Molecular and clonal evolution of primary lesions vs. brain metastasis in EGFR-mutated NSCLC: a retrospective cohort study

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) constituting approximately 85% of all cases, and is a major driver of cancer mortality rates globally (1-4). Approximately 10–40% of NSCLC patients display epidermal growth factor receptor (EGFR) activating mutations (5), and advances in molecular profiling have transformed the management of those patients by enabling targeted therapies (5,6). Despite these therapeutic breakthroughs, brain metastases (BM) occur in 20–40% of NSCLC patients, representing a significant clinical challenge due to poor prognosis and limited treatment options (4,7). The emergence of resistance mechanisms and the inability of many systemic therapies to cross the blood-brain barrier (BBB) further complicates disease management (3,8).

Genomic alterations, including EGFR, ALK, and RET rearrangements, activate oncogenic pathways such as MAPK and PI3K/AKT, contributing to cell proliferation, survival, and metastasis (3,5). These pathways are implicated in therapy resistance, highlighting the need for novel therapeutic approaches (6,9). Previous studies suggest that EGFR-positive tumors have a higher propensity for BM, often leading to more aggressive clinical behavior (4,7,10). Likewise, evidence suggests that EGFR mutations are prevalent in NSCLC and influence metastatic behavior, with specific variants such as exon 19 deletions and L858R mutations associated with distinct clinical outcomes and treatment responses (11).

Osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI) with high brain uptake, even in subjects with an intact BBB (12), is more efficacious than other EGFR-TKIs, exhibiting high rates of intracranial responses and reducing the risk of central nervous system (CNS) progressions (13); nevertheless, progressive disease occurs almost invariably in patients with advanced disease, and therapeutic options remain suboptimal (14). Radiation therapy (RT) has been a cornerstone for treating BM due to the limited CNS penetration of chemotherapy (CT) caused by the BBB. For decades, whole-brain RT was the standard approach, with more advanced techniques, such as stereotactic radiosurgery, now offering more targeted options (10).

This study aimed to evaluate the clinical and molecular characteristics of lung adenocarcinoma patients with metastatic and symptomatic brain involvement treated at different comprehensive cancer centers in Latin America. By analyzing paired comprehensive genomic profiles of primary tumors and dominant metastatic sites, along with subsequent molecular assessments after disease progression, this study aimed to explore associations between genomic alterations, clinical characteristics, and survival outcomes. This study also sought to characterize treatment strategies and their impact on disease progression and overall survival (OS) in this population. We present this article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-502/rc).

Methods

This retrospective cohort study included patients diagnosed with lung adenocarcinoma presenting with metastatic and symptomatic BM. Comprehensive genomic profiling was performed on the primary lung lesion and additionally on the dominant metastatic site after neurosurgical resection for symptomatic relief or biopsy for primary diagnosis, prior to confirmation of lung cancer. Additionally, molecular assessments were conducted after disease progression using tissue-based analysis or liquid biopsy. All evaluations used the FoundationOne CDx platform (15) to assess microsatellite instability, tumor mutational burden (TMB), single-nucleotide variants, and copy number variants. Patients were enrolled in the study from August 2019 to March 2024. Clinical and demographic variables as well as genomic characteristics were collected from a centralized, encrypted, and anonymized database at the Cancer Treatment and Research Center (CTIC) in Bogotá, Colombia. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Kayre Institutional Ethics Committee with the unique identification COD-FORD-ID-0028 on February 22, 2022. No consent to participate was needed for participation as it is a retrospective study.

First-line treatment consisted of osimertinib (80 mg PO daily) as systemic therapy for controlling brain lesions. All patients underwent stereotactic radiosurgery as definitive treatment for CNS metastases or as consolidation following neurosurgical excision (18 to 25 Gy in one to three fractions). Later lines of treatment were decided based on the molecular components identified at disease progression, at the discretion of the treating physicians, and the availability of medications. Patient clinical outcomes were measured as the overall response rate (ORR), progression-free survival (PFS), and OS. Investigator assessments used RECIST version 1.1, to evaluate ORR and responses in leptomeningeal, brain parenchyma (termed intracranial response), and extracranial lesions. PFS was defined as the time from the first administration of osimertinib to disease progression or death from any cause. OS was defined as the time from neurosurgical resection or biopsy to death from any cause.

Statistical analysis

Descriptive statistics were used to characterize the variables, employing central tendency and dispersion metrics based on each variable’s nature. Inferential statistics were applied to test the hypotheses regarding the associations between clinical characteristics, molecular profiling, and outcomes. Survival analysis was conducted using the Kaplan-Meier method.

Results

Thirty patients were included in the analysis. Of these, 22 (73%) were female, with a median age of 55 years. Nearly all patients (97%) had a history of smoking (mean 1.6 pack-years (standard deviation ±0.34 pack-years), with only one individual being a lifelong nonsmoker. At diagnosis, CNS involvement was present in 27 patients (90%), while 3 additional patients experienced CNS involvement during disease progression. The median number of BM was 2 (range: 1–3). The patient characteristics and metastatic locations are summarized in Table 1.

Molecular characteristics

Paired molecular analyses revealed notable differences between primary lung tumors and BM. Regarding genomic profiling, 14 patients (47%) exhibited exon 19 deletions, whereas the remaining patients had L858R mutations. TP53 was the most frequently co-mutated gene in the primary lesions, found in 16 cases (53.3%), followed by RBM10 mutations in seven patients (23%), with 3 cases of co-occurrence with TP53. Other notable alterations included mutations in STK11 (n=2, 7%), GNAS (n=4, 13%), and RB1 (n=1, 3%). The mean TMB in the primary lesions was 3.2 mut/Mb. The mutational co-occurrence in primary tumors is shown in Figure 1.

Figure 1 Mutational distributions and co-occurrences among primary tumors. EGFR, epidermal growth factor receptor.

When comparing primary lesions to BM, all patients showed an increase in TMB in the latter, with a median of 8.5 mut/Mb; furthermore, EGFR mutations were lost in nine of those patients (30%), distributed nearly equally between exon 19 deletions and L858R mutations. Among the co-mutations in BM, PIK3CA/PTEN/AKT pathway alterations, absent in primary lesions, were newly found in 10 cases (33%). Additional gains included BRAF V600E mutations (n=1, 3%), non-V600 BRAF mutations (n=3, 10%), and RB1 alterations (n=4, 13%). Amplification of various genes was detected in 19 patients (63%). RBM10 mutations were lost in all but two cases (71%). Molecular profiling between primary lesions and BM, including the gain and loss of function alterations, is presented in Figure 2.

Figure 2 Molecular profiling of gained and lost alterations between primary lesions and brain metastases. EGFR, epidermal growth factor receptor.

Clinical outcomes

The ORR was 70%, with 10% of the patients achieving a complete response. Stable disease was observed in nine patients (30%). The only variable significantly associated with response was bone metastatic involvement at diagnosis, with patients exhibiting bone metastases being less likely to respond [odds ratio (OR) =0.06; 95% confidence interval (CI): 0.005–0.69; P=0.024]. Intracranial response was achieved in all patients.

Progression and survival

Progression-related findings included MET amplification in 5 patients (16.7%) and small cell transformation in 2 patients (6.7%). HER2 amplification and EGFR C797S mutations were seen in one patient each (3.4%). PIK3CA alterations were detected in 4 patients (13.3%), while BRAF V600E mutations were identified in 2 patients (6.7%). Six patients showed alterations exclusively within BM, suggesting that progressive lesions originated from brain tumor cell lines.

At disease progression, patients with small cell transformation were treated with carboplatin, etoposide, and osimertinib (n=2, 6.7%). The most common second-line therapies were carboplatin, paclitaxel, bevacizumab, and atezolizumab (n=12, 40%), followed by carboplatin, pemetrexed, and osimertinib (n=11, 36.7%). Treatment for BRAF V600E-mutated cases included dabrafenib plus trametinib or vemurafenib (n=2, 6.7%). Other therapies included osimertinib combined with bevacizumab (n=2, 6.7%), with one patient receiving osimertinib and gefitinib for the C797S mutation (3.4%).

The median OS for the entire cohort was 54 months [95% CI: 49–not reached (NR)], as shown in Figure 3. Median PFS was 20.5 months (95% CI: 17.1–26.2 months), and PFS to second progression (PFS2) was 32.2 months (95% CI: 27.7–39.2 months), presented in Figure 4A,4B. Neither OS nor PFS was significantly affected by metastatic sites, TP53 or RBM10 mutations, EGFR mutation type, or the presence of alterations in PIK3CA or TP53. However, EGFR loss was associated with shorter PFS (17 months; 95% CI: 15.1–NR vs. 21.3 months; 95% CI: 19.1–33.8; P=0.02; HR =2.84) and PFS2 (29 months; 95% CI: 25.5–NR vs. 37.5 months; 95% CI: 27.7–44.1; P=0.019; HR =2.89). Additionally, patients with L858R mutations had shorter PFS2 (27.6 months; 95% CI: 25.6–39.2) compared to those with exon 19 deletions (37.8 months; 95% CI: 30.5–48.1; P=0.022; HR =2.48). Survival outcomes did not differ significantly between carboplatin/ pemetrexed/ bevacizumab/ atezolizumab and carboplatin/pemetrexed/osimertinib as second-line therapy (P=0.2 for both PFS and PFS2).

Figure 3 Survival analysis of the cohort. Median overall survival. Median is outlined with a black dashed line.

Figure 4 Survival analysis of the cohort. (A) Median progression-free survival curve. (B) Median progression-free survival to second progression curve. Median is outlined with a black dashed line.

Discussion

The proportion of lung cancer in nonsmokers is increasing and is frequently linked to driver-mutation in genes such as EGFR and ALK. Although most patients with EGFR-mutated advanced NSCLC have a notable initial response to treatment with 3rd-generation EGFR-TKIs, real-world survival estimates show that less than 20% of patients will survive after 5 years (16,17). One important reason is clonal evolution in response to standard therapy (17). however, early clinical and molecular variables, such as BM, liver metastasis, and tumor shedding, in addition to TP53 co-mutation, result in more aggressive biological behavior (18,19).

The results of this study offer critical insights into the clinical and molecular characteristics of lung adenocarcinoma in patients with metastatic and symptomatic brain involvement. Among the most notable findings, the median OS for the cohort reached 54 months (95% CI: 49–NR), highlighting the potential long-term benefits of targeted systemic and CNS-directed therapies in this high-risk population. PFS was 20.5 months (95% CI: 17.1–26.2 months), while PFS2 extended to 32.2 months (95% CI: 27.7–39.2 months), showing promising efficacy. Comprehensive genomic profiling was central to identifying actionable alterations in this cohort. Next-generation sequencing (NGS) revealed significant heterogeneity between the primary and metastatic lesions, particularly in the CNS. This finding highlights the importance of biopsy and molecular analysis of metastatic sites in guiding treatment decisions. Site-dependent tumor mutations, especially in the CNS, contribute to differential therapeutic responses and the development of resistance, emphasizing the necessity for personalized therapeutic approaches (9,20).

A high proportion of patients in our cohort were light smokers. Smoking history, mostly in heavy and current smokers, deeply impacts EGFR mutation prevalence, survival curves, and the mutational landscape. Tseng et al. describe that PFS and OS were lower in patients with a large smoking history (more than 15 pack-years) compared with less than 15 pack-years (21). Meanwhile, patients with fewer pack-years and former smokers had similar survival curves to of those never smokers. Similarly, Wang et al. reported that doubling smoking pack-years was associated with an increase in TMB and that there was a significant dose-response association between smoking history and genetic alterations in different trials (22).

Numerous clinical trials have evaluated the impact of multiple pharmacological combinations with EGFR-TKIs to synergistically restrict the development of acquired resistance in patients with previously untreated EGFR-mutated NSCLC. In the phase III MARIPOSA and FLAURA2 studies, up-scaling upfront therapy (lazertinib plus amivantamab in the former and osimertinib plus CT in the latter) resulted in significantly longer PFS than did osimertinib, which has been the standard of care; furthermore, there was a statistically significant and clinically meaningful reduction in mortality in both trials (23). Importantly, in the osimertinib arms in both trials, median PFS in patients with BM receiving osimertinib monotherapy was under 14 months (24); these survival metrics underscore the importance of integrating comprehensive molecular profiling and tailored therapeutic strategies to improve outcomes in patients with advanced disease.

Another critical observation in our study was the remarkable spatial heterogeneity between primary lung cancer and its counterpart, the metastatic brain tumor. The latter had a higher TMB; it was more likely to have osimertinib-resistant mutations and a loss of up to 30% of the EGFR-driver mutations.

In addition to TMB, multiple biomarkers predict the greatest clinical benefit of implementing immune checkpoint blockers (ICB), including PD-L1 and tumor-infiltrating lymphocytes (TILS). Our trial’s findings align with those of The Cancer Genome Atlas (TCGA) (25) and Real-World data (26); TMB in patients with EGFR classical mutations was comparatively lower than that in the EGFR-wild type (27). Whilst the present study found an increase in median TMB in BM, it remains unclear which TMB threshold could predict ICB efficacy in patients with NSCLC and EGFR mutations (28). ICBs have significantly improved outcomes for patients with advanced driver-negative NSCLC and some driver mutations with immunologically hot tumors (KRAS G12C, BRAF V600E, MET Ex14skipping mutations) (29,30). Regarding EGFR mutations, in a network meta-analysis including 12 randomized trials in patients with progressive disease on a TKI, the addition of either an ICB or ICB plus antiangiogenic therapy to CT improved PFS relative to CT alone (HR 0.77 and 0.54, respectively) (31); also, in a recent single randomized trial, among 322 such patients, the addition of Ivonescimab, a bispecific antibody targeting programmed death 1 and vascular endothelial growth factor, to CT significantly improved PFS (7.1 vs. 4.8 months) (32). The link between high PD-L1/TMB expression could be manifold. Patients with a strong PD L1 expression had insufficient response to Osimertinib monotherapy and exhibited primary resistance in two large cohorts of patients with treatment-naïve advanced EGFR-mutant NSCLC (33,34); mechanistic studies demonstrated that upregulation of PD-L1 was critical in inducing autophagy through the mitogen-activated protein kinase (MAPK) signaling pathway, which was beneficial for tumor progression and the development of EGFR-TKI resistance (35). A recent analysis of 339 NSCLC patients with hybrid capture-based NGS revealed a significant mutation rate of the PI3K signaling pathway in the EGFR-sensitive mutation group with high PD-L1 expression (38% vs. 12%, P<0.001) and high TMB group (31% vs. 13%, P<0.05). Notably, PI3K signaling pathway mutations may be responsible for inducing primary resistance to EGFR-TKIs in NSCLC patients with EGFR-sensitive mutations, high PD-L1 expression, or high TMB (36).

A molecular perspective considers primary resistance to front-line osimertinib mainly about the molecular heterogeneity of EGFR mutations and to co-existing molecular alterations in other genes; however, loss of EGFR mutation in synchronous metastatic niches has rarely been described, emphasizing the importance of upfront or delayed locoregional therapies to face this intricate phenomenon. Furthermore, loss of activating EGFR-mutant genes has been described as a mechanism of resistance to EGFR-TKI in vitro and in vivo (37,38) with constitutive activation of EGFR downstream signaling, PI3K/AKT, even after loss of the mutated EGFR gene (39). Parallel tissue biopsies and circulating tumor DNA (ctDNA) samples are the cornerstones for tracking clonal dynamics.

Historically, it has been argued that in patients with BM disease, the primary resistance mechanism is pharmacological due to low drug penetration of the BBB; however, the present study underscores different resistance mechanisms that explain aggressive behavior. Further analysis of this cohort revealed unique disease progression patterns. Patients with alternative driver mutations exhibit shorter PFS than those with single alterations, particularly in cases involving BM. This finding aligns with earlier studies suggesting complex driver mutations may lead to more aggressive clinical phenotypes (4,7). The correlation between driver mutations and metastatic patterns warrants additional research to elucidate the underlying biological mechanisms. Bastianos et al. reported branched evolution from a common ancestor and an independent evolutionary trajectory explaining the discrepancy in oncogenic alterations between BM and primary tumors. Intralesional and interlesional heterogeneity (by sampling distinct BM) analysis revealed that subclones sampled in these lesions were more related to one another than to those detected in the primary tumor sample (40). In addition, comprehensive pan-cancer analysis in BM at single-cell resolution revealed differences in the tumor ecosystem between these and primary tumors. Metastatic cancer cells adopt a neural-like cell state, suggesting that cells may preexist and are more prone to home in certain metastatic niches. In addition, patients with EGFR mutations exhibited lower M1, higher M2 state of myeloid cells, and more M-type cells, suggesting a stronger immunosuppressive and tumor-promoting state in the tumor microenvironment in EGFR-mutant patients, but potentially a benefit from myeloid-targeted immune therapies (41).

From a therapeutic perspective, these findings underscore the need for innovative strategies to address the dual challenges of systemic and CNS disease control. Combining first-line Osimertinib with stereotactic radiosurgery demonstrated effective CNS control for BM. However, despite these interventions, multifocal BM was observed in 43.3% of patients at diagnosis, with 26.6% presenting with two lesions and 16.6% with three or more lesions. The development of fourth-generation anti-EGFR, such as BBT-207, JIN-A02, and HS10504, with enhanced CNS penetration and a broader spectrum for EGFR-mutation coverage (42), will offer promising avenues for future clinical trials. Similarly, approaches with combinations of inhibitors may provide synergistic effects, prevent the emergence of resistance mutations, and narrow the spectrum and complexity of acquired resistance (43). Preclinical studies investigating these combinations are essential to inform subsequent clinical strategies.

Ablative CNS treatment with stereotactic radiosurgery or consolidation therapy following neurosurgical excision was associated with favorable intracranial control. This finding reinforces the importance of integrating advanced radiotherapeutic techniques into multidisciplinary care plans for patients with CNS metastases (10,44). Nevertheless, the limitations of current systemic therapies, particularly in controlling multifocal BM and preventing further progression, remain to be addressed. This study also emphasized the importance of longitudinal follow-up and real-world data collection to assess treatment durability and resistance evolution. Incorporating liquid biopsy approaches for detecting emerging resistance mutations in ctDNA could provide dynamic insights into disease progression and inform timely therapeutic modifications (3,9).

While these findings provide valuable insights, they must be interpreted within the context of certain limitations. This study has limitations inherent to its retrospective design and relatively small sample size, typical of highly selected cohorts with paired tissue samples. Selection bias may have influenced the cohort, as only patients with available paired tissue samples and symptomatic BM requiring neurosurgical intervention or biopsy were included. Additionally, real-world variability across participating Latin American centers may limit the generalizability of these findings to broader EGFR-mutated NSCLC populations.

While this study provides valuable insights into the clinical and molecular characteristics of lung adenocarcinoma patients with metastatic brain involvement from several Latin American comprehensive cancer centers (Mexico, Colombia, Brazil, and Chile), it is crucial to acknowledge the potential geographic and ethnic specificities of patient populations and treatment practices. Future multicenter studies involving a broader representation of global regions would be beneficial to further generalize these findings.

The observed mutational profiles and treatment responses, particularly the high prevalence of certain EGFR mutations and the efficacy of osimertinib, align with the reported trends in diverse populations. However, the specific co-mutations and resistance mechanisms identified warrant further investigation in other geographical cohorts to assess their broader applicability and potential influence on therapeutic strategies. The retrospective nature of this study and the relatively small sample size (n=30), while providing real-world data, inherently introduce potential biases related to data collection and treatment decisions. Future prospective studies with larger cohorts are needed to validate these findings and to establish more definitive cause-and-effect relationships.

The consistent use of the FoundationOne CDx platform for genomic profiling enhances the internal consistency of the molecular data. However, variations in local treatment protocols (e.g., specific CT regimens, RT techniques beyond SRS) and physician discretion in subsequent lines of therapy, while reflecting real-world practice, could introduce variability. Future studies with more standardized treatment algorithms are practically feasible and could further strengthen the generalizability of the treatment-related outcomes.

Conclusions

This study highlights lung adenocarcinoma’s clinical and molecular complexity with metastatic brain involvement, emphasizing the critical role of comprehensive genomic profiling and CNS-directed strategies. Genomic analysis of BM provides a window of opportunity to identify potentially clinically relevant additional alterations that are not detected in primary tumors. The incorporation of tailored systemic therapies with advanced local treatments has the potential to significantly improve the survival outcomes in this challenging patient population. Future efforts should address therapeutic resistance and enhance CNS-specific efficacy to optimize care for patients with advanced lung adenocarcinoma.

Acknowledgments

We thank the patients, their family members, healthcare professionals, and the supporting staff of the Luis Carlos Sarmiento Angulo Cancer Treatment and Research Center (Colombia), Instituto Nacional de Cancerología - INCaN (Mexico), Fleming Institute (Argentina), and Cancer Research and Treatment Center (Costa Rica) for their participation and support.

This study was presented at the Latin American Lung Cancer Meeting held in Bogotá, Colombia, between November 14 and 15, 2024 and was awarded the Young Investigator Award.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-502/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-502/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-502/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-502/coif). A.R.P. received payments from Johnson & Johnson for lectures about EGFR mutated lung cancer, biology and treatment. J.Z. reports payments for lectures and educational activities from Adium, Amgen, AstraZeneca, Bayer, BMS, Eli Lilly, Pfizer, MSD, Novartis, Janssen, Johnson & Johnson, Roche, Abbot; receipt of equipment, materials, drugs, medical writing, gifts or other services from Astra, Pfizer, Roche, Abbot, Johnson & Johnson, BMS, MSD, Adium. O.A. received grants from Astra Zeneca, Boehringer Ingelheim, and Roche; consulting fees from Pfizer, Lilly, Merck, Bristol Myers Squibb, Astra Zeneca, Boehringer Ingelheim, and Roche. L.V. reports consulting fees from Astra Zeneca; payment or honoraria from Astra Zeneca, MSD, Medtronic and Johnson & Johnson; travel support from Astra Zeneca and MSD; and is on the Advisory Board of MSD. S.M. reports honoraria for educational events and lectures from MSD, AstraZeneca, Johnson & Johnson, Medtronic. C.C. received speaker fees from AstraZeneca. A.G. reports honoraria for educational lectures from MSD, BMS, AstraZeneca, and Varian; support for attending a meeting – ASCO GU from Johnson & Johnson; and is Vice President of the Colombian Association of Radiation Oncology since 2023. J.O. has received support to attend (including travel fare and hotel) to medical meetings from Astra Zeneca. J.R. received payment or honoraria from Astra Zeneca and Amgen; meeting support from Thermo Fisher and Sophia genetics; is on the Advisory Board for J&J and is Scientific Director in Foundation for Applied Clinical and Molecular Cancer Research. C.R. reports consulting fees from Novocure and AIRC; institutional fees for advisory board membership from AstraZeneca, Imagene, MedStar, Amgen, Boehringer-Ingelheim, Hoffmann-La Roche Ltd, Janssen Pharmaceutical, NeoGenomics, Pfizer, Inc. and Regeneron; support for attending meetings from ASCO, ESO, ISLB, IASLC, AIOM, AIRC, Boehringer-Ingelheim, PrecisCA, OncoHost, OneCell, TACTICS; participation on a Data Safety Monitoring Board or Advisory Board of Roche, Abbvie, Eli Lilly, CORED, MEC, AstraZeneca, OncoHost, OneCell, Centro Pfizer-Universidad de Granada-Junta de Andalucía de Genómica e Investigación Oncológica (GENYO), External Advisor of the School of Public Health, University of Granada, Spain, Scientific Committee of the Fondazione Siciliana di Oncologia, and non-renumerated analysis of liquid biopsies in a lung cancer trial for Guardant Health; and is president of ISLB and board member of IASLC; peceipt of equipment, materials, drugs, medical writing, gifts or other service from OneCell. A.F.C. reports grants from Roche, BI, Novartis, Foundation Medicine, Astra, QQF, Roche Diagnostics, Amgen, Bayer, CCF, Idylla; consulting fees from Roche, BI, Pfizer, Novartis, Celldex, Astra, Astellas, Amgen, MSD, Merck Serono, EISAI, Merck Serono, Jannsen, BioNTech, Amgen, Flatiron Health, Teva Pharma, Takeda, FICMAC; payment or honoraria from Johnson & Johnson, Roche, BI, Pfizer, Novartis, Astra, Amgen, BMS, MSD, EISAI, TEVA, Novartis, Merck Serono, Takeda, Mirati; payment for expert testimony Roche, BI, Novartis, BMS, Abbie, Celldex, Idylla, Thermo Fisher, Illumina, Amgen, Eli Lilly, Guardant Health, Regeneron, Bayer, Eli Lilly, Biocartis, Pharma Mar, Pfizer; support for attending meetings from Roche, BI, Novartis, BMS, Abbie, Celldex, Idylla, Thermo Fisher, Illumina, Amgen, Eli Lilly, Guardant Health, Regeneron, Bayer, Eli Lilly, Biocartis, Pfizer; participation on a Data Safety Monitoring Board or Advisory Board of Roche, Johnson & Johnson, Astra Zeneca; is a board member of IASLC and ASCO; receipt of equipment, materials, drugs, medical writing, gifts or other services from Roche, Rochem Biocare, Thermo Fisher, Illumina. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Kayre Institutional Ethics Committee with the unique identification COD-FORD-ID-0028 on February 22, 2022. No consent to participate was needed for participation as it is a retrospective study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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