Subset Of Lung Adenocarcinomas Research Articles

Abstract 1746: KRAS mutant lung adenocarcinoma is associated with distinct mutational signature profiles

Abstract Background: Lung cancer is the 3rd most common cancer in the UK and leading cause of cancer mortality worldwide. Somatic RAS mutations are the most common oncogenes in human cancer, and KRAS mutations are the largest molecular subset of lung adenocarcinoma (LUAD). Each cancer genome accumulates a unique combination of somatic mutations, and these mutational signatures provide a genetic imprint of mutational processes that have occurred during tumorigenesis. Mutational signatures provide information on tumor aetiology/maintenance and could highlight potential therapeutic targets. This is the largest study to date to profile KRAS mutant LUAD mutational signatures using whole genome sequencing (WGS) data. Methods: Paired tumor and germline WGS data were obtained through the 100 000 Genomes Project (Genomics England). KRAS mutations were profiled using Ensembl Variant Effect Predictor (VEP) v109.0. Single base substitution (SBS), doublet base substitution (DBS), small insertion and deletions (ID) and copy number (CN) mutational signature extraction was performed using SigProfiler (COSMIC version 3.3). Statistical analysis and plotting were performed in R; a two-tailed p-value <0.05 was considered statistically significant. Results: 680 LUAD patients were included; 301 KRAS mutant (44%), 379 KRAS wild type (63%). Common KRAS variants were present, including G12C (38.9%), G12V (15.9%) and G12D (14.0%). 17 SBS, 9 DBS, 10 ID and 9 CN signatures were extracted and present in both KRAS mutant and wild type cohorts. KRAS mutant samples were enriched in smoking related signatures (SBS4, DBS2, ID3; odds (OR) ratio 5.54, 3.80, 4.97 respectively, p <0.001) and diploid CN signature (CN1, OR 1.41, p = 0.03). Conversely, KRAS mutant samples were less likely to have clocklike (ID5, OR 0.61, p = 0.012; ID8, OR 0.44, p <0.001), APOBEC (SBS2, OR 0.49, p <0.001; SBS13; 0.50, p <0.001), chromosomal instability (CN9, OR 0.63, p = 0.019), loss of heterozygosity (CN14, OR 0.47, p = 0.015), or homologous recombination deficiency (CN17, OR 0.58, p <0.001) signatures. Discussion: These results show distinct mutational signature profiles between KRAS mutant and wild type LUAD. As previously described, KRAS mutant LUAD is dominated by smoking related signatures; however, the majority of remaining signatures were more common in the KRAS wild type cohort where there was a statistically significant difference. This data suggests different mutational processes are observed in KRAS mutant compared to wild type LUAD. KRAS mutations are commonly clonal and thought to initiate tumorigenesis: timing signatures relative to KRAS mutations is necessary to further our understanding of KRAS cancer evolution and the subject of future work. Combined with clinical data integration, mutational signatures could provide prognostic information and inform predictive models. Citation Format: Laura Woodhouse, Aliah Hawari, Andreas Gruber, Colin Lindsay, David Wedge. KRAS mutant lung adenocarcinoma is associated with distinct mutational signature profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1746.

Lung adenocarcinoma patients with ROS1-rearranged tumors by sex and smoking intensity

BackgroundROS1 rearrangements (ROS1+) define a distinct molecular subset of lung adenocarcinomas. ROS1 + tumors are known to occur more in never-smokers, but the frequency and outcome of ROS1 positivity by sex and smoking intensity are not clearly documented. Patients and methodsThis patient cohort study included all never- (<100 cigarettes lifetime) and light- (100 cigarettes-20 pack-years) smokers, and a sample of heavy-smokers. ROS1 + rates by sex and smoking intensity were compared within and beyond our study. Survival outcomes were analyzed using Kaplan-Meier curves and Cox proportional hazards models. ResultsOf the 571 total patients, ROS1 + was detected in 24 (4.2%): 6.4% in men and 3.0% in women; 5.1% in never-, 5.7% in light-, and 1.8% in heavy-smokers (P=0.05). Among the 209 stage IIIB-IV patients, men had much higher ROS1 + rate (11.1%) not only than women (1.7%, P=0.004) in our study, but also than men (0.4%–1.8%) in 8 published studies (Ps = 0.0019–0.0001). ROS1+ rates were similar between never- (9.3%) and light-smokers (8.1%) and significantly lower in heavy-smokers (1.2%, P=0.017), a finding confirmed by 6 published studies (Ps = 0.041–0.0001). Overall survival of ROS1 + patients were significantly better than the ROS1- (P=0.023) mainly due to targeted therapy. Among patients who exhibited resistance to crizotinib, follow-up treatment of entrectinib and lorlatinib showed remarkable survival benefits. ConclusionsThe ROS1 + rates were higher in men than in women, and similar in never- and light-smokers, more pronounced in stage IIIB-IV patients. Newer-generation ALK/ROS1-targeted drugs showed efficacy in a cohort of crizotinib resistant ROS1 + patients. These results, when validated, could assist efficiently accruing ROS1 + patients.

Clinicopathological features of SMARCA4-deficient lung adenocarcinoma: a study of 42 cases

Objective: To investigate the clinicopathological characteristics and genetic mutations of SMARCA4-deficient lung adenocarcinoma. Methods: From January 2021 to April 2023 in the First Affiliated Hospital of Zhengzhou University, 42 cases of SMARCA4-deficienct lung adenocarcinoma were diagnosed and now analyzed. All cases were retrospectively studied using hematoxylin-eosin staining and immunohistochemistry. The clinicopathological features were reviewed. Next-generation sequencing (NGS) was performed to investigate the mutations of related genes. Results: Among the 42 cases, there were 35 biopsy and 7 surgical specimens. There were 38 males and 4 females. The male to female ratio was 9.5∶1.0, with an age range from 42 to 78 years. Thirty-three patients were smokers. Overall, 4 cases (9.5%), 2 cases (4.7%), 18 cases (42.9%) and 18 cases (42.9%) were at stages Ⅰ, Ⅱ, Ⅲ, and Ⅳ, respectively. Microscopically, all the cases were non-mucinous adenocarcinoma, without lepidic pattern. The morphology was diverse. Rhabdomyoid cells, tumor giant cells and tumor necrosis were present. Most of the tumor cells had eosinophilic cytoplasm and occasionally clear cytoplasm. Defined cell borders and variable cytoplasmic hyaline secretory globules could be found. Inflammatory cells infiltrated the tumor stroma. Immunohistochemistry showed 29 cases (69.0%, 29/42) expressed TTF1, 10 cases (40.0%, 10/25) expressed Napsin A, and 20 cases (100.0%, 20/20) expressed INI1. Forty cases (95.2%, 40/42) showed BRG1 loss in all tumor cells, while 2 cases (4.8%, 2/42) had partial BRG1 loss. PD-L1 (22C3) was positive in 59.2% of the cases (16/27). NGS revealed mutations in EGFR, ROS1, MET, RET and KRAS. Six cases (6/8) showed SMARCA4 mutation, while some cases were accompanied by mutations of TP53 (7/15), STK11 (4/8), and KEAP1 (1/8). Driver gene mutations were more common in women (P<0.05). Patients were followed up for 1-25 months. Four patients died and 20 patients' diseases progressed. Conclusions: SMARCA4-deficient lung adenocarcinoma lacks characteristic morphology. Most of them express TTF1 and harbor driver gene mutations. It is necessary to identify this subset of lung adenocarcinoma by carrying out BRG1 stain routinely on lung adenocarcinoma. These patients can then be identified and benefit from targeted therapies.

Abstract A036: A subset of lung adenocarcinomas defined by high-level ERBB2 amplification is vulnerable to HER2-targeted therapy

Abstract Background: Most lung adenocarcinomas (LUAD) are driven by activation of receptor tyrosine kinases (RTK). HER2 (encoded by ERBB2) is an RTK capable of ligand-independent dimerization and signaling when overexpressed. ERBB2 amplification (ERBB2amp) is a common cause of HER2 overexpression. While activating ERBB2 mutations (e.g., ERBB2 exon 20 YVMA insertions) are targetable drivers of LUAD, the role of ERBB2amp in lung cancer (LC) is unclear and there is no FDA-approved therapy for this indication in LC. Here, we report the clinicopathological and molecular characteristics of LUAD with high-level ERBB2amp and investigate the efficacy of HER2-targeted agents in LC models with ERBB2amp. We also demonstrate the clinical benefit of trastuzumab-deruxtecan (T-DXd) in a patient with ERBB2amp LUAD. Design: An institutional database of hybrid capture DNA NGS panel targeting 290-447 genes (v1-v3.1) was interrogated for LUAD with high-level ERBB2amp (defined as at least 6 calculated gene copies by NGS). HER2 immunohistochemistry (IHC) was performed on 29 available cases and scored according to ASCO/CAP scoring criteria. HER2 IHC scores of 2+ and 3+ were considered overexpression. Two LC cell lines, H2170 and CALU3 were identified as ERBB2amp and their responses to HER2 inhibitors were characterized with Western blotting, viability, and apoptosis assays. CALU3 and H2170 xenograft models were also established and treated with HER2 inhibitors or T-DXd. Results: ERBB2amp was identified in 31/3,684 (0.8%) LUAD patients. HER2 overexpression was present in 24 out of 29 evaluable cases (79.3%). Eighteen cases (58%) showed high-level ERBB2amp with no concurrent drivers and were more likely to be male (P = 0.001) and have a history of tobacco use (P = 0.001), compared to patients with high-level ERBB2amp with a concurrent driver mutation (activating ERBB2 SNV/indels, activating EGFR and MET mutations, RET fusion). H2170 and CALU3 cells displayed overexpression of HER2 and phospho-HER2, and growth of these cells showed sub-nanomolar sensitivity to trastuzumab and T-DXd, and low nanomolar sensitivity to afatinib. T-DXd induced apoptosis in both cell lines (assessed by activation of caspase 3/7 and elevation of BIM and cleaved PARP levels). While trastuzumab and afatinib significantly slowed down growth of H2170 and CALU3 xenografts, only T-DXd achieved tumor shrinkage equivalent to partial (H2170) or complete response (CALU3) in all animals. We describe the case of a LUAD patient with a RET rearrangement who acquired high-level ERBB2amp after progression on selpercatinib and was treated with T-DXd, with evidence of disease stability and clinical benefit. Conclusion: High-level ERBB2 amplification detected by NGS reliably predicts HER2 overexpression in lung adenocarcinoma. LUAD with ERBB2 amplification and no concurrent oncogenic drivers is a rare event that occurs predominantly in male smokers. This data can inform future trials investigating the utility of HER2-targeting agents in this population. Citation Format: Igor Odintsov, Maisam Makarem, Sara E. Bachert, Tom Zhang, Romel Somwar, Pasi A. Jänne, Lynette M. Sholl. A subset of lung adenocarcinomas defined by high-level ERBB2 amplification is vulnerable to HER2-targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A036.

MLK4 promotes glucose metabolism in lung adenocarcinoma through CREB-mediated activation of phosphoenolpyruvate carboxykinase and is regulated by KLF5

MLK4, a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, has been implicated in cancer progression. However, its role in lung adenocarcinoma has not been characterized. Here, we showed that MLK4 was overexpressed in a significant subset of lung adenocarcinoma, associated with a worse prognosis, and exerted an oncogenic function in vitro and in vivo. Bioinformatics analyses of clinical datasets identified phosphoenolpyruvate carboxykinase 1 (PCK1) as a novel target of MLK4. We validated that MLK4 regulated PCK1 expression at transcriptional level, by phosphorylating the transcription factor CREB, which in turn mediated PCK1 expression. We further demonstrated that PCK1 is an oncogenic factor in lung adenocarcinoma. Given the importance of PCK1 in the regulation of cellular metabolism, we next deciphered the metabolic effects of MLK4. Metabolic and mass spectrometry analyses showed that MLK4 knockdown led to significant reduction of glycolysis and decreased levels of glycolytic pathway metabolites including phosphoenolpyruvate and lactate. Finally, the promoter analysis of MLK4 unravelled a binding site of transcription factor KLF5, which in turn, positively regulated MLK4 expression in lung adenocarcinoma. In summary, we have revealed a KLF5-MLK4-PCK1 signalling pathway involved in lung tumorigenesis and established an unusual link between MAP3K signalling and cancer metabolism.

Expression of Paired box 9 defines an aggressive subset of lung adenocarcinoma preferentially occurring in smokers.

A distinct subset of lung adenocarcinomas (LADs), arising from a series of peripheral lung cells defined as the terminal respiratory unit (TRU), is characterized by thyroid transcription factor 1 (TTF-1) expression. The clinical relevance of transcription factors (TFs) other than TTF-1 remains unknown in LAD and was explored in the present study. Seventy-one LAD samples were subjected to high-throughput transcriptome screening of LAD using cap analysis gene expression (CAGE) sequencing data; CAGE provides genome-wide expression levels of the transcription start sites (TSSs). In total, 1083 invasive LAD samples were subjected to immunohistochemical examination for paired box 9 (PAX9) and TTF-1 expression levels. PAX9 is an endoderm development-associated TF that most strongly and inversely correlates with the expression of TTF-1 TSS subsets. Immunohistochemically, PAX9 expression was restricted to the nuclei of ciliated epithelial and basal cells in the bronchi and bronchioles and the nuclei of epithelial cells of the bronchial glands; moreover, PAX9 expression was observed in 304 LADs (28%). PAX9-positive LADs were significantly associated with heavy smoking, non-lepidic subtype, EGFR wild-type tumors, and PD-L1 expression (all p < 0.0001). All these characteristics were opposite to those of TRU-type LADs with TTF-1 expression. PAX9 expression was an independent prognostic factor for decreased overall survival (p = 0.022). Our results revealed that PAX9 expression defines an aggressive subset of LADs preferentially occurring in smokers that may arise from bronchial or bronchiolar cells.

Protein kinase Cι mediates immunosuppression in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most prevalent form of non-small cell lung cancer (NSCLC) and a leading cause of cancer death. Immune checkpoint inhibitors (ICIs) of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signaling induce tumor regressions in a subset of LUAD, but many LUAD tumors exhibit resistance to ICI therapy. Here, we identified Prkci as a major determinant of response to ICI in a syngeneic mouse model of oncogenic mutant Kras/Trp53 loss (KP)-driven LUAD. Protein kinase Cι (PKCι)-dependent KP tumors exhibited resistance to anti-PD-1 antibody therapy (α-PD-1), whereas KP tumors in which Prkci was genetically deleted (KPI tumors) were highly responsive. Prkci-dependent resistance to α-PD-1 was characterized by enhanced infiltration of myeloid-derived suppressor cells (MDSCs) and decreased infiltration of CD8+ T cells in response to α-PD-1. Mechanistically, Prkci regulated YAP1-dependent expression of Cxcl5, which served to attract MDSCs to KP tumors. The PKCι inhibitor auranofin inhibited KP tumor growth and sensitized these tumors to α-PD-1, whereas expression of either Prkci or its downstream effector Cxcl5 in KPI tumors induced intratumoral infiltration of MDSCs and resistance to α-PD-1. PRKCI expression in tumors of patients with LUAD correlated with genomic signatures indicative of high YAP1-mediated transcription, elevated MDSC infiltration and low CD8+ T cell infiltration, and with elevated CXCL5/6 expression. Last, PKCι-YAP1 signaling was a biomarker associated with poor response to ICI in patients with LUAD. Our data indicate that immunosuppressive PKCι-YAP1-CXCL5 signaling is a key determinant of response to ICI, and pharmacologic inhibition of PKCι may improve therapeutic response to ICI in patients with LUAD.

Co-Mutation of FAT3 and LRP1B in Lung Adenocarcinoma Defines a Unique Subset Correlated With the Efficacy of Immunotherapy.

Immunotherapy based on immune checkpoint inhibitors (ICIs) have demonstrated remarkable survival benefits and gained regulatory approval in non-small cell lung cancer (NSCLC) patients without an actionable driver mutation, but currently there is no well-established standard for how to screen the most suitable population for ICIs treatment. Here, we conducted a comprehensive analysis of the somatic mutation landscape of lung adenocarcinoma (LUAD) samples. After the stepwise screening of high-frequency mutated genes, two genes with prominent significance, FAT3 and LRP1B, were finally screened out. Through further analysis, we discovered that the co-mutation of FAT3 and LRP1B was associated with an earlier age of onset and occurred more frequently in Black/African American. Furthermore, co-mutation defines a unique subgroup of lung adenocarcinoma that can increase tumor mutational burden (TMB), boost cytotoxicity and tumor immunogenicity, and facilitate lymphocyte infiltration. The results of gene set enrichment analysis (GSEA) indicated that co-mutation can influence tumorigenesis through a variety of mechanisms. More strikingly, the subset of LUAD with co-mutation of FAT3 and LRP1B exhibited significantly prolonged immunotherapy progression free survival (PFS). In summary, co-mutation of FAT3 and LRP1B is a promising useful biomarker for predicting the efficacy of immunotherapy, which can improve the clinical efficiency of practicing precision medicine in lung adenocarcinoma patients.

ROS1 rearrangements in lung adenocarcinomas are defined by diffuse strong immunohistochemical expression of ROS1

A small subset of lung adenocarcinomas harbour ROS1 gene arrangements and are amenable to tyrosine kinase inhibitor therapy. Current practice in Australia involves screening for ROS1 rearrangements in adenocarcinomas using ROS1 immunohistochemistry (IHC) followed by confirmatory molecular testing such as fluorescence in situ hybridisation (FISH), if other known genetic driver alterations are absent. The best threshold to determine ROS1 IHC positivity is not well defined, however, and this study aims to determine the optimal threshold for ROS1 IHC screening to identify ROS1-rearranged lung adenocarcinomas. A total of 177 lung adenocarcinomas tested for a ROS1 rearrangement by FISH at our institution between 2017 and 2020 due to presence of ROS1 IHC staining were included in the study. ROS1 IHC staining was assessed by scoring the staining intensity (0, 1, 2, or 3+) and multiplying by the percentage of positive cells to generate an H-score. IHC H-scores were compared with FISH. Of 177 cases, 32 (18%) were ROS1 FISH-positive and 145 (82%) were negative. FISH-positive cases had a median H-score of 300 (range 200-300; mean 290.3) and negative cases had a median H-score of 40 (range 0-300; mean 63). All FISH-positive cases showed strong and diffuse IHC positivity. Using a threshold H-score of 200, the sensitivity of identifying ROS1 rearrangements was 100% and the specificity was 95% amongst cases referred with ROS1 IHC positivity. Adenocarcinomas with a FISH-confirmed ROS1 rearrangement demonstrate diffuse, strong (2-3+) IHC staining. Cases with weak, patchy ROS1 IHC staining are not associated with ROS1 rearrangements and in these cases FISH testing is of little to no utility.

Aggressive early-stage lung adenocarcinoma is characterized by epithelial cell plasticity with acquirement of stem-like traits and immune evasion phenotype.

Lung adenocarcinoma (LUAD) is the main non-small-cell lung cancer diagnosed in ~40-50% of all lung cancer cases. Despite the improvements in early detection and personalized medicine, even a sizable fraction of patients with early-stage LUAD would experience disease relapses and adverse prognosis. Previous reports indicated the existence of LUAD molecular subtypes characterized by specific gene expression and mutational profiles, and correlating with prognosis. However, the biological and molecular features of such subtypes have not been further explored. Consequently, the mechanisms driving the emergence of aggressive LUAD remained unclear. Here, we adopted a multi-tiered approach ranging from molecular to functional characterization of LUAD and used it on multiple cohorts of patients (for a total of 1227 patients) and LUAD cell lines. We investigated the tumor transcriptome and the mutational and immune gene expression profiles, and we used LUAD cell lines for cancer cell phenotypic screening. We found that loss of lung cell lineage and gain of stem cell-like characteristics, along with mutator and immune evasion phenotypes, explain the aggressive behavior of a specific subset of lung adenocarcinoma that we called C1-LUAD, including early-stage disease. This subset can be identified using a 10-gene prognostic signature. Poor prognosis patients appear to have this specific molecular lung adenocarcinoma subtype which is characterized by peculiar molecular and biological features. Our data support the hypothesis that transformed lung stem/progenitor cells and/or reprogrammed epithelial cells with CSC characteristics are hallmarks of this aggressive disease. Such discoveries suggest alternative, more aggressive, therapeutic strategies for early-stage C1-LUAD.

P76.33 Concurrent EGFR and KRAS Mutations in Lung Adenocarcinoma: A Single Institution Case Series

Lung adenocarcinomas with molecular mutations in both the EGFR and KRAS genes are a distinct group of patients. EGFR mutant lung cancers most commonly occur in nonsmokers, respond well to tyrosine kinase inhibitor (TKI) therapy, and have a higher relative overall survival rate. In contrast, KRAS mutant lung cancers occur more commonly in patients with smoking history, do not respond to TKI therapy, and have a lower overall relative survival rate. Historically, EGFR and KRAS mutations were thought to be mutually exclusive however in this case series we present multiple cases of lung adenocarcinoma with both EGFR and KRAS mutations. From a single institution database of patients with non-small cell lung cancer (NSCLC) from 2010 – 2019, cases with concurrent EGFR and KRAS mutations were extracted. Clinical characteristics of these patient cases were reviewed, including sex, age, smoking history, stage, treatment. In addition, pathologic features were evaluated including mutations and histologic features. Follow up data including treatment was recorded. From this database 92 out of 1025 (9%) patients had EGFR mutations and 4 out of the 92 EGFR mutated cancers had both KRAS and EGFR mutations (4%). All 4 patients were between ages 55 and 67, and 3 out of 4 (75%) patients were male. 2 out of 4 (50%) patients presented with metastatic disease, while the remainder presented with localized disease. All 4 patients had KRAS mutations on codon 12 (c.34_35delinsAA and p.G12N, c.35G>A and p.G12D, p.G12S, c.34G>A and p.G12S), and 2 of these had additional mutations on exon 13 (c.38_39delinsCT and p.G13A, and p.G13D). Two patients had EGFR exon 21 mutation (c.2573T>G, p.L858R, and c.2573T>G, p.L858R), one patient had exon 19 mutation (c.2240_2254del, p.L747_T751del), and one patient had exon 18 mutation (c.2180A>G, p.Y727C). Two patients had an additional MET amplification. One patient (25%) had PDL-1 expression >1%. One of the 2 patients with localized disease had lobectomy, while the other patient underwent radiation therapy. Both patients are alive and have no recurrence of disease. One of the 2 patients with metastatic disease underwent resection of brain metastases and SBRT, while the other underwent conservative management for bone metastases. Due to patient preference, neither patient received chemotherapy or immunotherapy. One patient died and the other remains in hospice care. Patients with concomitant EGFR and KRAS mutations may be a distinct subset of lung adenocarcinoma. EGFR and KRAS should no longer be considered mutually exclusive mutations. Clinical questions remain on the efficacy of treating concomitant EGFR and KRAS mutations with TKI therapy. Further studies are necessary to determine the most effective therapeutic strategies to address this unique subset of lung cancer patients. KRAS inhibitors are currently under study.

Genomic Evolution in a Patient With Lung Adenocarcinoma With a Germline EGFR T790M Mutation

IntroductionA subset of lung adenocarcinomas (ADs) has been found to have somatic activating mutations in the tyrosine kinase domain of the EGFR gene, associated with response to EGFR tyrosine kinase inhibitor therapy. Rare germline mutations within this domain, including EGFR T790M, have been associated with genetic susceptibility to lung ADs. Using high-throughput sequencing, we elucidate the genomic evolution in tissues from a patient with lung AD carrying a germline EGFR T790M mutation. MethodsWe performed microdissection, targeted panel, and whole-exome sequencing to molecularly characterize multiple foci of atypical adenomatous hyperplasia (AAH), in situ and invasive components of AD, normal lung tissue, and whole blood from the patient. Normal lung tissue was analyzed for potential acquired somatic genome alterations (“field effect”). ResultsAll lesions harbored a secondary somatic EGFR mutation, either L858R or L861Q, in addition to the germline T790M mutation. Clear overlap was observed between the somatic profiles of in situ and invasive AD components, confirming clonal relatedness. AAH lesions shared few to no somatic alterations with the AD, suggesting clonal independence. No robust evidence of field effect was identified in the normal lung tissue. ConclusionsSomatic EGFR mutations are early events in the pathogenesis of lung ADs arising in the context of germline EGFR T790M. Synchronous AAH lesions seem to be independent. Stepwise genomic evolution is observed in association with invasiveness of the neoplastic cell population.

Abstract PO-118: Novel mechanisms of EGFR-TKI resistance and clonal evolution of lung adenocarcinoma by overexpression of a cell adhesion molecule, CADM1

Abstract Evolution of cancer and generation of its heterogeneity are most dynamically demonstrated in the process of cancer cells acquiring resistance to molecular targeted therapy. Lung adenocarcinoma is the best solid tumor to which molecular targeted therapy is established and clinically available. However, tumors resistant against targeted drugs develop frequently, providing the biggest challenge in current treatment of lung adenocarcinoma. The tumors resistant to the initial treatment also show more malignant features, such as invasion and metastasis. Thus, it is interesting to investigate possible mechanisms to link EGFR-TKI resistance to malignant progression in lung adenocarcinoma. A cell adhesion molecule, CADM1, is originally identified as a tumor suppressor in lung adenocarcinoma. CADM1 is involved in the formation and maintenance of an epithelial cell structure. On the other hand, CADM1 is frequently inactivated by promoter methylation in various cancers in their advanced stages. In lung adenocarcinoma, CADM1 was expressed in the cell-cell attachment sites in lepidic growth components, whereas CADM1 expression was frequently lost in invasive lesions, conferring heterogenous features to cancer tissue. We have also reported that CADM1 inhibits HGF-induced epithelial mesenchymal transition of canine kidney cells, MDCK, by associating with MET and intervening with MET signaling on the cell membrane. Here, we demonstrate significant expression of CADM1 in a lung adenocarcinoma cell, HCC827, but not in its gefitinib-resistant derivatives, HCC827GR5 or GR6, which express significant amounts of phosphorylated MET (p-MET). Introducing CADM1 expression into HCC827GR5 restores the sensitivity to gefitinib in association with decreasing amount of pMET. In contrast, CADM1 is not effective at restoring gefitinib sensitivity in PC9 cells carrying T790M mutation of EGFR. Dox-inducible CADM1 expression in HCC827GR5 also showed restoration of gefitinib sensitivity both in vivo and in vitro. Finally, intra-tumoral injection of adenoviruses expressing CADM1, but not control adenoviruses, significantly suppressed subcutaneous tumor formation only when treated with gefitinib to nude mice. These results suggest that exogenous expression of CADM1 could provide a unique approach to overcome EGFR-TKI resistance of lung adenocarcinoma caused by MET overexpression. Inversely, loss of CADM1 and cancer cell adhesion could promote EGFR-TKI resistance caused by MET overexpression. Considering that CADM1 expression is lost selectively and clonally in microinvasive lesions of lung adenocarcinoma, disruption of cancer cell adhesion would be involved in generation of cell clones resistant to molecular targeted drugs and resultant heterogeneity in a subset of lung adenocarcinoma. Citation Format: Takehiro Tsuchiya, Hideki Kuwano, Takeshi Ito, Masayoshi Nagata, Taketo Kawai, Yumi Tsuboi, Daisuke Matsubara, Isami Okamoto, Kenji Tamura, Jun Nakajima, Motoi Oba, Yoshinori Murakami. Novel mechanisms of EGFR-TKI resistance and clonal evolution of lung adenocarcinoma by overexpression of a cell adhesion molecule, CADM1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-118.

Allele-Specific Role of ERBB2 in the Oncogenic Function of EGFR L861Q in EGFR-Mutant Lung Cancers.

Unlike common EGFR mutations, many less common EGFR mutations remain poorly characterized in terms of oncogenic function and drug sensitivity. Here, we characterize the subset of lung adenocarcinoma harboring EGFR L861Q through both preclinical and clinical investigations. We reviewed clinical and genomic data from patients with EGFR-mutant lung cancer. We established cells expressing EGFR mutations and performed functional analysis of L861Q in comparison with common EGFR mutations. Among the patients with lung cancer, 3.4% (47 of 1367) possess an EGFR L861Q mutation. Of the patients with L861Q, 23.4% (11 of 47) had a concurrent exon 18 mutation (typically involving G719). Invitro studies revealed that the oncogenic activity of L861Q is dependent on asymmetric dimerization. Cells expressing L861Q were less sensitive to EGFR-specific inhibitors compared with cells expressing L858R but were similarly sensitive to pan-ERBB inhibitors. In cells expressing L861Q, ERBB2 phosphorylation was markedly higher compared with cells expressing L858R, and an enhanced interaction between EGFR and ERBB2 was observed in coimmunoprecipitation studies. In addition, treatment with osimertinib enhanced expression of the antiapoptotic protein MCL1, and knockdown of ERBB2 suppressed the expression of MCL1 in L861Q, raising the possibility of differential allele-specific cross-phosphorylation of ERBB2. Moreover, compared with EGFR-specific inhibitors, pan-ERBB inhibitors exerted superior growth inhibitory effects on cells expressing compound L861Q/G719X mutations. Our results suggest that ERBB2 plays a previously unrecognized role in EGFR L861Q-driven tumorigenesis, and pan-ERBB inhibitors are likely to be more effective than selective EGFR tyrosine kinase inhibitors in this setting.

Epigenetic Scanning of KEAP1 CpG Sites Uncovers New Molecular-Driven Patterns in Lung Adeno and Squamous Cell Carcinomas.

Background: The KEAP1/NRF2 (Kelch-like ECH-associated protein 1/nuclear factor erythroid 2–related factor 2) pathway modulates detoxification processes and participates in the resistance of solid tumors to therapy. Scientific evidence about the presence of genetic and epigenetic abnormalities of the KEAP1 gene was firstly reported in non-small-cell lung cancer (NSCLC) and then described in other tumors. At present, the prognostic role of aberrant methylation at cytosine-guanine dinucleotide (CpG) sites of the KEAP1 gene promoter is debated in NSCLC, and its correlation with transcriptional changes and protein levels remains to be defined in large sample cohorts. Methods: We evaluated and compared multiple KEAP1 omics data (methylation, transcript, and protein expression levels) from The Cancer Genome Atlas (TCGA) to explore the role of CpGs located in different portions of KEAP1 and the correlation between methylation, transcription, and protein levels. Data from two subsets of lung adenocarcinoma (LUAD, n = 617) and lung squamous cell carcinoma (LUSC, n = 571) cohorts of NSCLC patients with different disease stages were evaluated. Results: We found that the methylation levels of many KEAP1 CpGs at various promoter and intragenic locations showed a significant inverse correlation with the transcript levels. Interestingly, these results were limited to the KRAS wild-type LUSC and LUAD cohorts, whereas in LUAD the effect of the epigenetic silencing of KEAP1 on its transcription was also observed in the EGFR mutated subpopulation. Conclusions: These results support the idea that the prognostic role of KEAP1 CpG sites warrants more in-depth investigation and that the impact of their changes in methylation levels may differ among specific NSCLC histologies and molecular backgrounds. Moreover, the observed impact of epigenetic silencing on KEAP1 expression in specific KRAS and EGFR settings may suggest a potential role of KEAP1 methylation as a predictive marker for NSCLC patients for whom anti-EGFR treatments are considered.

TENET 2.0: Identification of key transcriptional regulators and enhancers in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related death and lung adenocarcinoma is its most common subtype. Although genetic alterations have been identified as drivers in subsets of lung adenocarcinoma, they do not fully explain tumor development. Epigenetic alterations have been implicated in the pathogenesis of tumors. To identify epigenetic alterations driving lung adenocarcinoma, we used an improved version of the Tracing Enhancer Networks using Epigenetic Traits method (TENET 2.0) in primary normal lung and lung adenocarcinoma cells. We found over 32,000 enhancers that appear differentially activated between normal lung and lung adenocarcinoma. Among the identified transcriptional regulators inactivated in lung adenocarcinoma vs. normal lung, NKX2-1 was linked to a large number of silenced enhancers. Among the activated transcriptional regulators identified, CENPA, FOXM1, and MYBL2 were linked to numerous cancer-specific enhancers. High expression of CENPA, FOXM1, and MYBL2 is particularly observed in a subgroup of lung adenocarcinomas and is associated with poor patient survival. Notably, CENPA, FOXM1, and MYBL2 are also key regulators of cancer-specific enhancers in breast adenocarcinoma of the basal subtype, but they are associated with distinct sets of activated enhancers. We identified individual lung adenocarcinoma enhancers linked to CENPA, FOXM1, or MYBL2 that were associated with poor patient survival. Knockdown experiments of FOXM1 and MYBL2 suggest that these factors regulate genes involved in controlling cell cycle progression and cell division. For example, we found that expression of TK1, a potential target gene of a MYBL2-linked enhancer, is associated with poor patient survival. Identification and characterization of key transcriptional regulators and associated enhancers in lung adenocarcinoma provides important insights into the deregulation of lung adenocarcinoma epigenomes, highlighting novel potential targets for clinical intervention.

HER2 Exon 20 Insertion Mutations in Lung Adenocarcinoma: Case Series and Response to Pyrotinib.

HER2 mutations have emerged as oncogenic driver gene mutations in non-small cell lung cancer (NSCLC), which have not been described in detail like other driver gene mutations. Here, 295 patients with advanced lung adenocarcinoma were retrospectively screened for HER2 mutations using next-generation sequencing (NGS), and the positive cases were validated by Sanger sequencing. We identified five cases with HER2 exon 20 insertions, representing 1.7% of 295 lung adenocarcinomas. Among them, four different subtypes of HER2 exon 20 insertions were identified, including a rare subtype G778_S779insCPG never reported before with a partial response (PR) to pyrotinib and progression-free survival (PFS) of 12.8 months. Our findings reveal that HER2 exon 20 insertion mutations were detected in a small subset of lung adenocarcinomas. Given the different drug sensitivities, determining the mutation subtype by next-generation sequencing at the time of diagnosis might make sense.

Vulnerability of drug-resistant EML4-ALK rearranged lung cancer to transcriptional inhibition.

A subset of lung adenocarcinomas is driven by the EML4‐ALK translocation. Even though ALK inhibitors in the clinic lead to excellent initial responses, acquired resistance to these inhibitors due to on‐target mutations or parallel pathway alterations is a major clinical challenge. Exploring these mechanisms of resistance, we found that EML4‐ALK cells parental or resistant to crizotinib, ceritinib or alectinib are remarkably sensitive to inhibition of CDK7/12 with THZ1 and CDK9 with alvocidib or dinaciclib. These compounds robustly induce apoptosis through transcriptional inhibition and downregulation of anti‐apoptotic genes. Importantly, alvocidib reduced tumour progression in xenograft mouse models. In summary, our study takes advantage of the transcriptional addiction hypothesis to propose a new treatment strategy for a subset of patients with acquired resistance to first‐, second‐ and third‐generation ALK inhibitors.

ASCL1 promotes tumor progression through cell-autonomous signaling and immune modulation in a subset of lung adenocarcinoma.

The master regulator of neuroendocrine differentiation, achaete-scute complex homolog 1 (ASCL1) defines a subgroup of lung adenocarcinoma. However, the mechanistic role of ASCL1 in lung tumorigenesis and its relation to the immune microenvironment is principally unknown. Here, the immune landscape of ASCL1-positive lung adenocarcinomas was characterized by immunohistochemistry. Furthermore, ASCL1 was transduced in mouse lung adenocarcinoma cell lines and comparative RNA-sequencing and secretome analyses were performed. The effects of ASCL1 on tumorigenesis were explored in an orthotopic syngeneic transplantation model. ASCL1-positive lung adenocarcinomas revealed lower infiltration of CD8+, CD4+, CD20+, and FOXP3+ lymphocytes and CD163+ macrophages indicating an immune desert phenotype. Ectopic ASCL1 upregulated cyclin transcript levels, stimulated cell proliferation, and enhanced tumor growth in mice. ASCL1 suppressed secretion of chemokines, including CCL20, CXCL2, CXCL10, and CXCL16, indicating effects on immune cell trafficking. In accordance with lower lymphocytes infiltration, ASCL1-positive lung adenocarcinomas demonstrated lower abundance of CXCR3-and CCR6-expressing cells. In conclusion, ASCL1 mediates its tumor-promoting effect not only through cell-autonomous signaling but also by modulating chemokine production and immune responses. These findings suggest that ASCL1-positive tumors represent a clinically relevant lung cancer entity.

P2.03-26 Elucidating Mechanisms of Resistance to Targeted Therapies in Mutant EGFR or KRAS Driven Lung Adenocarcinoma Harboring Dual Loss of p53 and RB1

Inactivation of the two canonical tumor suppressors, p53 and RB1, is a genetic hallmark of small-cell lung cancer (SCLC). In contrast, lung adenocarcinomas (LUADs) preferentially harbor alterations in the p16 pathway over RB1. Nonetheless, despite being rare, concurrent loss of p53 and RB1 occurs in a subset of LUADs and this is hypothesized to be necessary for the histological transformation of LUAD to SCLC, observed during treatment with tyrosine kinase inhibitors (TKIs). However, whether the dual loss of p53 and RB1 is sufficient for this histological transformation remains unknown. Furthermore, loss of RB1 in LUADs with EGFR mutations is associated with poor response to TKIs in the absence of SCLC transformation. Here, we aimed to explore how loss of p53/RB1 affects the biology of p16 pathway-altered LUAD, particularly in the context of acquired resistance mechanisms to targeted therapies. Four TP53-mutated LUAD cell lines were used: two EGFR mutation-positive (PC9 and H1975) and two KRAS mutation-positive (H1792 and H358). All these cell lines possess p16 pathway alterations: p16 (CDKN2A) mutations in PC9 and H1975, CDK4 amplification in H1792, and silenced p16 in H358. Inactivation of RB1 was carried out using CRISPR-Cas9 and RB1 knockout monoclonal cells were established. Cell proliferative and clonogenic abilities were assessed. In addition, osimertinib-resistant (PC9 and H1975) and trametinib-resistant (H1792 and H358) cells were generated (initial high dose and/or stepwise dose escalation methods). Acquired resistance mechanisms were evaluated by MSK-IMPACT profiling. Two RB1 knockout clones were established for each cell line. No advantageous effects were observed for proliferative and clonogenic abilities after RB1 knockout. Although loss of p53 and RB1 has been reported to result in lineage shift in prostate cancer through the upregulation of SOX2, deregulation of SOX2 expression was not observed upon RB1 knockout in the TP53-mutant LUAD cells. In addition, although loss of RB1 caused a modest reduction in osimertinib and trametinib sensitivities of H1975 and H358 cells, respectively, no effect was observed in PC9 and H1792 cells. After becoming resistant to osimertinib or trametinib, transformation to SCLC was not observed. Individual resistance mechanisms are currently being assessed by MSK-IMPACT. Dual loss of p53 and RB1 caused no advantageous effects in TP53-mutated and p16 pathway-altered LUAD cells and did not initiate transformation to SCLC as a resistance mechanism to targeted therapies. TP53/RB1 deficiency-related acquired resistance mechanisms to osimertinib or trametinib will be further explored and presented.