Vascular growth patterns correlate with histological subtypes in human lung adenocarcinoma.

MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras(G12D)-induced tumor formation in the Men1(f/f);K-Ras(G12D/+);Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.

SummaryLung cancer is an extremely heterogeneous disease, and its treatment remains one of the most challenging tasks in medicine. Few existing laboratory lung cancer models can faithfully recapitulate the diversity of the disease and predict therapy response. Here, we establish 12 patient-derived organoids from the most common lung cancer subtype, lung adenocarcinoma (LADC). Extensive gene and histopathology profiling show that the tumor organoids retain the histological architectures, genomic landscapes, and gene expression profiles of their parental tumors. Patient-derived lung cancer organoids are amenable for biomarker identification and high-throughput drug screening in vitro. This study should enable the generation of patient-derived lung cancer organoid lines, which can be used to further the understanding of lung cancer pathophysiology and to assess drug response in personalized medicine.

Is Lung Cancer in the Nonsmoker a Different Disease?

Recombinant human erythropoietins (rHuEPOs) are used to treat cancer-related anemia. Recent preclinical studies and clinical trials, however, have raised concerns about the potential tumor-promoting effects of these drugs. Because the clinical significance of erythropoietin receptor (EPOR) signaling in human non-small cell lung cancer (NSCLC) also remains controversial, our aim was to study whether EPO treatment modifies tumor growth and if EPOR expression has an impact on the clinical behavior of this malignancy. A total of 43 patients with stage III–IV adenocarcinoma (ADC) and complete clinicopathological data were included. EPOR expression in human ADC samples and cell lines was measured by quantitative real-time polymerase chain reaction. Effects of exogenous rHuEPOα were studied on human lung ADC cell lines in vitro. In vivo growth of human ADC xenografts treated with rHuEPOα with or without chemotherapy was also assessed. In vivo tumor and endothelial cell (EC) proliferation was determined by 5-bromo-2’-deoxy-uridine (BrdU) incorporation and immunofluorescent labeling. Although EPOR mRNA was expressed in all of the three investigated ADC cell lines, rHuEPOα treatment (either alone or in combination with gemcitabine) did not alter ADC cell proliferation in vitro. However, rHuEPOα significantly decreased tumor cell proliferation and growth of human H1975 lung ADC xenografts. At the same time, rHuEPOα treatment of H1975 tumors resulted in accelerated tumor endothelial cell proliferation. Moreover, in patients with advanced stage lung ADC, high intratumoral EPOR mRNA levels were associated with significantly increased overall survival. This study reveals high EPOR level as a potential novel positive prognostic marker in human lung ADC.

Aberrant Stratifin Overexpression Is Regulated by Tumor-Associated CpG Demethylation in Lung Adenocarcinoma

Introduction to 2021 WHO Classification of Thoracic Tumors

Background: Lung cancer is a leading cause of death. Despite advancements in early diagnosis and treatment, most patients are diagnosed at later stages, and the average 5-year survival rate is &amp;lt; 30%. Lung adenocarcinoma (LUAD) is the most common non-small cell lung cancer (NSCLC), representing about 40% of cases, followed by lung squamous cell carcinoma (LUSC) at 25%. The biological patterns and molecular characteristics of LUAD and LUSC exhibit differences. Changes in DNA methylation levels of various genes have been observed in lung cancer subtypes, yet research on differences in DNA methylation patterns between LUAD and LUSC is limited. Methods: The methylation microarray dataset (GSE39279, Illumina HumanMethylation450 BeadChip) from Gene Expression Omnibus at the National Center for Biotechnology Information was used. The dataset comprised 444 NSCLC samples derived from tumor tissues, of which 322 were LUAD and 122 were LUSC. We used the Champ (Chip Analysis Methylation Pipeline) package to identify differentially methylated probes (DMPs, Benjamini-Hochberg adjusted p-value &amp;lt; 0.05) and genes representing specific biological pathways between LUAD and LUSC. Singular Value Decomposition regression analysis was used to estimate the impact of age, sex, and smoking status and then adjusted for the covariates with p-values &amp;lt; 0.05. We used the eFORGE TF database to calculate the Find Individual Motif Occurrences (FIMO) p-values for the overlap with transcription factor binding sites. The Gene Expression Profiling Interactive Analysis database was used to measure the gene expression levels of LUAD and LUSC samples in the Genotype-Tissue Expression and the Cancer Genome Atlas Program portals. Results: In total, 223,007 DMPs were identified by comparing LUAD and LUAC, with 130,610 hypomethylated and 92,467 hypermethylated. Using the absolute difference of β values between groups ≥ 0.3, we identified 15 DMPs, 11 hypomethylated and four hypermethylated. The DMPs on promoter regions were all hypomethylated in LUSC compared to LUAD (cg00415665 and cg22997040 in ZHX2, cg27649037 in ST18, cg20691436 in CALML3, and cg24580076 in C7orf20). We also identified DMPs based on the importance scores (&amp;gt; 0.1) from a 5-fold cross-validation random forest analysis. The two DMPs in the ZHX2 gene had the highest importance scores (&amp;gt; 8.0). Among those, cg00415665 was covered by the Zfp161_secondary motif (FIMO p-value: &amp;lt; 10-5). The average expression level of ZHX2 in LUSC (transcripts per million, TPM=9.7) was higher than in LUAD (TPM=8.2). Conclusions: We characterized the methylation landscape of NSCLCs by histological subtypes and identified that the methylation pattern of cg00415665 in ZHX2, a tumor suppressor gene, was significantly associated with the histological subtype of NSCLC. Further investigation of these findings will provide additional insight into the biology and genetics of LUAD and LUSC. Citation Format: Hyeyeun Lim, Christopher I. Amos, Jinyoung Byun, Aaron P. Aaron. Comparing the methylation profiles between lung adenocarcinoma and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2106.

P3.01-022 Impact of Histologic Subtype and Spread through Air Spaces (STAS) in Stage III (N2) Lung Adenocarcinoma: Topic: Morphology

Lung adenocarcinoma is classified morphologically into five histological subtypes according to the WHO classification. While each histological subtype correlates with a distinct prognosis, the molecular basis has not been fully elucidated. Here we conducted DNA methylation analysis of 30 lung adenocarcinoma cases annotated with the predominant histological subtypes and three normal lung cases using the Infinium BeadChip. Unsupervised hierarchical clustering analysis revealed three subgroups with different methylation levels: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME). Micropapillary pattern (MPP)-predominant cases and those with MPP components were significantly enriched in HME (p = 0.02 and p = 0.03, respectively). HME cases showed a significantly poor prognosis for recurrence-free survival (p < 0.001) and overall survival (p = 0.006). We identified 365 HME marker genes specifically hypermethylated in HME cases with enrichment of "cell morphogenesis" related genes; 305 IME marker genes hypermethylated in HME and IME, but not in LME, with enrichment "embryonic organ morphogenesis"-related genes; 257 Common marker genes hypermethylated commonly in all cancer cases, with enrichment of "regionalization"-related genes. We extracted surrogate markers for each epigenotype and designed pyrosequencing primers for five HME markers (TCERG1L, CXCL12, FAM181B, HOXA11, GAD2), three IME markers (TBX18, ZNF154, NWD2) and three Common markers (SCT, GJD2, BARHL2). DNA methylation profiling using Infinium data was validated by pyrosequencing, and HME cases defined by pyrosequencing results also showed the worse recurrence-free survival. In conclusion, lung adenocarcinomas are stratified into subtypes with distinct DNA methylation levels, and the high-methylation subtype correlated with MPP-predominant cases and those with MPP components and showed a poor prognosis.

BackgroundLung adenocarcinoma, the most common type of lung cancer, has a high level of morphologic heterogeneity and is composed of tumor cells of multiple histological subtypes. It has been reported that immune cell infiltration significantly impacts clinical outcomes of patients with lung adenocarcinoma. However, it is unclear whether histologic subtyping can reflect the tumor immune microenvironment, and whether histologic subtyping can be applied for therapeutic stratification of the current standard of care.MethodsWe inferred immune cell infiltration levels using a histological subtype-specific gene expression dataset. From differential gene expression analysis between different histological subtypes, we developed two gene signatures to computationally determine the relative abundance of lepidic and solid components (denoted as the L-score and S-score, respectively) in lung adenocarcinoma samples. These signatures enabled us to investigate the relationship between histological composition and clinical outcomes in lung adenocarcinoma using previously published datasets.ResultsWe found dramatic immunological differences among histological subtypes. Differential gene expression analysis showed that the lepidic and solid subtypes could be differentiated based on their gene expression patterns while the other subtypes shared similar gene expression patterns. Our results indicated that higher L-scores were associated with prolonged survival, and higher S-scores were associated with shortened survival. L-scores and S-scores were also correlated with global genomic features such as tumor mutation burdens and driver genomic events. Interestingly, we observed significantly decreased L-scores and increased S-scores in lung adenocarcinoma samples with EGFR gene amplification but not in samples with EGFR gene mutations. In lung cancer cell lines, we observed significant correlations between L-scores and cell sensitivity to a number of targeted drugs including EGFR inhibitors. Moreover, lung cancer patients with higher L-scores were more likely to benefit from immune checkpoint blockade therapy.ConclusionsOur findings provided further insights into evaluating histology composition in lung adenocarcinoma. The established signatures reflected that lepidic and solid subtypes in lung adenocarcinoma would be associated with prognosis, genomic features, and responses to targeted therapy and immunotherapy. The signatures therefore suggested potential clinical translation in predicting patient survival and treatment responses. In addition, our framework can be applied to other types of cancer with heterogeneous histological subtypes.

The determination of biomarkers in the blood specific for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) is crucial for the selection of effective treatment strategies and the prediction of prognosis. The purpose of the present study was to analyze the differentially expressed genes (DEGs) in LUSC and LUAD from The Cancer Genome Atlas (TCGA) database. In order to identify the potential biomarkers for non-small cell lung cancer (NSCLC) for clinical diagnosis, bioinformatics was used to analyze the DEGs of two subtypes of NSCLC, LUAD and LUSC. Exosomes were isolated from the serum of patients with LUAD or LUSC and identified using transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. A total of four differential exosomal mRNAs were selected for validation with serum samples from 70 patients with NSCLC via reverse transcription-quantitative polymerase chain reaction. Receiver operating characteristic curves were established to evaluate the clinical diagnostic value of four DEGs for patients with LUAD and LUSC. The analysis based on TCGA data revealed the DEGs in LUSC and LUAD: A total of 1,619 genes were differentially expressed in patients with LUSC and LUAD. DEGs analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that inflammation-related signaling pathways, such as complement pathways, and multiple autoimmune diseases, such as systemic lupus erythematosus and asthma were mainly enriched in LUAD. The cell cycle, Hippo signaling pathway, Rap1 signaling pathway and Wnt signaling pathway were the main signaling pathways enriched in LUSC. The combination of tumor protein P63 (TP63), keratin 5 (KRT5), CEA cell adhesion molecule 6 (CEACAM6) and surfactant protein B (SFTPB) improved the specificity and sensitivity in the diagnosis of different lung cancer subtypes. Exosomal TP63, KRT5, CEACAM6 and SFTPB mRNAs can thus be used as biomarkers to differentiate between LUSC and LUAD, and may provide a novel strategy for their differential diagnosis and treatment.

Lung cancer has the highest cancer-related mortality worldwide. Lung adenocarcinoma (LUAD) is the most common histological subtype of non-small cell lung cancer (NSCLC). Chromatin licensing and DNA replication factor 1 (CDT1), a key regulator of cell cycle control and replication in eukaryotic cells, has been implicated in various cancer-related processes. Given its significant role in cancer, the focus on CDT1 in this study is justified as it holds promise as a potential biomarker or therapeutic target for cancer treatment. However, its prognostic value in lung adenocarcinoma (LUAD) remains unclear. Bioinformatics analysis was conducted using data obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were utilized to predict biological processes and signaling pathways, respectively. The LinkedOmics database was employed to identify differentially expressed genes (DEGs) associated with CDT1. Nomograms and Kaplan-Meier plots were generated to assess the survival rates of patients with lung adenocarcinoma (LUAD). To determine the RNA and protein expression levels of CDT1 in LUAD and adjacent normal tissues, quantitative polymerase chain reaction (qPCR) and immunohistochemistry techniques were employed, respectively. CDT1 was upregulated in the vast majority of cancer tissues, based on pan-cancer analysis in TCGA and GEO datasets, as to lung cancer, the level of CDT1 expression was much higher in LUAD tissue than in healthy lung tissue. Our clinical data supported these findings. In our study, we used a specific cutoff value to dichotomize the patient samples into high and low CDT1 expression groups. The Kaplan-Meier survival curve revealed poor survival rates in CDT1 high expression group than the low expression group. To determine if CDT1 expression was an independent risk factor in LUAD patients, univariate and multivariate Cox regression analyses were performed. The result showed that CDT1 was a potential novel prognosis factor for LUAD patients, whose prognosis was poorer when CDT1 expression was higher. Based on functional enrichment analysis, highly expressed DEGs of CDT1-high patients were predicted to be involved in the cell cycle. According to our analysis of immune infiltration, CDT1 exhibited a strong correlation with specific immune cell subsets and was found to be a significant predictor of poor survival in patients with LUAD. Our research found that CDT1 was upregulated in LUAD and that high CDT1 expression predicted poor prognosis. We comprehensively and systematically analyzed the expression level in the datasets as well as in our own clinical samples, we also evaluated the prognostic and diagnostic value of CDT1, and finally, the potential mechanisms of CDT1 in the progression of LUAD. These results suggested that CDT1 may be a prognostic marker and therapeutic target for LUAD.

The histologic heterogeneity of lung adenocarcinoma is well known. Many histologic subtypes have been described, and recently their prognostic and predictive value has emerged. Laser capture microdissection may aid in the isolation of cancer cells from distinct subtypes of lung adenocarcinoma, thus enabling the description of their specific molecular features. Characterization of epidermal growth factor receptor (EGFR) mutations in histologic subtypes of lung adenocarcinoma has become an important issue. The purpose of this study was to analyze EGFR mutations in exons 18-21 in single histologic subtypes of lung adenocarcinoma after laser capture microdissection. A revision and reclassification of a series of 208 non-small cell lung cancers was conducted, and 62 adenocarcinomas with a total of 119 histologic component subtypes were identified. Laser capture microdissection of each subtype was performed. EGFR mutations in exons 18-21 were detected using polymerase chain reaction single-strand conformation polymorphism and direct DNA sequencing. EGFR mutations were detected only in 3 out of the 62 adenocarcinomas analyzed. Two adenocarcinomas harbored EGFR mutations in exon 19 (the E746-T751 deletion VA insertion and the LREAT deletion) and one adenocarcinoma the EGFR exon 21 L858R missense point mutation. EGFR mutations were observed in all component subtypes. This suggests that, in a patient with lung adenocarcinoma, EGFR mutations are not associated with particular component histologic subtypes and probably occur at an early stage of tumorigenesis. Notably, 2 out of the 3 mutated adenocarcinomas had a bronchioloalveolar component, whereas the third mutated adenocarcinoma had a papillary subtype. Although we detected EGFR mutations only in 3 out of 62 adenocarcinomas and EGFR mutations were present in every subtype of each mutated adenocarcinoma, our research might represent a basis for further studies in characterizing molecular profiles of different component subtypes of lung adenocarcinoma.

Molecular biomarkers are reshaping patient stratification and treatment decisions, yet their precise use and best implementation remain uncertain. Intratumor heterogeneity (ITH), an area of increasing research interest with prognostic value across various conditions, lacks defined clinical relevance in certain non-small cell lung cancer (NSCLC) subtypes. Exploring the relationship between ITH and tumor mutational burden (TMB) is crucial, as their interplay might reveal distinct patient subgroups. This study evaluates how the ITH-TMB dynamic affects prognosis across the two main histological subtypes of NSCLC, squamous cell and adenocarcinoma, with a specific focus on early-stage cases to address their highly unmet clinical needs. We stratify a cohort of 741 early-stage NSCLC patients from The Cancer Genome Atlas (TCGA) based on ITH and TMB and evaluate differences in clinical outcomes. Additionally, we compare driver mutations and the tumor microenvironment (TME) between high and low ITH groups. In lung squamous cell carcinoma (LUSC), high ITH predicts an extended progression-free survival (PFS) (median: 21 vs. 14 months, P=0.01), while in lung adenocarcinoma (LUAD), high ITH predicts a reduced PFS (median: 15 vs. 20 months, P=0.04). This relationship is driven by the low TMB subset of patients. Additionally, we found that CD8 T cells were enriched in better-performing subgroups, regardless of histologic subtype or ITH status. There are significant differences in clinical outcomes, driver mutations, and the TME between high and low ITH groups among early-stage NSCLC patients. These differences may have treatment implications, necessitating further validation in other NSCLC datasets.

Introduction: Although histological subtype in lung adenocarcinoma has been reported as a poor prognostic factor in several studies, its utility has not yet been revealed as an adaptation criterion of postoperative adjuvant chemotherapy. Methods: Four hundred ninety-four lung adenocarcinoma patients were enrolled in this retrospective study. A subanalysis was performed in 420 lung adenocarcinoma patients with pathological stage 0–I disease for risk factors of postoperative recurrence. Results: Maximum standardized uptake value (SUV_max) (p < 0.01), pathological stage ≥II (p < 0.04), and adjuvant chemotherapy (p < 0.01) were risk factors for recurrence in the multivariate analysis, whereas histological subtype was not a significant factor for recurrence at all stages. In the subanalysis, univariate analysis showed that carcinoembryonic antigen expression (p < 0.01), prognostic nutrition index (p = 0.03), SUV_max (p < 0.01), lymphatic invasion (p < 0.01), vascular invasion (p < 0.01), grade 3–4 differentiation (p < 0.01), pathological stage ≥IA3 (p < 0.01), and histological subtype (p = 0.03) were significant risk factors of recurrence. SUV_max (p < 0.01) was the only risk factor for recurrence in the multivariate analysis, whereas histological subtype was not (p = 0.07). Relapse-free survival (RFS) was significantly worse in the micropapillary- and solid-predominant subtype groups than in the other subtypes (p = 0.01). On the other hand, RFS with or without uracil-tegafur as adjuvant chemotherapy in lung micropapillary- or solid-predominant adenocarcinoma patients with pathological stage IA-IB disease was not significantly different. Conclusion: This study suggested that histological subtypes, such as micropapillary- or solid-predominant pattern, are risk factors for recurrence in pathological stage 0-I lung adenocarcinoma and may be necessary adjuvant chemotherapy instead of uracil-tegafur.