, 2020 (Gondos et al. 2020. *Journal of Clinical Oncology* 38, no. 15_suppl 9592–9592).

**Data Sharing Statement:** The data that support the findings of this study have been originated by Flatiron Health, Inc. These de-identified data may be made available upon request, and are subject to a license agreement with Flatiron Health; interested researchers should contact DataAccess@flatiron.com to determine licensing terms.

### *CRediT authorship contribution statement*

**Luis Paz-Ares:** Conceptualization, Supervision, Validation, Investi-gation, Visualization, Writing – original draft, Project administration,

Writing – review & editing. **Adam Gondos:** Conceptualization, Data curation, Software, Formal analysis, Supervision, Validation, Investi-gation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review & editing. **Diego Saldana:** Conceptu-alization, Data curation, Software, Formal analysis, Validation, Investi-gation, Visualization, Methodology, Project administration, Writing – review & editing. **Marlene Thomas:** Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Project administration, Writing – review & editing. **Celine Mascaux:** Concep-tualization, Validation, Investigation, Writing – review & editing. **Lukas Bubendorf:** Conceptualization, Validation, Investigation, Writing – review & editing. **Fabrice Barlesi:** Conceptualization, Validation, Investi-gation, Writing – original draft, Writing – review & editing.

### Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: **LPA:** has received a research grant from Bristol Myers Squibb, Astra-Zeneca and PharmaMar, and personal fees from MSD, Bristol Myers Squibb, F. Hoffmann-La Roche Ltd, AstraZeneca, Eli Lilly, Merck, Novartis, Amgen, Pfizer, Takeda, Blueprint, Bayer, PharmaMar, Ipsen, AbbVie, GSK, Janssen, Boehringer Ingelheim, Sanofi, Tesaro, Mirati, Altum sequencing and Genomica. **AG:** Employed by and has stocks or other ownership in F. Hoffmann-La Roche Ltd. **DS:** Employed by F. Hoffmann-La Roche Ltd (previously) and Novartis, has stocks or other ownership in Novartis and has received research funding (institution) from F. Hoffmann-La Roche Ltd and Novartis. **MT:** Employed by and has a leadership role in F. Hoffmann-La Roche Ltd, and has stocks or other ownership, and holds patents, royalties, other intellectual property (self) with F. Hoffmann-La Roche Ltd. **CM:** Has received personal fees for consulting or advisory boards from F. Hoffmann-La Roche Ltd, Astra-Zeneca, Kephren, Bristol Myers Squibb, MSD and Pfizer, and has received travel and accommodation expenses from F. Hoffmann-La Roche Ltd, AstraZeneca, Takeda and Boehringer Ingelheim. **LB:** has received a research grant from MSD and personal fees for advisory boards from AstraZeneca, AbbVie, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly, MSD and Pfizer. **FB:** has received personal fees from AstraZeneca, Bayer, Bristol Myers Squibb, Boehringer Ingelheim, Eli Lilly Oncology, F. Hoffmann-La Roche Ltd, Novartis, Merck, MSD, Pierre Fabre, Pfizer and Takeda. All authors received research support in the form of third-party medical writing assistance for this manuscript, provided by F. Hoffmann-La Roche Ltd.

### Acknowledgements

**Funding:** This analysis was sponsored by F. Hoffmann-La Roche Ltd (Basel Switzerland). Data were analysed by employees at the sponsor, F. Hoffmann-La Roche Ltd. Authors employed by the study sponsor contributed to the study design; management, analysis and interpretation of the data; and preparation, review and approval of the manuscript, as well as the decision to submit the manuscript for publication.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.lungcan.2022.01.021.

### References

[1] L.V. Sequist, J.-H. Yang, N. Yamamoto, K. O'Byrne, V. Hirsh, T. Mok, S.L. Geater, S. Orlov, C.-M. Tsai, M. Boyer, W.-C. Su, J. Bennouna, T. Kato, V. Gorbunova, K. H. Lee, R. Shah, D. Massey, V. Zazulina, M. Shahidi, M. Schuler, Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations, J. Clin. Oncol. 31 (27) (2013) 3327–3334.

[2] T. Mitsudomi, S. Morita, Y. Yatabe, S. Negoro, I. Okamoto, J. Tsurutani, T. Seto, M. Satouchi, H. Tada, T. Hirashima, K. Asami, N. Katakami, M. Takada,
H. Yoshioka, K. Shibata, S. Kudoh, E. Shimizu, H. Saito, S. Toyooka, K. Nakagawa, M. Fukuoka, West Japan Oncology Group, Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): An open label, randomised phase 3 trial, Lancet Oncol. 11 (2) (2010) 121–128.

[3] R. Rosell, E. Carcerine, R. Gervais, A. Vergnenegre, B. Massuti, E. Felip, R. Palmero, R. Garcia-Gomez, C. Pallares, J.M. Sanchez, R. Porta, M. Cobo, P. Garrido, F. Longo, T. Moran, A. Insa, F. De Marinis, R. Corre, I. Bover, A. Illiano, E. Dansin, J. de Castro, M. Milleta, N. Reguart, G. Altavilla, U. Jimenez, M. Provencio, M.A. Moreno, J. Terrasa, J. Muñoz-Langa, J. Valdivia, D. Isla, M. Domine, O. Molinier, J. Mazieres, N. Baize, R. Garcia-Campelo, G. Robinet, D. Rodriguez-Abreu, J. Lopez-Vivanco, V. Gubbia, L. Ferrera-Galdado, P. Bombaron, R. Bernabe, A. Bearz, A. Artal, E. Cortesi, C. Rolfo, M. Sanchez-Ronco, A. Drozdowskyj, C. Queralt, I. de Aguirre, J.L. Ramirez, J.J. Sanchez, M. A. Molina, M. Taron, L. Paz-Ares, Spanish Lung Cancer Group in collaboration with Groupe Français de Pneumo-Cancérologie et Associazione Italiana Oncologia Toracica, Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): A multicentre, open-label, randomised phase 3 trial, Lancet Oncol. 13 (3) (2012) 239–246.

[4] T.S. Mok, Y. Cheng, X. Zhou, K.H. Lee, K. Nakagawa, S. Niho, M. Lee, R. Linke, R. Rosell, J. Corral, M.R. Migliorino, A. Pluzanski, E.I. Sbar, T. Wang, J.L. White, Y.-L. Wu, Improvement in overall survival in a randomized study that compared dacomitinib versus gefitinib in patients with advanced non-small-cell lung cancer and EGFR-activating mutations, J. Clin. Oncol. 36 (22) (2018) 2244–2250.

[5] D.R. Camidge, H.R. Kim, M.-J. Ahn, J.-H. Yang, J.-Y. Han, J.-S. Lee, M.J. Hochmair, J.-C. Li, G.-C. Chang, K.H. Lee, C. Gridelli, A. Delmonte, R. Garcia Campelo, D.-W. Kim, A. Bearz, F. Griesinger, A. Morabito, E. Felip, R. Califano, S. Ghosh, A. Spira, S.N. Gettinger, M. Tiseo, M. Gupta, D. Kerstein, S. Popat, Brigitainin versus crizotinib in ALK-positive non-small-cell lung cancer, N. Engl. J. Med. 379 (21) (2018) 2027–2039.

[6] A.T. Shaw, G.J. Riely, Y.J. Bang, D.W. Kim, D.R. Camidge, B.J. Solomon, M. Varella-Garcia, A.J. Iafrate, G.I. Shapiro, T. Usari, S.C. Wang, K.D. Wilner, J. W. Clark, S.I. Ou, Crizotinib in ROS1-rearranged advanced non-small-cell lung cancer (NSCLC): Updated results, including overall survival, from PROFILE 1001, Ann. Oncol. 30 (7) (2019) 1121–1126.

[7] D. Planchard, B. Besse, H.J.M. Groen, P.J. Souquet, E. Quox, C.S. Baik, F. Barlesi, T.M. Kim, J. Mazieres, S. Novello, J.R. Rigas, A. Upalawanna, A.M. D'Amelio Jr., P. Zhang, B. Mookerjee, B.E. Johnson, Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: An open-label, multicentre phase 2 trial, Lancet Oncol. 17 (7) (2016) 984–993.

[8] A. Drilon, T.W. Laetsch, S. Kummar, S.G. Du Boyst, U.N. Lassen, G.D. Demetri, M. Nathenson, R.C. Doebele, A.F. Farago, A.S. Pappo, B. Turpin, A. Dowlati, M. S. Brose, L. Mascarenhas, N. Federman, J. Berlin, W.S. El-Deiry, C. Baik, J. Deeken, V. Boni, R. Nagasubramanian, M. Taylor, E.R. Rudzinski, F. Meric-Bernstam, D.P. S. Sohal, P.C. Ma, L.E. Raez, J.F. Hechtman, R. Benayed, M. Ladanyi, B.B. Tuch, K. Ebata, S. Cruickshank, N.C. Ku, M.C. Cox, D.S. Hawkins, D.S. Hong, D. M. Hyman, Efficacy of larotrectinib in TRK fusion-positive cancers in adults and children, N. Engl. J. Med. 378 (8) (2018) 731–739.

[9] E.R. Malone, M. Oliva, P.J.B. Sabatini, T.L. Stockley, L.L. Siu, Molecular profiling for precision cancer therapies, Genome Med. 12 (1) (2020) 8.

[10] N.I. Lindeman, P.T. Cagle, D.L. Aisner, M.E. Arcila, M.B. Beasley, E.H. Bernicker, C. Colasacco, S. Dacic, F.R. Hirsch, K. Kerr, D.J. Kwiatkowski, M. Ladanyi, J. A. Nowak, L. Sholl, R. Temple-Smolkin, B. Solomon, L.H. Souter, E. Thunnissen, M. S. Tsao, C.B. Ventura, M.W. Wynes, Y. Yatabe, Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: Guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology, Arch. Pathol. Lab. Med. 142 (3) (2018) 321–346.

[11] National Comprehensive Cancer Network (NCCN). NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®): Non-Small Cell Lung Cancer. Version 7.2021. https://www.nccn.org/professionals/physician_gls/PDF/nscll.pdf. (Accessed 25 November 2021).

[12] European Society for Medical Oncology (ESMO). Metastatic non-small cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up, 2020. https://www.esmo.org/content/download/347819/6934778/1/ESMO-CP-G-mNSCLC-15SEPT2020.pdf (Accessed August 2021).

[13] A. De Geer, J. Eriksson, H.W. Finnern, A cross-country review of data collected on non-small cell lung cancer (NSCLC) patients in cancer registries, databases, retrospective and non-randomized prospective studies, J. Med. Econ. 16 (1) (2013) 134–149.

[14] G.M. Frampton, A. Fichtenholtz, G.A. Otto, K. Wang, S.R. Downing, J. He, M. Schnall-Levin, J. White, E.M. Sanford, P. An, J. Sun, F. Juhn, K. Brennan, K. Iwanik, A. Maillet, J. Buell, E. White, M. Zhao, S. Balasubramanian, S. Terzic, T. Richards, V. Banning, L. Garcia, K. Mahoney, Z. Zwirko, A. Donahue, H. Beltran, J.M. Mosquera, M.A. Rubin, S. Dogan, C.V. Hedvat, M.F. Berger, L. Pusztai, M. Lechner, C. Boshoff, M. Jarosz, C. Vietz, A. Parker, V.A. Miller, J.S. Ross, J. Curran, M.T. Cronin, P.J. Stephens, D. Lipson, R. Yelensky, Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing, Nat. Biotechnol. 31 (11) (2013) 1023–1031.

[15] US Food and Drug Administration (FDA), Center for Devices and Radiological Health (CDRH), FoundationOne®CDX: Summary of safety and effectiveness data (SSED), 2019. https://www.accessdata.fda.gov/cdrh_docs/pdf17/P170019S006B.pdf. (Accessed 25 November 2021).

[16] T.A. Clark, J.H. Chung, M. Kennedy, J.D. Hughes, N. Chennagiri, D.S. Lieber, B. Fendler, L. Young, M. Zhao, M. Coyne, V. Breese, G. Young, A. Donahue, D. Pavlick, A. Tsiros, T. Brennan, S. Zhong, T. Mughal, M. Bailey, J. He, S. Roels, G. M. Frampton, J.M. Spoerke, S. Gendreau, M. Lackner, E. Schleifman, E. Peters, J. S. Ross, S.M. Ali, V.A. Miller, J.P. Gregg, P.J. Stephens, A. Welsh, G.A. Otto, D. Lipson, Analytical validation of a hybrid capture-based next-generation sequencing clinical assay for genomic profiling of cell-free circulating tumor DNA, J. Mol. Diagn. 20 (5) (2018) 686–702.

[17] M.E. Gutierrez, K. Choi, R.B. Lanman, E.J. Licita, S.M. Skrzypczak, R. Pe Benito, T. Wu, S. Arunajadai, S. Kaur, H. Harper, A.L. Pecora, E.V. Schultz, S.L. Goldberg, Genomic profiling of advanced non-small cell lung cancer in community settings: Gaps and opportunities, Clin. Lung Cancer 18 (6) (2017) 651–659.

[18] C. Lim, M.S. Tsao, L.W. Le, F.A. Shepherd, R. Feld, R.L. Burkes, G. Liu, S. Kamel-Reid, D. Hwang, J. Tanguay, G. da Cunha Santos, N.B. Leigh, Biomarker testing and time to treatment decision in patients with advanced nonsmall-cell lung cancer, Ann. Oncol. 26 (7) (2015) 1415–1421.

[19] D.H. Lee, M.-S. Tsao, K.-O. Kambartel, H. Isobe, M.-S. Huang, C.H. Barrios, A. Khattak, F. de Marinis, S. Kothari, A. Arunachalam, X. Cao, T. Burke, A. Valladares, J. de Castro, A. Ahmad, Molecular testing and treatment patterns for patients with advanced non-small cell lung cancer: PIVOTAL observational study, PLoS ONE 13 (8) (2018) e0202865, https://doi.org/10.1371/journal.pone.0202865.

[20] Friends of Cancer Research, Trends in the molecular diagnosis of lung cancer, 2015. https://www.focr.org/sites/default/files/pdf/FINAL%202017%20Friends%20NSCLC%20White%20Paper.pdf. (Accessed August 2021).

[21] N.A. Pennell, M.E. Arcila, D.R. Gandara, H. West, Biomarker testing for patients with advanced non-small cell lung cancer: Real-world issues and tough choices, Am. Soc. Clin. Oncol. Educ. Book. (39) (2019) 531–542, https://doi.org/10.1200/EDBK_237863.

[22] Centers for Medicare & Medicare Services, Decision memo for next generation sequencing (NGS) for cancer beneficiaries with advanced cancer (CAG-00450N). 2018. https://www.cms.gov/medicare-coverage-database/details/nca-decisi-on-memo.aspx?NCAId=290. (Accessed August 2021).

[23] Food and Drug Administration (FDA), Oncomine™ Dx Target Test: Summary of safety and effectiveness data, 2017. https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160045B.pdf. (Accessed August 2021).

[24] Food and Drug Administration (FDA), Evaluation of automatic class III designation for MSK-impact, 2017. https://www.accessdata.fda.gov/cdrh_docs/reviews/den170058.pdf. (Accessed August 2021).

[25] D. Su, D. Zhang, K. Chen, J. Lu, J. Wu, X. Cao, L. Ying, Q. Jin, Y. Ye, Z. Xie, L. Xiong, W. Mao, F. Li, High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods, J. Exp. Clin. Res. 36 (1) (2017) 121.

[26] L. Nong, Z. Zhang, Y. Xiong, Y. Zheng, X. Li, D. Li, Q. He, T. Li, Comparison of next-generation sequencing and immunohistochemistry analysis for targeted therapy-related genomic status in lung cancer patients, J. Thorac. Dis. 11 (12) (2019) 4992–5003.

[27] S. Ramalingam, J. Vansteenkiste, D. Planchard, B.C. Cho, J.E. Gray, Y. Ohe, L. Zhou, Reungwetwattana, Y. Cheng, F. Chewaskulyong, R. Shah, M. Cobo, K. H. Lee, P. Cheema, M. Tiseo, T. John, M.-C. Lin, F. Imamura, T. Kurata, A. Todd, R. Hodge, M. Saggese, Y. Rukazenkov, J.-C. Soria, FLAURA Investigators, Overall survival with osimertinib in untreated, EGFR-mutated advanced NSCLC, N. Engl. J. Med. 382 (1) (2020) 41–50.

[28] J.-C. Soria, Y. Ohe, J. Vansteenkiste, T. Reungwetwattana, B. Chewaskulyong, K. H. Lee, A. Dechaphunkul, F. Imamura, N. Nogami, T. Kurata, I. Okamoto, C. Zhou, B.C. Cho, Y. Cheng, E.K. Cho, P.J. Voon, D. Planchard, W.-C. Su, J.E. Gray, S.-M. Lee, R. Hodge, M. Marotti, Y. Rukazenkov, S.S. Ramalingam, FLAURA Investigators, Osimertinib in untreated EGFR-mutated advanced non-small-cell lung cancer, N. Engl. J. Med. 378 (2) (2018) 113–125.

[29] S. Peters, D.R. Camidge, A.T. Shaw, S. Gadgeel, J.S. Ahn, D.-W. Kim, S.-H. Ou, M. Pérol, R. Dzadiuszko, R. Rosell, A. Zeiter, E. Mitry, S. Golding, B. Balas, J. Noe, P.N. Morcos, T. Mok, ALEX Trial Investigators, Alectinib versus crizotinib in untreated ALK-positive non-small-cell lung cancer, N. Engl. J. Med. 377 (9) (2017) 829–838.

[30] S. Novello, J. Mazières, I.J. Oh, J. de Castro, M.R. Migliorino, Å. Helland, R. Dzadiuszko, F. Griesinger, A. Kotb, A. Zeiter, A. Cardona, B. Balas, H. K. Johannsdottir, A. Das-Gupta, J. Wolf, Alectinib versus chemotherapy in crizotinib-pretreated anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer: Results from the phase III ALUR study, Ann. Oncol. 29 (6) (2018) 1409–1416.

[31] S.M. Lim, H.R. Kim, J.-S. Lee, K.H. Lee, Y.-G. Lee, Y.J. Min, E.K. Cho, S.S. Lee, B.-S. Kim, M.Y. Choi, H.S. Shim, J.-H. Chung, Y. La Choi, M.J. Lee, M. Kim, J.-H. Kim, S.M. Ali, M.-J. Ahn, B.C. Cho, Open-label, multicenter, phase II study of ceritinib in patients with non-small-cell lung cancer harboring ROS1 rearrangement, J. Clin. Oncol. 35 (23) (2017) 2613–2618.

[32] G. Singal, P.G. Miller, V. Agarwala, G. Li, G. Kaushik, D. Backenroth, A. Gossai, G. M. Frampton, A.Z. Torres, E.M. Lehnert, D. Bourque, C. O'Connell, B. Bowser, T. Caron, E. Baydur, K. Seidl-Rathkopf, I. Ivanov, G. Alpha-Cobb, A. Guria, J. He, S. Frank, A.C. Nunnally, M. Bailey, A. Jaskiw, D. Feuchtabaum, N. Nussbaum, A. P. Abernethy, V.A. Miller, Association of patient characteristics and tumor genomics with clinical outcomes among patients with non-small cell lung cancer using a clinicogenomic database, JAMA 321 (14) (2019) 1391–1399.

[33] M.S. Leapman, C.J. Presley, W. Zhu, P.R. Soulos, K.B. Adelson, R.A. Miksad, D. J. Boffa, C.P. Gross, Association of programmed cell death ligand 1 expression status with receipt of immune checkpoint inhibitors in patients with advanced non-small cell lung cancer, JAMA Netw. Open 3 (6) (2020) e0207205, https://doi.org/10.1001/jamanetworkopen.2020.7205.
[34] H. Chen, R. Luthra, R.S. Goswami, R.R. Singh, S. Roy-Chowdhuri, Analysis of pre-analytic factors affecting the success of clinical next-generation sequencing of solid organ malignancies, Cancers (Basel) 7 (3) (2015) 1699–1715.

[35] M. Dietel, L. Bubendorf, A.-M.-C. Dingemans, C. Dooms, G. Elmberger, R.C. García, K.M. Kerr, E. Lim, F. López-Ríos, E. Thunnissen, P.E. Van Schil, M. von Laffert, Diagnostic procedures for non-small-cell lung cancer (NSCLC): Recommendations of the European Expert Group, Thorax 71 (2) (2016) 177–184.

[36] K. Brice, L.E. Raez, K. Dumais, D. Wietecha, A. Lopez-Cohen, P. Izquierdo, L. Drusbosky, H.I. Dada, H.W. Powery, Liquid biopsies for the front line therapy decision in non-small cell lung cancer, J. Clin. Oncol. 39 (15 Suppl) (2021) Abstract e21103.

[37] R. Madison, A.B. Schrock, E. Castellanos, J.P. Gregg, J. Snider, S.M. Ali, V. A. Miller, G. Singal, B.M. Alexander, J.M. Venstrom, J.H. Chung, Retrospective analysis of real-world data to determine clinical outcomes of patients with advanced non-small cell lung cancer following cell-free circulating tumor DNA genomic profiling, Lung Cancer 148 (2020) 69–78.

[38] R. Woodhouse, M. Li, J. Hughes, D. Delfosse, J. Skoletsky, P. Ma, W. Meng, N. Dewal, C. Milbury, T. Clark, A. Donahue, D. Stover, M. Kennedy, J. Dacpano-Komansky, C. Burns, C. Vietz, B. Alexander, P. Hegde, L. Dennis, P. Ha, Clinical and analytical validation of FoundationOne Liquid CDx, a novel 324-Gene cfDNA-based comprehensive genomic profiling assay for cancers of solid tumor origin, PLoS ONE 15 (9) (2020) e0237802, https://doi.org/10.1371/journal.pone.0237802.

[39] C. Rolfo, P.C. Mack, G.V. Scagliotti, P. Baas, F. Barlesi, T.G. Bivona, R.S. Herbst, T. S. Mok, N. Peled, R. Pirker, L.E. Raez, M. Reck, J.W. Riess, L.V. Sequist, F. A. Shepherd, L.M. Sholl, D.S.W. Tan, H.A. Wakelee, M.W. Wistuba II, D.P. Wynes, F.R. Carbone, D.R.G. Hirsch, Liquid biopsy for advanced non-small cell lung cancer (NSCLC): A statement paper from the IASLC, J. Thorac. Oncol. 13 (9) (2018) 1248–1268.

[40] N.B. Leighl, R.D. Page, V.M. Raymond, D.B. Daniel, S.G. Divers, K.L. Reckamp, M. A. Villalona-Calero, D. Dix, J.I. Odegaard, R.B. Lanman, V. A. Papadimitrakopoulou, Clinical utility of comprehensive cell-free DNA analysis to identify genomic biomarkers in patients with newly diagnosed metastatic non-small cell lung cancer, Clin. Cancer Res. 25 (15) (2019) 4691–4700.

[41] R. Dziadziuszko, T. Mok, S. Peters, J.Y. Han, J. Alatorre-Alexander, N. Leighl, V. Sriuranpong, M. Pérol, G. de Castro Junior, E. Nadal, F. de Marinis, O. A. Frontera, D.S.W. Tan, D.H. Lee, H.R. Kim, M. Yan, T. Riehl, E. Schleifman, S. M. Paul, S. Mocci, R. Patel, Z.J. Assaf, D.S. Shames, M.S. Mathisen, S.M. Gadgeel, Blood first assay screening trial (BFAST) in treatment-naïve advanced or metastatic NSCLC: Initial results of the phase 2 ALK-positive cohort, J. Thorac. Oncol. 16 (12) (2021) 2040–2050.

[42] M. Makarem, N.B. Leighl, Molecular testing for lung adenocarcinoma: Is it time to adopt a “plasma-first” approach? Cancer 126 (14) (2020) 3176–3180.

[43] Foundation Medicine, FDA approves Foundation Medicine's FoundationOne®Liquid CDx, a comprehensive pan-tumor liquid biopsy test with multiple companion diagnostic indications for patients with advanced cancer [press release]. 26 April, 2018, 2020. https://www.foundationmedicine.com/press-releases/991ce685-7dbf-400a-aa33-eb4f9d411ed3#:~:text=to%20Press%20Releases,Foundation%20Medicine's%20New%20Liquid%20Biopsy%20Assay%20Granted%20Breakthrough%20Device,U.S.%20Food%20and%20Drug%20Administration&text=The%20new%20assay%20will%20include,tumor%20mutational%20burden%20(bTMB). (Accessed August 2021).

[44] Food and Drug Administration (FDA), FDA approves first liquid biopsy next-generation sequencing companion diagnostic test [press release]. 7 August, 2020, 2020. https://www.fda.gov/news-events/press-announcements/fda-approves-first-liquid-biopsy-next-generation-sequencing-companion-diagnostic-test. (Accessed August 2021).

[45] P.C. Mack, K.C. Banks, C.R. Espenschied, R.A. Burich, O.A. Zill, C.E. Lee, J. W. Riess, S.A. Mortimer, A. Talasaz, R.B. Lanman, D.R. Gandara, Spectrum of driver mutations and clinical impact of circulating tumor DNA analysis in non-small cell lung cancer: Analysis of over 8000 cases, Cancer 126 (14) (2020) 3219–3228.

[46] C. Aggarwal, J.C. Thompson, T.A. Black, S.I. Katz, R. Fan, S.S. Yee, A.L. Chien, T. L. Evans, J.M. Bauml, E.W. Alley, C.A. Ciunci, A.T. Berman, R.B. Cohen, D. B. Lieberman, K.S. Majmundar, S.L. Savitch, J.J.D. Morrissette, W.-T. Hwang, K.S. J. Elenitoba-Johnson, C.J. Langer, E.L. Carpenter, Clinical implications of plasma-based genotyping with the delivery of personalized therapy in metastatic non-small cell lung cancer, JAMA Oncol. 5 (2) (2019) 173–180.

[47] A.F. Farago, L.P. Le, Z. Zheng, A. Muzikansky, A. Drilon, M. Patel, T.M. Bauer, S. V. Liu, S.-H. Ou, D. Jackman, D.B. Costa, P.S. Multani, G.G. Li, Z. Hornby, E. Chow-Maneval, D. Luo, J.E. Lim, A.J. Iafrate, A.T. Shaw, Durable clinical response to entrectinib in NTRK1-rearranged non-small cell lung cancer, J. Thorac. Oncol. 10 (12) (2015) 1670–1674.