EGFR-mutant Lung Adenocarcinoma Research Articles

Thyroid transcription factor 1 (TTF-1) negativity as a predictor of unfavorable response to EGFR-TKI therapy in advanced lung adenocarcinoma patients with EGFR mutations.

The absence of thyroid transcription factor 1 (TTF-1) is associated with a lower frequency of epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma (LUAD). The aim of this study was to assess the impact of TTF-1 expression on the clinical response to EGFR-tyrosine kinase inhibitor (TKI) treatment in patients with advanced LUAD. The data of patients with advanced LUAD who were admitted to the Beijing Tiantan Hospital and Peking University Cancer Hospital (China) between April 2009 and May 2023 was retrospectively analyzed. A total of 227 patients diagnosed with advanced LUAD were included, of which 28.2% (64/227) had TTF-1-negative adenocarcinoma, while 54.6% (124/227) harbored EGFR mutations. Negative TTF-1 expression significantly correlated with male sex (68.8% vs. 42.3%, p < 0.001), history of heavy smoking (57.8% vs. 36.2%, p = 0.003), poorly differentiated tumors (86.5% vs. 43.2%, p < 0.001), and lower frequency of EGFR mutations (26.6% vs. 65.6%, p < 0.001) compared with TTF-1 positivity. Multivariable logistic regression showed that low prevalence of EGFR mutations (p < 0.001) and male sex (p = 0.006) were independent predictive factors for the negative expression of TTF-1. Patients lacking TTF-1 also exhibited worse overall response rate (ORR; 23.5% vs. 54.2%, p = 0.019), disease control rate (DCR; 58.8% vs. 89.7%, p = 0.003), and median progression-free survival (PFS; 2.9 vs. 11.6 months, p < 0.001) following treatment with EGFR-TKIs compared to the TTF-1-positive patients with EGFR mutations. Patients with TTF-1-negative and EGFR-mutant LUAD show a diminished response to EGFR-TKIs.

Dual inhibition of KIF11 and BCL2L1 induces apoptosis in lung adenocarcinoma cells

EGFR-mutant lung adenocarcinoma (LUAD) mostly depends on EGFR for survival and consequently responds well to EGFR inhibitors. However, resistance to the drugs develops almost universally during treatment. We previously demonstrated that EGFR-mutant LUAD cell lines, HCC827 and H1975, have subpopulations of cells, which we termed HCC827 GR2 and H1975 WR7 cells, that can thrive independently of EGFR signaling. These EGFR-independent EGFR-mutant cancer cells are difficult to treat because they lack sensitivity to EGFR inhibitors. Therefore, the development of novel strategies to target EGFR–independent EGFR-mutant LUAD is particularly important. We found that high expression of kinesin family member 11 (KIF11) correlated with poor survival in patients with LUAD. We also observed that KIF11 silencing caused cell cycle arrest at G2/M in HCC827 GR2 and H1975 WR7 cells. Furthermore, dual silencing of KIF11 plus BCL2L1, an anti-apoptotic BCL2 family member, in these two EGFR-independent sublines resulted in marked apoptosis levels. Dual inhibition of KIF11 plus BCL2L1 also induced apoptosis in HCC827 and H1975 parental cells and a KRAS-mutant LUAD cell line, H441. These findings collectively suggest that dual inhibition of KIF11 plus BCL2L1 may be a new approach for the treatment of LUAD.

Mammalian SWI/SNF chromatin remodeling complexes promote tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer.

Acquired resistance to tyrosine kinase inhibitors (TKI), such as osimertinib used to treat EGFR-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-genetic mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures of osimertinib sensitive and resistant EGFR-mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization, we identify both shared and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant models to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and suggest potential utility of mSWI/SNF inhibitors in TKI-resistant lung cancers.

Knowledge-based mechanistic modeling accurately predicts disease progression with gefitinib in EGFR-mutant lung adenocarcinoma

Lung adenocarcinoma (LUAD) is associated with a low survival rate at advanced stages. Although the development of targeted therapies has improved outcomes in LUAD patients with identified and specific genetic alterations, such as activating mutations on the epidermal growth factor receptor gene (EGFR), the emergence of tumor resistance eventually occurs in all patients and this is driving the development of new therapies. In this paper, we present the In Silico EGFR-mutant LUAD (ISELA) model that links LUAD patients’ individual characteristics, including tumor genetic heterogeneity, to tumor size evolution and tumor progression over time under first generation EGFR tyrosine kinase inhibitor gefitinib. This translational mechanistic model gathers extensive knowledge on LUAD and was calibrated on multiple scales, including in vitro, human tumor xenograft mouse and human, reproducing more than 90% of the experimental data identified. Moreover, with 98.5% coverage and 99.4% negative logrank tests, the model accurately reproduced the time to progression from the Lux-Lung 7 clinical trial, which was unused in calibration, thus supporting the model high predictive value. This knowledge-based mechanistic model could be a valuable tool in the development of new therapies targeting EGFR-mutant LUAD as a foundation for the generation of synthetic control arms.

Clinical Impact of Genomic and Pathway Alterations in Stage I EGFR-Mutant Lung Adenocarcinoma.

We investigated the clinical impact of genomic and pathway alterations in stage I epidermal growth factor receptor (EGFR)-mutant lung adenocarcinomas, which have a high recurrence rate despite complete surgical resection. Out of the initial cohort of 257 patients with completely resected stage I EGFR-mutant lung adenocarcinoma, tumor samples from 105 patients were subjected to analysis using large-panel next-generation sequencing. We analyzed 11 canonical oncogenic pathways and determined the number of pathway alterations (NPA). Survival analyses were performed based on co-occurring alterations and NPA in three patient groups: all patients, patients with International Association for the Study of Lung Cancer (IASLC) pathology grade 2, and patients with recurrent tumors treated with EGFR-tyrosine kinase inhibitor (TKI). In the univariate analysis, pathological stage, IASLC grade, TP53 mutation, NPA, phosphoinositide 3-kinase pathway, p53 pathway, and cell cycle pathway exhibited significant associations with worse recurrence-free survival (RFS). Moreover, RPS6KB1 or EGFR amplifications were linked to a poorer RFS. Multivariate analysis revealed that pathologic stage, IASLC grade, and cell cycle pathway alteration were independent poor prognostic factors for RFS (p=0.002, p < 0.001, and p=0.006, respectively). In the grade 2 subgroup, higher NPA was independently associated with worse RFS (p=0.003). Additionally, in patients with recurrence treated with EGFR-TKIs, co-occurring TP53 mutations were linked to shorter progression-free survival (p=0.025). Genomic and pathway alterations, particularly cell cycle alterations, high NPA, and TP53 mutations, were associated with worse clinical outcomes in stage I EGFR-mutant lung adenocarcinoma. These findings may have implications for risk stratification and the development of new therapeutic strategies in early-stage EGFR-mutant lung cancer patients.

Genetic dissection of intratumor heterogeneity of PD-L1 expression in EGFR-mutated lung adenocarcinoma.

In this study, we investigated the association between PD-L1 expression in tumor cells and underlying genetic mutations, which was analyzed in detail using laser microdissection and next-generation sequencing analysis. To investigate whether driver mutations are involved in the background of PD-L1 expression, the EGFR major activating mutation was selected as the most frequent driver mutation. Surgical resection specimens were used to extract sufficient amounts of nucleic acids for analysis, and the high tumor proportion score (TPS:100%) and low (TPS: 0%) PD-L1-expressing parts of the tumor were each laser microdissected to examine the association between PD-L1 expression heterogeneity and genetic mutations within the same tumor. The association between PD-L1 heterogeneity and gene mutations within the same tumor was investigated. Analysis showed no association between PD-L1 expression heterogeneity and genetic variants, which were found to be almost identical. However, PD-L1 expression was found to be associated with the number of tumor infiltrating lymphocytes (TILs) present in the tumor, which may be related to whether or not lymphocytes can infiltrate into the tumor depending on the tumor histological type (solid pattern, lepidic pattern, etc.) and other factors.

De Novo and Histologically Transformed Small-Cell Lung Cancer Is Sensitive to Lurbinectedin Treatment Through the Modulation of EMT and NOTCH Signaling Pathways.

Small-cell lung cancer (SCLC) is a high-grade neuroendocrine tumor with dismal prognosis and limited treatment options. Lurbinectedin, conditionally approved as a second-line treatment for metastatic SCLC, drives clinical responses in about 35% of patients, and the overall survival (OS) of those who benefit from it remains very low (∼9.3 months). This finding highlights the need to develop improved mechanistic insight and predictive biomarkers of response. We used human and patient-derived xenograft (PDX)-derived SCLC cell lines to evaluate the effect of lurbinectedin in vitro. We also demonstrate the antitumor effect of lurbinectedin in multiple de novo and transformed SCLC PDX models. Changes in gene and protein expression pre- and post-lurbinectedin treatment was assessed by RNA sequencing and Western blot analysis. Lurbinectedin markedly reduced cell viability in the majority of SCLC models with the best response on POU2F3-driven SCLC cells. We further demonstrate that lurbinectedin, either as a single agent or in combination with osimertinib, causes an appreciable antitumor response in multiple models of EGFR-mutant lung adenocarcinoma with histologic transformation to SCLC. Transcriptomic analysis identified induction of apoptosis, repression of epithelial-mesenchymal transition, modulation of PI3K/AKT, NOTCH signaling associated with lurbinectedin response in de novo, and transformed SCLC models. Our study provides a mechanistic insight into lurbinectedin response in SCLC and the first demonstration that lurbinectedin is a potential therapeutic target after SCLC transformation.

Differential Prognostic Value of Vascular Invasion in Resected Lung Adenocarcinomas According to Epidermal Growth Factor Receptor Mutational Status

BackgroundIt is unclear whether all patients with stage IB to IIIA epidermal growth factor receptor (EGFR)-mutant adenocarcinoma should receive adjuvant osimertinib. We investigated the prognostic value of vascular invasion for risk stratification according to EGFR mutational status. Materials and MethodsThis retrospective study evaluated patients with stage IB to IIIA lung adenocarcinoma resected between 2011 and 2016 at a tertiary care center. The study outcome was overall survival (OS). The prognostic value of vascular invasion was analyzed using the adjusted log-rank test and multivariable Cox regression with clinico-pathological factors as covariates. A sensitivity analysis, which included the presence of ground-glass opacity on CT scans as an additional covariate, and subgroup analyses according to the pathological stage were performed. ResultsIn total, 272 patients were included (146 women; median age, 66 years [interquartile range: 58, 72 years]; 128 EGFR-mutant adenocarcinomas). The 5-year OS rate was 90.8% (95% CI: 84.0%, 98.1%) in EGFR-mutant, vascular invasion-absent lung adenocarcinomas, which was higher than in other subgroups (P < .05). Vascular invasion was an independent, negative prognostic factor in EGFR-mutant lung adenocarcinomas (adjusted log-rank test, P = .02; adjusted hazard ratio, 3.01; 95% CI: 1.30, 7.02; P = .01). However, the prognosis of EGFR wild-type adenocarcinomas was not associated with the presence of vascular invasion (adjusted log-rank test, P = .95; adjusted hazard ratio, 1.32; 95% CI: 0.74, 2.34; P = .35). Similar results were observed in the sensitivity analysis and subgroup analyses. ConclusionsVascular invasion-absent, EGFR-mutant, resected lung adenocarcinomas showed a very good prognosis, and vascular invasion had a differential prognostic value according to EGFR mutational status.

An oncogenic CTNNB1 mutation is predictive of post-operative recurrence-free survival in an EGFR-mutant lung adenocarcinoma.

The Wnt/β-catenin pathway is known to be frequently dysregulated in various human malignancies. Alterations in the genes encoding the components of Wnt/β-catenin pathway have also been described in lung adenocarcinoma. Notably however, the clinical impacts of Wnt/β-catenin pathway alterations in lung adenocarcinoma have not been fully evaluated to date. We here investigated the prognostic implications of single gene variations in 174 cases of surgically resected lung adenocarcinoma tested using targeted next-generation sequencing. Screening of the prognostic impact of single gene alterations identified an association between CTNNB1 mutation and poor recurrence-free survival in EGFR-mutant LUADs. Based on these results, the entire cohort was stratified into three groups in accordance with the mutational status of Wnt/β-catenin pathway genes (i.e. oncogenic CTNNB1 mutation [CTNNB1-ONC], other Wnt/β-catenin pathway gene mutations [Wnt/β-catenin-OTHER], and wild type for Wnt/β-catenin pathway genes [Wnt/β-catenin-WT]). The clinicopathologic characteristics and survival outcomes of these groups were then compared. Oncogenic CTNNB1 and other Wnt/β-catenin pathway gene mutations were identified in 10 (5.7%) and 14 cases (8.0%), respectively. The CTNNB1-ONC group cases displayed histopathologic features of conventional non-mucinous adenocarcinoma with no significant differences from those of the other groups. Using β-catenin immunohistochemistry, we found that the CTNNB1-ONC group displayed aberrant nuclear staining more frequently, but only in 60% of the samples. The LUADs harboring a CTNNB1-ONC exhibited significantly poorer RFS outcomes than the other groups, regardless of the β-catenin IHC status. This was a pronounced finding in the EGFR-mutant LUADs only in subgroup analysis, which was then confirmed by multivariate analysis. Nevertheless, no significant OS differences between these Wnt/β-catenin groups were evident. Hence, oncogenic CTNNB1 mutations may be found in about 6% of lung adenocarcinomas and may predict post-operative recurrence in EGFR-mutant LUADs. Aberrant nuclear β-catenin staining on IHC appears to be insufficient as a surrogate marker of an oncogenic CTNNB1 mutation.

E-cadherin expression in the tumor microenvironment of advanced epidermal growth factor receptor-mutant lung adenocarcinoma and the association with prognosis

BackgroundThe expression of programmed death-ligand 1 (PD-L1), tumor-infiltrating lymphocytes (TILs), E-cadherin, and vimentin in lung cancer tumor microenvironment is known to impact patient survival or response to therapy. The expression of these biomarkers may also differ between primary lung tumors and brain metastatic tumors. In this study, we investigated the interaction between these biomarkers in lung tumors with or without concomitant brain metastasis and the interaction with paired brain metastatic tumors.MethodsThe study included 48 patients with stage IV epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma. Sixteen of the forty-eight patients were diagnosed with brain metastasis, while the remaining thirty-two were not. All sixteen patients with brain metastasis had brain tumors. The expression of PD-L1, TILs (CD8+ T lymphocytes and FOXP3+ regulatory T lymphocytes), E-cadherin, and vimentin were evaluated using immunohistochemical (IHC) staining.ResultsPatients with brain metastasis exhibited a higher frequency of exon 19 deletion and uncommon EGFR mutations, a higher lung tumor vimentin score, worse progression-free survival (PFS), and overall survival (OS) than patients without brain metastasis. IHC staining showed no difference between paired lung and brain tumors. Patients with low PD-L1 expression had better PFS and OS. After multivariate analysis, higher body mass index, the presence of brain metastasis, bone metastasis, and uncommon EGFR mutations were correlated with worse PFS, while the presence of brain metastasis and high lung tumor E-cadherin score was associated with worse OS.ConclusionsIn patients with stage IV EGFR-mutant lung adenocarcinoma, high E-cadherin expression in the lung tumor might be associated with worse OS. Vimentin expression in the lung tumor was positively related to the risk of brain metastasis.

Etoposide/platinum plus anlotinib for patients with transformed small-cell lung cancer from EGFR-mutant lung adenocarcinoma after EGFR-TKI resistance: a retrospective and observational study.

The histological conversion of lung adenocarcinoma (LUAD) into small-cell lung cancer (SCLC) is an important resistance mechanism for epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI)-resistant LUAD. Anlotinib has been recommended as the third-line treatment for SCLC patients. The efficacy of etoposide/platinum (EP) as the main treatment is very limited for patients with transformed SCLC. However, little is known about EP plus anlotinib for transformed SCLC. The present study retrospectively explored the clinical response to EP combined with anlotinib in patients with transformed SCLC from LUAD after EGFR-TKI failure. A total of 10 patients who underwent SCLC transformation from EGFR-TKI-resistant LUAD were retrospectively reviewed from September 1, 2019, to December 31, 2022, in three regional hospitals. All of the patients were treated with the combination regimen of EP and anlotinib for four to six cycles, followed by anlotinib maintenance therapy. The clinical efficacy indices including objective response rate (ORR), disease control rate (DCR), median progression-free survival (mPFS), median overall survival (mOS), and toxicities were evaluated. The median time from EGFR-TKI treatment to SCLC conversion was 20.1 ± 2.76 months (17-24 months). Genetic examination after transformation showed that 90% of the patients retained their original EGFR gene mutations. Additional driver genes were found, including BRAF mutation (10%), PIK3CA mutation (20%), RB1 loss (50%), and TP53 mutation (60%). The ORR and DCR were 80% and 100%, respectively. The mPFS was 9.0 months (95% CI, 7.9-10.1 months), and the mOS was 14.0 months (95% CI, 12.0-15.9 months). Less than 10% of grade 3 toxicities were observed, and no grade 4 toxicity and death events were reported. The EP plus anlotinib regimen appears to be a promising and safe strategy in transformed SCLC patients after EGFR-TKI resistance, which warrants further investigation.

Concurrent EGFR wild-type tongue squamous cell carcinoma and EGFR-mutant lung adenocarcinoma and response to osimertinib.

e18074 Background: Epidermal growth factor receptor (EGFR) mutation is most commonly oncogenic driver in lung adenocarcinoma with 50% incidence in Asians.Osimertinib is the third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), which has been widely used in metastatic EGFR-mutant non-small cell lung cancer (NSCLC) and has significantly improved outcomes. At present, the treatment of HNSCC mainly relies on surgery or chemoradiotherapy. EGFR is overexpressed in more than 90% of head and neck squamous cell carcinoma (HNSCC). Targeted therapy for HNSCC is mainly monoclonal antibodies, while the application of small molecule inhibitors is limited. The EGFR-TKI can only be applied in subsequent-line, when disease progression on or after platinum therapy. However, the efficacy of EGFR-TKI in patients with both NSCLC and HNSCC remains unclear. Methods: We searched all patients with EGFR-mutation metastatic non-small cell lung cancer and HNSCC who used EGFR-TKI in the Second Xiangya Hospital. Tumor sections were assessed immunohistochemically using PD-L1 and EGFR expression. EGFR amplification was detected by fluorescence in situ hybridization (FISH). We collected clinical characteristics and treatment histories for all patients including time to progression on EGFR-TKI. Time to progression on EGFR-TKI was defined as time from start of EGFR-TKI to time of radiographic RECIST progression. The efficacy and prognosis were evaluated based on the RECIST (version 1.1). Results: We found an advanced lung adenocarcinoma patient who harbored an EGFR-mutation and concurrently had tongue squamous cell carcinoma with EGFR wild-type.The tongue tumor tissue sample for EGFR expression was positive by immunohistochemistry and EGFR amplification was negative by FISH. NGS showed inactive mutation of patched1 (PTCH1) and EGFR wild-type.PET-CT scans demonstrated a partial response (PR) of the lung tumor and tongue cancer after two months of treatment of osimertinib. The lung tumor maintian partial response until now (nearly 30 months). And the tongue tumor reached the first progression- free survival (PFS) at 21 months. Conclusions: The patient with NSCLC and tongue cancer who exhibited objective response to EGFR-TKI, osimertinib monotherapy. The result indicates a clear response of tongue cancer to osimertinib and the potential for the use of osimertinib in tongue cancer. However, with the specific mechanism largely unknown, it is necessary to study further the effect of osimertinib on HNSCC.

Tumor-agnostic genomic and clinical analysis of solid tumors with BRAF fusions.

3146 Background: Approximately 15% of cancers harbor BRAF alterations, of which ~5% are BRAF fusions. BRAF fusions are class II alterations that have worse outcomes than class I V600E alterations. While prior investigational therapies for BRAF fusions had disappointing outcomes, novel therapies are in clinical trials, underscoring the need to better characterize these tumors. Methods: Data from adult and pediatric patients with BRAF fusion+ cancers identified between January 2014 and November 2022 utilizing a center-wide next generation sequencing (NGS) program of &gt;99,000 sequenced samples were analyzed. DNA-based hybrid capture tumor NGS (MSK-IMPACT), ctDNA targeted NGS (MSK-ACCESS), and/or RNA anchored multiplex PCR tumor NGS (MSK-Fusion) were used. Fusions were manually reviewed and considered to have oncogenic potential if they had an in-frame protein fusion involving a non-BRAF partner gene and an intact BRAF kinase domain (exons 11-18). Fusions not previously reported in OncoKB, COSMIC, TCGA, NIH gene, Fusion GDB2, and PubMED were classified as novel. All cases underwent clinical data curation including baseline demographic, tumor characteristics, and treatment histories. Results: 212 patients (0.2%) with BRAF-fusion positive solid tumors were identified. 194 were identified by DNA-based tumor NGS (96 had sufficient tissue for confirmatory RNA-based NGS, all of which were positive). Six were identified by ctDNA NGS and 12 by RNA-based tumor NGS only (5 had insufficient tissue for DNA-based tumor NGS). 83 unique 5’ fusion partners were found, of which 42 were novel. The most frequent tumor types were pilocytic astrocytoma (n=30, 14%), prostate CA (n=28, 13%), melanoma (n=24, 11%), lung CA (n=21, 10%), and colon CA (n=15, 7%). 44% of patients with pilocytic astrocytoma had BRAF fusions, of which 90% were BRAF-KIAA1549. Concomitant alterations (≥10% frequency) included TP53 (24%), TERT (18%), CDKN2A deletions (13%), and CDKN2B deletions (12%). 20% (n=43) were treated with MAPK-pathway directed therapies spanning multiple histologies and lines of therapies. Of the 212 patients, 17 had acquired BRAF fusions after targeted therapy for another oncogene ( EGFR mutation (n=11), BRAF V600E (n=4), FGFR fusion (n=1), NTRK fusion (n=1)). The majority of patients with acquired BRAF fusions had EGFR-mutant lung adenocarcinoma (n=11, 76%); the median time from EGFR targeted therapy initiation to BRAF fusion detection was 25 months (range 16-38 months). Conclusions: A wide variety of adult and pediatric solid tumors harbored de novo BRAF fusions. Complementary RNA sequencing optimized fusion identification in many cases. Multiple novel fusion partners were found. Acquired BRAF fusions were identified after targeted therapy for a variety of distinct oncogenes, the majority of which were EGFR mutations.

Revealing distinct immune landscape in EGFR mutant and EGFR wild type lung adenocarcinoma by using single-cell RNA sequencing.

e21008 Background: Non-small cell lung cancer (NSCLC) with EGFR mutations displays heterogeneity in the extent of intratumoral immunocytes infiltration compared to those with EGFR wild types, which may explain their variable responsiveness to PD-1 blockade. Here, we performed single-cell RNA sequencing (scRNA-seq) to unveil the immunescape and heterogeneity of lung adenocarcinoma (LUAD) between EGFR mutant (EGFR-mut) patients and wild type (EGFR-wt) patients. Methods: Eleven EGFR-mut and 7 EGFR-wt NSCLC tumor samples were analyzed. Immune cells (CD45+) were enriched through fluorescence-activated cell sorting (FACS) and subjected to single-cell RNA sequencing (scRNA-seq) using the 10X Genomics platform. Sequencing reads were normalized and analyzed using the R/Seurat package. Cellular components of each sample were determined based on known marker genes. Wilcox test was used to compare promotion by subtype composition and immune cell infiltrates. Results: Different immune cell compositions were observed between EGFR-mut and EGFR-wt LUAD tumor samples. We found EGFR-wt tumors are marked by an enrichment of CCL15+ tumor-associated macrophages(TAMs), which display pro-inflammatory properties and high expressions of TNF-a-signaling genes. In contrast, EGFR-mut tumors exhibit enrichment of SPARCL1+TAMs, which are involved in epithelial-mesenchymal transition (EMT) and angiogenesis. EGFR-wt tumors exhibited enrichment of CD8-Teff-MX1 and Gamma delta T cells, both of which displayed higher cytotoxic and naive features. Moreover, the high expression of Gamma delta T corresponds with a better clinical prognosis. In contrast, CD8-Teff-XCL1 and NKT-FGFBP2 were found enriched in EGFR-mut tumors, both of which contributed to higher exhaustion and decreased cytotoxic function. Additionally, NK-IL32 was found enriched in EGFR-mut tumors, showing a downregulated T cell receptor signaling and stronger exhausted features. The high expression of this subcluster correlated with poor outcomes in LUAD patients harboring EGFR mutation. Conclusions: Our study provided a deeper understanding of the immune landscape of EGFR-mut and EGFR-wt LUAD and gained better insights into the immunological mechanisms involved in the response to ICIs. The results of our study could be instructive for the development of future immunotherapy strategies for NSCLC patients harboring EGFR mutations.

Clinical trial versus standard of care in EGFR-wild type and ALK- negative lung adenocarcinoma: A propensity-matched analysis.

e21116 Background: Clinical trials have been a vital role for survival improvement in recurrent or metastatic lung cancer. Recent advances in targeted therapy for EGFR-mutant or ALK-positive lung adenocarcinoma dramatically increased survival. However, EGFR-wild type and ALK negative lung adenocarcinoma still had relatively poor survival outcome. Clinical trial of targeted therapy for rare driver mutation or immunotherapy was introduced in 2015. Moreover, next-generation sequencing was reimbursed in 2017 in Korea. This study evaluated the benefit of clinical trials for EGFR-wild type and ALK negative lung adenocarcinoma in recent 5 years. Methods: This study included patients with recurrent or metastatic lung adenocarcinoma who received systemic treatment between March 2017 and June 2022 at Samsung Medical Center. We compared overall survival in group with clinical trial and the standard of care (SOC) by using propensity score matching (PSM) (1:1). We used cox proportional hazard models to calculate the hazard ratio (HR) and 95% confidence interval while accounting for clustering among matched pairs. Results: Of total, 1,686 EGFR-wild type and ALK negative lung adenocarcinoma were included for final analysis. Among them, 1,380 received SOC only and 306 patients enrolled at least one clinical trial. In a group of clinical trial, 41.5%, 29.1%, and 23,2% of patients were enrolled for first-, second-, and third-line treatment, respectively. To achieve the balance between the SOC group and the clinical trial group, 8 baseline characteristics (age, sex, smoking history, ECOG performance status, brain metastasis, liver metastasis, bone metastasis, and rare driver mutation) were included. After PSM, 612 patients were matched to group of the SOC (n = 306) and the group of clinical trial (n = 306). Among the clinical trial, 27.8% was a clinical trial of targeted therapy and 72.2% was a clinical trial of immunotherapy or others. Median follow-up duration was 32.8 months (range, 5.0–64.4). Median overall survival was 17.1 months (95% CI, 13.2–21.4) for SOC and 27.3 months (95% CI, 22.1–32.4) for group of clinical trial (HR = 0.71 (95% CI, 0.58–0.88, P = 0.002). Conclusions: This study showed the performance of clinical trials for patients with EGFR-wild type and ALK-negative lung adenocarcinoma at tertiary hospital. After PSM, clinical trials showed survival benefits that reduced the risk of death by 29% in patients with EGFR-wild type and ALK-negative lung adenocarcinoma.

Differential expression analysis of circRUNX1 in patients with early resected EGFR-mutant NSCLC.

8529 Background: In the ADAURA study, patients with stage IB-IIIA of resected EGFR mutated lung adenocarcinoma receiving adjuvant osimertinib had a 2-year survival of 89% versus 52% in the placebo group (p&lt;0.001), however, predictive markers of response and recurrence are still needed. Circular RNA (circRNA) is a type of RNA which forms a covalently closed continuous loop. Some circRNAs have been reported as essential for tumor cell proliferation through functions distinct from the canonical linear RNA. In this project, we studied circRNA expression levels in formalin-fixed paraffin-embedded (FFPE) lung tumor samples in order to explore potential biomarkers that could predict the benefit of adjuvant EGFR TKIs in EGFR-mutant NSCLC patients. Methods: FFPE tumor samples from patients with EGFR-mutant, stage I-IIIB NSCLC (n=79) were collected. RNA was purified using the High Pure FFPET RNA Isolation Kit (Qiagen) and quantified by Nanodrop 2000 (Thermo Scientific). A total of 250 ng of RNA were subjected to overnight hybridization and downstream nCounter processing (NanoString) following manufacturer’s instructions for our circRNA custom panel. Differential expression analysis was performed using Excel (Microsoft) and Prism GraphPad (Dotmatics) software. Results: Analysis of the 79 samples included in the study shown a cluster of 3 circRNAs significantly upregulated (p&lt;0.05 and &gt;2 fold-change) in EGFR mutation-positive NSCLC relapsing withing the 36 months after resection, including circRUNX1, circFUT8 and circAASDH. Disease-free survival (DFS) was significantly shorter for the subgroup of patients with high versus low circRUNX1 expression. Additional multivariate analysis identified circRUNX1 expression and tumor stage as the only statically significant prognostic factors (circRUNX1 hazard ratio = 4.480, confidence interval = 2.172-9.239, P &lt; 0.001; pathological stage hazard ratio = 2.237, confidence interval = 1.183- 4.231, P = 0.008). Conclusions: CircRUNX1 could be a novel biomarker to predict the benefit of adjuvant EGFR TKIs with regards to DFS in EGFR-mutant resected NSCLC patients.

Implementation of a knowledge-based mechanistic model of EGFR-mutant lung adenocarcinoma that considers tumor heterogeneity and metastases to reproduce disease progression upon EGFR-TKI administration.

e21190 Background: 16,4% of lung adenocarcinomas (LUAD) are presenting a mutation in the epidermal growth factor receptor (EGFR), resulting in its constitutive activation and leading to uncontrolled cell proliferation [1]. Tyrosine kinase inhibitors (TKI) have been developed to inhibit EGFR activity but the presence of metastases and resistance mutations explain the lack of durable response to the treatment [2]. Knowledge-based mechanistic models reproducing existing clinical trials, based on population characteristics, can be used to help the design of future clinical trials. In particular, they can inform on the best responders to given treatments. Methods: We developed a physiologically based pharmacokinetic model of osimertinib, a 3rd generation TKI, to account for the distribution of the drug in the primary tumor and metastases after oral administration. This model was then combined to a pathophysiological mechanistic model of EGFR-mutant LUAD to represent the impact of osimertinib on the signals arising from EGFR activation. The combined model outputs the evolution of the primary tumor and each metastasis to allow the evaluation of the patient progression according to the RECIST criteria. Furthermore, each tumor in the model is composed of several subclones each possessing their own set of mutations and therefore responding differently to the treatment. Data from the clinical trials FLAURA and AURA3, in which osimertinib was given respectively as first and second line, were used to calibrate the model. Visual predictive checks as well as statistical tests were performed to ensure the proper behavior of the model. Results: The model successfully reproduced the time to progression in an EGFR mutant LUAD population treated with osimertinib as first line or as second line. In addition, the model reproduced the causes of progression according to the RECIST criteria and the sites of apparition of new metastases (in lung, brain, liver and bone). Conclusions: Reproduction of real world data brings credibility to the model. This is essential to use the model as an investigational tool to provide relevant insights, potentially on the best responders to osimertinib. After validation with additional clinical patient level data, the model could be used to create synthetic control arms in upcoming clinical trials. This would grant an improved analysis of covariate relationships with the comparison of an investigational treatment to the standard of care osimertinib administered as first or second line. It would also reduce the number of patients needed in the trial.

Routine Clinically Detected Increased ROS1 Transcripts Are Related With ROS1 Expression by Immunohistochemistry and Associated With EGFR Mutations in Lung Adenocarcinoma

IntroductionTranslocations of the ROS1 gene were found to drive tumorigenesis in 1% to 2% of lung adenocarcinoma. In clinical practice, ROS1 rearrangements are often screened by immunohistochemistry (IHC) before confirmation with either fluorescence in situ hybridization or molecular techniques. This screening test leads to a non-negligible number of cases that have equivocal or positive ROS1 IHC, without ROS1 translocation. MethodsIn this study, we have analyzed retrospectively 1021 cases of nonsquamous NSCLC having both ROS1 IHC and molecular analysis using next-generation sequencing. ResultsROS1 IHC was negative in 938 cases (91.9%), equivocal in 65 cases (6.4%), and positive in 18 cases (1.7%). Among these 83 equivocal or positive cases, only two were ROS1 rearranged, leading to a low predictive positive value of the IHC test (2%). ROS1-positive IHC was correlated with an increased mRNA ROS1 transcripts. Moreover, we have found a mean statistically significant relationship between ROS1 expression and EGFR gene mutations, suggesting a crosstalk mechanism between these oncogenic driver molecules. ConclusionThis study demonstrates that ROS1 IHC represents true ROS1 mRNA expression, and raises the question of a potential benefit of combined targeted therapy in EGFR-mutated NSCLC.

Diarylheptanoid 35d overcomes EGFR TKI resistance by inducing hsp70-mediated lysosomal degradation of EGFR in EGFR-mutant lung adenocarcinoma

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD) patients often respond to EGFR tyrosine kinase inhibitors (TKIs) initially but eventually develop resistance to TKIs. The switch of EGFR downstream signaling from TKI-sensitive to TKI-insensitive is a critical mechanism-driving resistance to TKIs. Identification of potential therapies to target EGFR effectively is a potential strategy to treat TKI-resistant LUADs. In this study, we developed a small molecule diarylheptanoid 35d, a curcumin derivative, that effectively suppressed EGFR protein expression, killed multiple TKI-resistant LUAD cells invitro, and suppressed tumor growth of EGFR-mutant LUAD xenografts with variant TKI-resistant mechanisms including EGFR C797S mutations invivo. Mechanically, 35d triggers heat shock protein 70-mediated lysosomal pathway through transcriptional activation of several components in the pathway, such as HSPA1B, to induce EGFR protein degradation. Interestingly, higher HSPA1B expression in LUAD tumors associated with longer survival of EGFR-mutant, TKI-treated patients, suggesting the role of HSPA1B on retarding TKI resistance and providing a rationale for combining 35d with EGFR TKIs. Our data showed that combination of 35d significantly inhibits tumor reprogression on osimertinib and prolongs mice survival. Overall, our results suggest 35d as a promising lead compound to suppress EGFR expression and provide important insights into the development of combination therapies for TKI-resistant LUADs, which could have translational potential for the treatment of this deadly disease.

Novel CDK12/13 Inhibitors AU-15506 and AU-16770 Are Potent Anti-Cancer Agents in EGFR Mutant Lung Adenocarcinoma with and without Osimertinib Resistance.

Osimertinib is a third-generation epidermal growth factor receptor and tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of lung adenocarcinoma patients harboring EGFR mutations. However, acquired resistance to this targeted therapy is inevitable, leading to disease relapse within a few years. Therefore, understanding the molecular mechanisms of osimertinib resistance and identifying novel targets to overcome such resistance are unmet needs of cancer patients. Here, we investigated the efficacy of two novel CDK12/13 inhibitors, AU-15506 and AU-16770, in osimertinib-resistant EGFR mutant lung adenocarcinoma cells in culture and xenograft models in vivo. We demonstrate that these drugs, either alone or in combination with osimertinib, are potent inhibitors of osimertinib-resistant as well as -sensitive lung adenocarcinoma cells in culture. Interestingly, only the CDK12/13 inhibitor in combination with osimertinib, although not as monotherapy, suppresses the growth of resistant tumors in xenograft models in vivo. Taken together, the results of this study suggest that inhibition of CDK12/13 in combination with osimertinib has the potential to overcome osimertinib resistance in EGFR mutant lung adenocarcinoma patients.