Abstract A037: Differences in BRCAness/PARP inhibitor response signatures and homologous recombination gene expression across lung adenocarcinoma and squamous cell carcinoma gene expression subtypes

Lung Adenocarcinoma and Squamous Cell Carcinoma Gene Expression Subtypes Demonstrate Significant Differences in Tumor Immune Landscape

Non-small cell lung cancer (NSCLC) remains the most frequent malignancy worldwide, and lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) represent two major subtypes. LINC00628 has been demonstrated to promote LUAD progression; however, its clinical role in NSCLC remains elusive. The aim of the present study was to analyze the expression of long intergenic non-protein coding RNA 628 (LINC00628) in NSCLC, including in the LUAD and LUSC subtypes. In addition, its roles in NSCLC development and prognosis were also examined. Data from The Cancer Genome Atlas (TCGA) database were first used to assess the expression and prognostic potential in both LUAD and LUSC, then LINC00628 expression in 128 NSCLC tissues was measured using reverse transcription-quantitative PCR. A receiver operating characteristic curve was used to assess the ability of LINC00628 to discriminate between patients with LUAD and LUSC. Kaplan-Meier curves were used to analyze the relationship between LINC00628 expression and the overall survival of patients. Cox regression analysis was used to explore the potential prognostic factors that might be independently associated with NSCLC overall survival. Both in silico and tissue analysis data indicated that the expression of LINC00628 was significantly upregulated in NSCLC tissue compared with matched normal controls (P<0.001). LINC00628 expression levels were also significantly higher in LUAD cases than in patients with LUSC (P<0.001). In addition, LINC00628 could discriminate LUAD from LUSC cases. The expression of LINC00628 was significantly associated with tumor size (P=0.013), histological type (P=0.009), lymph node metastasis (P=0.021) and TNM stage (P=0.008). Survival analysis based on data from both TCGA and patients included in the present study identified an association between LINC00628 and overall survival in LUAD, but this relationship was not observed in LUSC for TCGA data. Cox regression analysis demonstrated that high LINC00628 expression was associated with poor overall survival in patients with LUAD (P=0.001), but not in patients with LUSC (P=0.088). In conclusion, LINC00628 expression was upregulated in NSCLC and associated with patient prognosis. Patients with LUAD had higher LINC00628 expression levels than those with LUSC, and increased LINC00628 served as an independent prognostic factor in LUAD, but not LUSC.

Classification of tumors into subtypes can inform personalized approaches to treatment including the choice of targeted therapies. The two most common lung cancer histological subtypes, lung adenocarcinoma and lung squamous cell carcinoma, have been previously divided into transcriptional subtypes using microarray data, and corresponding signatures were subsequently used to classify RNA-seq data. Cross-platform unsupervised classification facilitates the identification of robust transcriptional subtypes by combining vast amounts of publicly available microarray and RNA-seq data. However, cross-platform classification is challenging because of intrinsic differences in data generated using the two gene expression profiling technologies. In this report, we show that robust gene expression subtypes can be identified in integrated data representing over 3500 normal and tumor lung samples profiled using two widely used platforms, Affymetrix HG-U133 Plus 2.0 Array and Illumina HiSeq RNA sequencing. We tested and analyzed consensus clustering for 384 combinations of data processing methods. The agreement between subtypes identified in single-platform and cross-platform normalized data was then evaluated using a variety of statistics. Results show that unsupervised learning can be achieved with combined microarray and RNA-seq data using selected preprocessing, cross-platform normalization, and unsupervised feature selection methods. Our analysis confirmed three lung adenocarcinoma transcriptional subtypes, but only two consistent subtypes in squamous cell carcinoma, as opposed to four subtypes previously identified. Further analysis showed that tumor subtypes were associated with distinct patterns of genomic alterations in genes coding for therapeutic targets. Importantly, by integrating quantitative proteomics data, we were able to identify tumor subtype biomarkers that effectively classify samples on the basis of both gene and protein expression. This study provides the basis for further integrative data analysis across gene and protein expression profiling platforms.

Hyaluronidase, hyaluronan synthase, E-cadherin and TGF-Β profile in lung adenocarcinoma subtypes and squamous cell carcinoma of smokers/nonsmokers

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.

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.

3077Background: Gene expression based subtyping in lung Adenocarcinoma (AD) and lung Squamous Cell Carcinoma (SQ) classifies AD and SQ tumors into distinct subtypes with variable biologic and clini...

BackgroundNon-small cell lung carcinoma (NSCLC) mainly includes lung squamous cell carcinoma and adenocarcinoma. This study aimed to investigate the difference between the expression of Cbl-b in lung squamous cell carcinoma and adenocarcinoma.Material/MethodsThe clinical features and survival data of NSCLC patients and Cbl-b mRNA (FPKM) were obtained from the TCGA database. Then, lung squamous cell carcinoma and adenocarcinoma cell lines were transfected with lentivirus-mediated RNA interference vector to knockdown the expression of Cbl-b. Next, a Transwell assay was performed to study the effect of Cbl-b shRNA on migration and invasion of lung squamous cell carcinoma and adenocarcinoma cells. Finally, Western blot analysis was performed to measure the expressions of PI3K, p-PI3K, AKT, p-AKT, ERK1/2, p-ERK1/2, GSK3β, p-GSK3β, mTOR, and p-mTOR protein in lung adenocarcinoma and squamous cell carcinoma cells.ResultsThe correlation of Cbl-b expression and OS was different between NSCLC adenocarcinoma and squamous carcinoma. After transfection, the expression of Cbl-b was inhibited in A549, H1975, and SW900 cells. Cbl-b shRNA promoted the migration and invasion of lung adenocarcinoma A549 and H1975 cells, but it inhibited the invasion of lung squamous cell carcinoma SW900 cells. In addition, Cbl-b regulated the expression of PI3K and ERK1/2-GSK3β pathway proteins in A549 and SW900 cells.ConclusionsThe OS of Cbl-b mRNA low expression in lung adenocarcinoma and squamous cell carcinoma was different. The difference in signal pathways may be one of the reasons for the difference in the correlation between Cbl-b expression and the survival rate of these 2 pathological types of lung cancer.

Mesothelin is a 40 kDa secreted glycoprotein expressed in normal mesothelial cells and over-expressed in several histological types of tumors. Detection of mesothelin by immunohistochemistry (IHC) may assist in the diagnosis of mesothelioma. Mesotheliomas are positive for mesothelin staining, but carcinomas of the lung may also be positive for this marker. Mesothelin positivity in adenocarcinoma of the lung reportedly ranges from 22% to 71% of cases depending upon the specific subtype. Mesothelin positivity in squamous cell carcinomas (SCC) of lung is reported to be 16% in non-keratinizing and 31% in keratinizing subtypes. We performed mesothelin IHC on a cohort of 50 lung carcinoma samples (16 adenocarcinomas and 34 SCC). Mesothelin expression was observed in 10 out of 16 (62.5%) lung adenocarcinoma samples, of which 8 showed greater than 10% of tumor cells with positive membrane staining of any intensity. Mesothelin expression was observed in 19 out of 34 (55.9%) lung SCC samples, of which only 3 samples showed greater than 10% of tumor cells with positive membrane staining of any intensity. These findings are in concordance with previous reports which show a higher prevalence of mesothelin protein expression in lung adenocarcinoma than in lung SCC. To correlate RNA expression with protein expression, we performed gene expression profiling on a subcohort of these lung cancer specimens. Out of 50 lung carcinoma samples, 28 samples (10 adenocarcinomas and 18 SCCs) provided adequate RNA yield for gene expression profiling for mesothelin. A total mean relative gene expression (mRGE) value of 13.64 with a standard deviation (SD) of ±3.71 was obtained for the adenocarcinoma samples and a mRGE value of 14.78 with a SD of 2.64 was obtained for the SCC samples. We next arbitrarily assigned samples with greater than 10% mesothelin stained cells as “positive” and the remainder as “negative”. Six negative adenocarcinoma samples yielded a mRGE value of 12.98 with a SD of ±3.84, and four positive adenocarcinoma samples yielded a mRGE value of 14.63 with SD of ±3.82. Sixteen negative SCC samples resulted in a mRGE 14.63 with SD of ± 2.75 and two positive SCC samples yielded a mRGE of 15.98 with a SD of ±1.37. There was no apparent correlation between mRGE values and IHC positivity. We also correlated gene expression with p53 mutation status. Sixteen wild type samples, composed of equal number of adenocarcinoma and SSC had a total mRGE of 14.29 with SD of ±3.32. Seven samples with p53 mutations had a total mRGE of 16.09 with SD ±1.93. These data indicate that mesothelin gene expression is not associated with p53 mutation status. Future studies with an increased number of samples may yield significant associations between protein expression, gene expression and mutation status. Citation Format: Jackson Wong, Dana Gaffney, Michael Sharp, Brenda Hertzog, Jayaprakash Karkera, Suso Platero, John Alvarez. Profiling mesothelin protein expression by immunohistochemistry and gene expression in adenocarcinoma and squamous cell carcinoma of lung. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4666. doi:10.1158/1538-7445.AM2014-4666

Transcriptome-based molecular subtyping of non–small cell lung cancer may predict response to immune checkpoint inhibitors

To analyze the differentially expressed genes (DEGs) between lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) with bioinformatics analysis and search for potential biomarkers for clinical diagnosis of nonsmall cell lung cancer (NSCLC). The gene expression profiling datasets of LUAD and LUSC were acquired. The transcriptome differences between LUAD and LUSC were identified using R language processing and t-test analysis. The differential expressions of the genes were shown by Venn diagram. The DEGs identified by GEO2R were analyzed with DAVID and Ingenuity Pathway Analysis (IPA) to identify the signaling pathways and biomarkers that could be used for differential diagnosis of LUAD and LUSC. The TCGA data and the biomarker expression data from clinical lung cancer samples were used to verify the differential expressions of the Osteoarthritis pathway and LXR/RXR between LUAD and LUSC. We further examined the differential expressions of miR-181 and its two target genes, WNT5A and MBD2, in 23 clinical specimens of lung squamous cell carcinoma and the paired adjacent tissues. GEO data analysis identified 851 DEGs (including 276 up-regulated and 575 down-regulated genes) in LUAD and 885 DEGs (including 406 up-regulated and 479 down-regulated genes) in LUSC. DAVID and IPA analysis revealed that leukocyte migration and inflammatory responses were more abundant in LUAD than in LUSC. Osteoarthritis pathway was inhibited in LUAD and activated in LUSC. IPA analysis showed that transcription factors (GATA4, RELA, YBX1, TP63 and MBD2), cytokines (WNT5A and IL1A) and microRNAs (miR-34a, miR-181b and miR-15a) differed significantly between LUAD and LUSC. miR-34a with IL-1A, miR-15a with YBX1, and miR-181b with WNT5A and MBD2 could serve as the paired microRNA and mRNA targets for differential diagnosis of NSCLC subtypes. Analysis of the clinical samples showed an increased expression of miR-181b-5p and the down-regulation of WNT5A, which could be used as molecular markers for the diagnosis of LUSC. Through transcriptome analysis, we identified candidate genes, paired microRNAs and pathways for differentiating LUAD and LUSC, and they can provide novel differential diagnosis and therapeutic strategies for LUAD and LUSC.

Based on 4D-CT, we aimed to characterize the pattern of morphological changes in lung tumors during respiration, and investigated its potential in non-invasively differentiating lung adenocarcinoma (AC) and squamous cell carcinoma (SCC).We applied a 3D surface analysis on 22 tumors (13 AC, 9 SCC) to investigate the tumor regional morphological fluctuations in response to respiration phases. Tumor surface vertices among ten respiratory phases were matched using surface-based registration, and the shape descriptors (ρ and detJ) were calculated and tracked across respiration stages in a regionally aligned scenario. Pair-wise group comparisons were performed between lung AC and SCC subtypes, in terms of ratios of maximal shape changes as well as correlation coefficients between tumor shape and respiratory stage indicators from the lung.AC type tumors had averaged larger surface measurements at exhale than at inhale, and these surface measurements were negatively correlated with lung volumes across respiratory stages. In contrast, SCC type tumors had averaged smaller surface measurements at exhale than at inhale, and the correlations with lung volumes were positive. The group differences in maximal shape changes as well as correlations were both statistically significant (p < 0.05).We developed a non-invasive lung tumor sub-type detection pipeline based on respiration-induced tumor surface deformation. Significant differences in deformation patterns were detected between lung AC and SCC. The derived surface measurements may potentially serve as a new non-invasive imaging biomarker of lung cancer subtypes.

Objective To investigate the clinical values and characteristics of whole body bone imaging (SPECT/CT) in detecting bone metastases in the preoperative patients with lung adenocarcinoma or squamous cell carcinoma for staging and determining the best treatment plan. Methods Eighty-two preoperative patients with primary pulmonary adenocarcinoma or squamous cell carcinoma performed 99Tcm-MDP SPECT/CT whole-body bone imaging. One week before surgery, parts of positive lesions performed MRI scan. The difference of the incidence of bone metastasis was analysed by χ2 test. Results In all 82 patients with lung cancer, there were 38 adenocarcinomas and 44 squamous cell carcinomas. Bone metastases were detected in 38 cases, the incidence rate was 46.3%. Of which, among lung adenocarcinoma, the incidence rate was 57.9% (22/38), and the incidence rate was 36.4% (16/44) in lung squamous cell carcinoma, and the difference was statistically significant (χ2=12.66, P=0.027). The most common area was bilateral ribs, followed by vertebra, pelvis, bones of the extremities and skull. Conclusion Lung adenocarcinoma compared with squamous cell carcinoma is prone to bone metastases, and bone metastases are more common in bilateral ribs. It has important value that whole body bone imaging in screening for bone metastases of pre-operative patients with lung cancer for staging and making the treatment plan. Key words: Lung neoplasms; Neoplasm metastasis; Diagnostic imaging; Single photon emission computed tomography/computed tomography

Objective To investigate the feasibility of differentiating lymph node metastases of four types of primary tumors (lymphoma, lung adenocarcinoma, lung squamous cell carcinoma and cholangiocarcinoma) using gemstone spectral imaging (GSI) . Methods Three cases with lymphoma (28 lymph node), five cases with lung adenocarcinoma(30 lymph node), four cases with lung squamous cell carcinoma(24 lymph node) and two cases with cholangiocarcinoma( 10 lymph node) were evaluated by germstona spectra imaging CT scans. Imaging protocol included unenhanced conventional CT scan (120 kVp) ,enhanced GSI (80/140 kVp) on arterial phase and conventional CT scan (120 kVp) on portal phase. CT attenuation values of lymph nodes in the monochromatic images at 11 sets of keV levels (40-140 keV, 10 keV step) and the iodine and water contents of these lymph nodes were measured. All results were analyzed with ANOVA and t test. Results The optimal monochromatic level was 70 keV for the optimal contrast-noise ratio (CNR) of metastatic lymphadenopathy. The CT attenuation values of metastatic lymphadenopathy were (81.36 ±9. 81 ), (58.33 ± 21.55 ), (56. 47 ± 10.62) and (73. 57 ±4. 43 ) HU,respectively, at 70 keV( F = 17.29, P ＜0. 01 ). There were significant differences in CT attenuation values between lymphoma and lung adenocarcinoma, between lymphoma and lung squamous cell carcinoma and between lung squamous cell carcinoma and cholangiocarcinoma (P ＜ 0. 05 ). The differences in CT attenuation values were significant between cholangiocarcinoma and lung squamous cell carcinoma, between cholangiocarcinoma and lymphoma ( P ＜ 0. 05 ). There was no difference in CT attenuation values at all 11 sets of keV levels between lung squamous cell carcinoma and lung adenocarcinoma ( P ＞ 0. 05 ). The iodine contents of lymphoma, lung adenocarcinoma, lung squamous cell carcinoma and cholangiocarcinoma were ( 1. 93 ± 0. 04 ), ( 1.16 ± 0. 15 ), ( 1.25 ± 0. 21 ) and ( 1.44 ± 0. 04 ) g/L, respectively. The water contents of lymphoma, lung adenocarcinoma, lung squamous cell carcinoma and cholangiocarcinoma were (1029.40 ± 20. 85), (1024.98 ± 11.19), (1022.12 ± 12. 94) and (1030.87 ± 10.10) g/L,respectively. Except between lung squamous cell carcinoma and lung adenocarcinoma, the differences in the iodine contents of metastatic lymphadenopathy were significant among tumors ( P ＜ 0. 05 ). There was no difference in the water contents of metastatic lymphadenopathy among tumors ( P ＞ 0. 05 ). Conclusions Although CT spectral imaging fails to differentiate metastatic lymphadenopathy of lung adenocarcinoma and lung squamous cell carcinoma, it is also a promising method of distinguishing metastatic lymphadenopathy of malignant tumors by CT attenuation values in monochromatic images and iodine contents in material density images. The optimal monochromatic level was determined to be at 70 keV for providing the optimal CNR of metastatic lymphadenopathy. Key words: Lymph nodes; Neoplasm metastasis; Tomography, X-ray computed

Objective: To analyze the clinical features and prognostic factors of different histological subtypes of lung adenocarcinoma. Methods: Data from 370 lung adenocarcinoma patients who underwent surgical resection for pathologically supported adenocarcinoma in our hospital between 2000 and 2003 were retro- spectively reviewed. The Kaplan-Meier method was used to estimate patient survival, and Cox’s proportional hazards model was performed for multivariate analysis. Results: The 5-year overall survival rate was 25.26%, and the mean survival time was 3.89 years. In multivariate analysis, histological subtype, incised margin residual, TNM stage, tumor size, and adjuvant chemotherapy were identified as independent survival predictors. The 5-year survival rate in bronchioloalveolar adenocarcinoma (BAC) patients was 41.30%, higher than in patients with other subtypes of lung adenocarcinoma (P=0.002). No significant difference was found in the prognosis among patients with different subtypes of adenocarcinoma without a BAC component. Conclusion: Ade-nocarcinoma with a BAC component is an independent subtype of lung adenocarcinoma. Its prognosis lies between those of BAC and adenocarcinoma without BAC. Histological subtype, incised margin residual, TNM stage, tumor size, and adjuvant chemotherapy are independent survival predictors.