Invasive Lung Adenocarcinoma Research Articles

Downregulation of AHNAK2 inhibits cell cycle of lung adenocarcinoma cells by interacting with RUVBL1.

Lung adenocarcinoma (LUAD) is the leading cause of death among cancer diseases. The tumorigenic functions of AHNAK2 in LUAD have attracted more attention in recent years, while there are few studies which have reported its high molecular weight. The mRNA-seq data of AHNAK2 and corresponding clinical data from UCSC Xena and GEO was analyzed. LUAD cell lines were transfected with sh-NC and sh-AHNAK2, and cell proliferation, migration and invasion were then detected by in vitro experiments. We performed RNA sequencing and mass spectrometry analysis to explore the downstream mechanism and interacting proteins of AHNAK2. Finally, western blot, cell cycle analysis and CO-IP were used to confirm our assumptions regarding previous experiments. Our study revealed that AHNAK2 expression was significantly higher in tumors than in normal lung tissues and higher AHNAK2 expression led to a poor prognosis, especially in patients with advanced tumors. AHNAK2 suppression via shRNA reduced the LUAD cell lines proliferation, migration and invasion and induced significant changes in DNA replication, NF-kappa B signaling pathway and cell cycle. AHNAK2 knockdown also caused G1/S phase cell cycle arrest, which could be attributed to the interaction of AHNAK2 and RUVBL1. In addition, the results from gene set enrichment analysis (GSEA) and RNA sequencing suggested that AHNAK2 probably plays a part in the mitotic cell cycle. AHNAK2 promotes proliferation, migration and invasion in LUAD and regulates the cell cycle via the interaction with RUVBL1. More studies of AHNAK2 are still needed to reveal its upstream mechanism.

Retracted] CtBP1 interacts with SOX2 to promote the growth, migration and invasion of lung adenocarcinoma.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that, for the western blots showing the CtBP1 and SOX2 bands in Fig.5C on p.74, the data were in fact the same, but flipped horizontally; moreover, two pairs of overlapping data panels were identified comparing between the cell invasion and assay data images shown in Figs.3E and 6C, such that these were likely to have been derived from the same original sources even though they were intended to show the results from differently performed experiments; similarly, the 'shSOX2/24h' and 'shCtBP1/24 h' data panels in Fig.6B showing the results of differently performed scratch‑wound assay experiments appeared to be overlapping, albeit with one of the panels being slightly rotated relative to the other. Finally, there were erroneous calculations included for the CtBP1 expression data shown in TableIII. Given the large number of apparent errors that were made during the assembly of various of the figures and TableIII in this paper, the Editor of OncologyReports has decided that this paper should be retracted from the Journal due to an overall lack of confidence in the presented data. After contacting the authors, they accepted the decision to retract this paper. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 42: 67‑78, 2019; DOI: 10.3892/or.2019.7142].

Establishment and visualization of a model based on high-resolution CT qualitative and quantitative features for prediction of micropapillary or solid components in invasive lung adenocarcinoma.

To predict the existence of micropapillary or solid components in invasive adenocarcinoma, a model was constructed using qualitative and quantitative features in high-resolution computed tomography (HRCT). Through pathological examinations, 176 lesions were divided into two groups depending on the presence or absence of micropapillary and/or solid components (MP/S): MP/S- group (n = 128) and MP/S + group (n = 48). Multivariate logistic regression analyses were used to identify independent predictors of the MP/S. Artificial intelligence (AI)-assisted diagnostic software was used to automatically identify the lesions and extract corresponding quantitative parameters on CT images. The qualitative, quantitative, and combined models were constructed according to the results of multivariate logistic regression analysis. The receiver operating characteristic (ROC) analysis was conducted to evaluate the discrimination capacity of the models with the area under the curve (AUC), sensitivity, and specificity calculated. The calibration and clinical utility of the three models were determined using the calibration curve and decision curve analysis (DCA), respectively. The combined model was visualized in a nomogram. The multivariate logistic regression analysis using both qualitative and quantitative features indicated that tumor shape (P = 0.029 OR = 4.89; 95% CI 1.175-20.379), pleural indentation (P = 0.039 OR = 1.91; 95% CI 0.791-4.631), and consolidation tumor ratios (CTR) (P < 0.001; OR = 1.05; 95% CI 1.036-1.070) were independent predictors for MP/S + . The areas under the curve (AUC) of the qualitative, quantitative, and combined models in predicting MP/S + were 0.844 (95% CI 0.778-0.909), 0.863 (95% CI 0.803-0.923), and 0.880 (95% CI 0.824-0.937). The combined model of AUC was the most superior and statistically better than qualitative model. The combined model could assist doctors to evaluate patient's prognoses and devise personalized diagnostic and treatment protocols for patients.

FZKA reverses gefitinib resistance by regulating EZH2/Snail/EGFR signaling pathway in lung adenocarcinoma

Ethnopharmacological relevanceFuzheng Kang-Ai (FZKA) decoction is mainly composed of 12 components with different types of herbs. In the last decade, FZKA has been used as an adjuvant treatment for lung cancer in clinical practice. Our previous studies have confirmed that FZKA shows a strong anti-cancer activity, significantly increases the clinical efficacy of gefitinib and reverses gefitinib resistance in non-small cell lung cancer (NSCLC). However, the molecular mechanism still needs to be further elucidated. Aim of the studyThe aim of this study was to investigate the role and mechanism by which FZKA inhibited the cell growth, proliferation and invasion of lung adenocarcinoma(LUAD) and reversed the acquired resistance of gefitinib for the therapy in LUAD. Materials and methodsCell viability assay and EDU assay were used for detecting of cell viability and cell proliferation. Transwell assay was performed to measure cell invasion. Western Blot and qRT-PCR were used for protein and gene expression test. The gene promoter activity was determined by dul-luciferase reporter assay. The in situ expression of protein was measured by cell immunofluorescence. Stabilized cell lines were established for stable overexpression of EZH2. Transient transfection assay was used for gene silence and overexpression. Xenograft tumors and bioluminescent imaging were used for in vivo experiments. ResultsFZKA significantly inhibited the cell viability, proliferation and cell invasion of LUAD, the combination of FZKA and gefitinib had a great synergy on the above processes. Moreover, FZKA significantly decreased EZH2 mRNA and protein expression, FZKA reversed the resistance of gefitinib by down-regulation of EZH2 protein. ERK1/2 kinase mediated the down-regulation of EZH2 reduced by FZKA. In addition, FZKA decreased the expression of Snail and EGFR by decreasing EZH2. Overexpression of Snail and EGFR significantly reversed the effect of FZKA-inhibited cell invasion and cell proliferation. More important, the combination of FZKA and gefitinib enhanced the inhibitory effect on EZH2, Snail and EGFR proteins. Furthermore, the growth inhibition and reversal of gefitinib resistance induced by FZKA were further validated in vivo. Finally, the expression and clinical correlation of EZH2,EGFR and Snail in cancer patients were further validated using bioinformatics analysis. ConclusionsFZKA significantly suppressed tumor progression and reversed gefitinib resistance by regulating the p-ERK1/2-EZH2-Snail/EGFR signaling pathway in LUAD.

Diagnostic sensitivity of solid volume measurement for pathological invasion in non-solid lung adenocarcinoma.

In the current tumor-node-metastasis (TNM) classification, the clinical T descriptor is defined by solid size (SS) on a computed tomography (CT) slice and the pathological one is done by invasive size (IS) in microscopic evaluations. We sometimes experience discrepancies in diagnosis of both descriptors. A volume analyzing application enables semi-automatic measurement of three-dimensional (3D) parameters in cases where there are discrepancies in diagnosing tumors' solid size and IS. In this study, we aimed to evaluate the association between 3D parameters and pathological invasion in non-solid small-sized lung adenocarcinomas. We enrolled 246 consecutive patients who underwent pulmonary resection at Shizuoka Cancer Center. Patients with lung adenocarcinomas that were radiologically non-solid, node-negative and sized ≤3 cm were eligible. We used a volume analyzing application to retrospectively measure 3D parameters of max and mean Hounsfield units (HUs) and solid volume (SV). The cut-off value of these parameters for diagnosing invasive adenocarcinoma (IAD) was set by describing receiver operating characteristic (ROC) curves. The correlation of IAD with these parameters was compared to its correlation with the SS. This study was not registered. Of 246 patients with adenocarcinoma, 183 (74.4%) had IADs. In multivariate analyses, the total size (TS) and SS were significantly associated with IAD (P=0.006, 0.001, respectively), whereas 3D parameters including SV were not (P=0.80). In radiological adenocarcinoma (2.1-3.0 cm), SV >300 mm3 diagnosed IAD with a higher sensitivity than that of the SS (0.93 and 0.83, respectively). TS >20 mm and SS >5 mm were well-correlated with IAD. SV measurement may complement the current computed tomographic diagnosis of IAD based on the SS (2.1-3.0 cm).

Tumor immune microenvironment (TIME) analysis of stage I lung adenocarcinoma with micropapillary pattern.

e20510 Background: Micropapillary is one of the most aggressive histologic subtypes of lung adenocarcinoma, which is considered a poor prognostic marker. Comprehensive analysis of tumor immune microenvironment (TIME) features of micropapillary of early invasive lung adenocarcinoma may provide better insight and facilitate the development of novel strategies for early detection and intervention. Methods: A total of 15 patients who underwent anatomical resection and were pathologically diagnosed with stage I micropapillary adenocarcinomas were included in this study. Gene expression profiles (GEPs) of tumor immune-related 289 genes were analyzed using NanoString nCounter. Differentially expressed genes(DEGs), estimation of TIME cell infiltration, and TIME signatures between micropapillary and non-micropapillary were assessed. Results: The number of micropapillary and non-micropapillary patients was 5 and 10. The comparison revealed CD69, IL6, CXCL8, CXCL2, POLR2A and so on were significantly upregulated in the micropapillary group compared with the non-micropapillary group, while HLA-DPA1, IFIT2, and other seven genes were down-regulated in the micropapillary group. These DEGs were mostly enriched in “positive regulation of cytokine production”， “T cell activation”， “receptor ligand activity，” and “signaling receptor activator activity” pathways according to GO enrichment analysis. The results of the KEGG analysis revealed that genes were significantly enriched in “IL-17 signaling pathway”, “TNF signaling pathway”, “cytokine-cytokine receptor interaction”, and so on. Meanwhile, different immune infiltration of the two groups was analyzed. Totally, there was a better TIME cell infiltration in the micropapillary group compared with the non-micropapillary group. Especially, the score of DCs and mast cells was significantly higher in micropapillary group. However, TIME signature scores were similar in both groups. Conclusions: Our study demonstrated that the TIME between micropapillary and non-micropapillary of stage I lung adenocarcinoma was different. Increased mast cells explained poor prognosis in lung adenocarcinoma with micropapillary pattern. A high level of DCs infiltration indicated that micropapillary adenocarcinoma might benefit from immunotherapy.

Evaluation of recurrence risk for patients with stage I invasive lung adenocarcinoma manifesting as solid nodules based on 18F-FDG PET/CT, imaging signs, and clinicopathological features

BackgroundStage I lung adenocarcinoma is a heterogeneous group. Previous studies have shown the prognostic evaluation value of PET/CT in this cohort; however, few studies focused on stage I invasive adenocarcinoma manifesting as solid nodules. This study aimed to evaluate the recurrence risk for patients with stage I invasive lung adenocarcinoma manifesting as solid nodules based on 18F-FDG PET/CT, CT imaging signs, and clinicopathological parameters.MethodsWe retrospectively enrolled 230 patients who underwent 18F-FDG PET/CT examination between January 2013 and July 2019. Metabolic parameters: maximum standard uptake value (SUVmax), mean standard uptake value, tumor metabolic volume (MTV), and total tumor glucose digestion were collected. Kaplan–Meier method was used to evaluate recurrence-free survival (RFS), and the multivariate Cox proportional hazards model was used to determine the independent risk factors associated with RFS. The time-dependent receiver operating characteristic curve (ROC) method was used to calculate the optimal cutoff value of metabolic parameters.ResultsThe 5-year RFS rate for all patients was 71.7%. Multivariate Cox analysis revealed that the International Association for the Study of Lung Cancer Pathology Committee (IASLC) pathologic grade 3 [Hazard ratio (HR), 3.96; 95% Confidence interval (CI), 1.11–14.09], the presence of cavity sign (HR 5.38; 95% CI 2.23–12.96), SUVmax (HR 1.23; 95% CI 1.13–1.33), and MTV (HR 1.05; 95% CI 1.01–1.08) were potential independent prognostic factors for RFS. Patients with IASLC grade 3, the presence of cavity sign, SUVmax > 3.9, or MTV > 5.4 cm3 were classified as high risk, while others were classified as low risk. There was a significant difference in RFS between the high-risk and low-risk groups (HR 6.04; 95% CI 2.17–16.82, P < 0.001), and the 5-year RFS rate was 94.1% for the low-risk group and 61.3% for the high-risk group.ConclusionsWe successfully evaluate the recurrence risk of patients with stage I invasive adenocarcinoma manifesting as solid nodules for the first time. The 5-year RFS rate in the high-risk group was significantly lower than in the low-risk group (61.3% vs. 94.1%). Our study may aid in optimizing therapeutic strategies and improving survival benefits for those patients.

Diagnostic Study of Multi-spectral Intelligent Analyzer in Diagnosis of the Infiltration Degree of Lung Adenocarcinoma

Lung cancer is one of the most common malignant tumors in the world. The accuracy of intraoperative frozen section (FS) in the diagnosis of lung adenocarcinoma infiltration cannot fully meet the clinical needs. The aim of this study is to explore the possibility of improving the diagnostic efficiency of FS in lung adenocarcinoma by using the original multi-spectral intelligent analyzer. Patients with pulmonary nodules who underwent surgery in the Department of Thoracic Surgery, Beijing Friendship Hospital, Capital Medical University from January 2021 to December 2022 were included in the study. The multispectral information of pulmonary nodule tissues and surrounding normal tissues were collected. A neural network model was established and the accuracy of the neural network diagnostic model was verified clinically. A total of 223 samples were collected in this study, 156 samples of primary lung adenocarcinoma were finally included, and a total of 1,560 sets of multispectral data were collected. The area under the curve (AUC) of spectral diagnosis in the test set (10% of the first 116 cases) of the neural network model was 0.955 (95%CI: 0.909-1.000, P<0.05), and the diagnostic accuracy was 95.69%. In the clinical validation group (the last 40 cases), the accuracy of spectral diagnosis and FS diagnosis were both 67.50% (27/40), and the AUC of the combination of the two was 0.949 (95%CI: 0.878-1.000, P<0.05), and the accuracy was 95.00% (38/40). The accuracy of the original multi-spectral intelligent analyzer in the diagnosis of lung invasive adenocarcinoma and non-invasive adenocarcinoma is equivalent to that of FS. The application of the original multi-spectral intelligent analyzer in the diagnosis of FS can improve the diagnostic accuracy and reduce the complexity of intraoperative lung cancer surgery plan. .

The prognostic significance of tumor budding and the expression of focal adhesion kinase and survivin in lung adenocarcinoma

ObjectivesAdenocarcinoma is the tumor group with the highest incidence among lung cancers with poor prognosis. Tumor budding (TB) is the migration of single tumor cells or small clusters of cells from the neoplastic epithelium to the invasive front of the tumor. Focal adhesion kinase (FAK) and survivin are considered as poor prognostic factors in several tumors. Hence, we investigated TB, FAK, and survivin expression in lung adenocarcinoma. MethodsThe study included 103 cases of lung adenocarcinoma in the resection materials. In tumoral tissues; TB was counted and scored in one high-power field (HPF), as low if <5 in 1 HPF and high if ≥5 in 1 HPF. FAK and survivin were studied immunohistochemically. ResultsThe mean number of TB in 1 HPF is 3.96 ± 2.8. Low-grade TB was observed in 45 (43.7 %) and high-grade TB was observed in 58 (56.3 %) patients. There was a positive correlation between TB and pT stage (p = 0.017), clinical stage (p = 0.002), lymphovascular invasion (p = 0.001), and perineural invasion (p = 0.045). The 4-year survival rate in patients was 90 % in those with low-grade TB and 60 % in those with high-grade TB (p = 0.001). FAK and survivin expressions were significantly increased in tumors with high-grade TB (p < 0.05). ConclusionA significant correlation was found between the grade of TB and pT stage, clinical stage, lymphovascular and perineural invasion in lung adenocarcinoma. TB can be considered as a histological parameter showing poor prognosis. It is thought that high expression of FAK and survivin also affect the prognosis in these patients by increasing TB.

Efficacy of Preoperative Biopsy in Predicting the Newly Proposed Histologic Grade of Resected Lung Adenocarcinoma

A novel histologic grading system for invasive lung adenocarcinomas (LUAD) has been newly proposed and adopted by the World Health Organization (WHO) classification. We aimed to evaluate the concordance of newly established grades between preoperative biopsy and surgically resected LUAD samples. Additionally, factors affecting the concordance rate and its prognostic impact were also analyzed. In this study, surgically resected specimens of 222 patients with invasive LUAD and their preoperative biopsies collected between January 2013 and December 2020 were used. We determined the histologic subtypes of preoperative biopsy and surgically resected specimens and classified them separately according to the novel WHO grading system. The overall concordance rate of the novel WHO grades between preoperative biopsy and surgically resected samples was 81.5%, which was higher than that of the predominant subtype. When stratified by grades, the concordance rate of grades 1 (well-differentiated, 84.2%) and 3 (poorly differentiated, 89.1%) was found to be superior compared to grade 2 (moderately differentiated, 66.2%). Overall, the concordance rate was not significantly different from biopsy characteristics, including the number of biopsy samples, biopsy sample size, and tumor area size. On the other hand, the concordance rate of grades 1 and 2 was significantly higher in tumors with smaller invasive diameters, and that of grade 3 was significantly higher in tumors with larger invasive diameters. Preoperative biopsy specimens can predict the novel WHO grades, especially grades 1 and 3 of surgically resected specimens, more accurately than the former grading system, regardless of preoperative biopsy or clinicopathologic characteristics.

A semiautomated radiomics model based on multimodal dual-layer spectral CT for preoperative discrimination of the invasiveness of pulmonary ground-glass nodules.

In recent years, spectral computed tomography (CT) has shown excellent performance in the diagnosis of ground-glass nodules (GGNs) invasiveness; however, no research has combined spectral multimodal data and radiomics analysis for comprehensive analysis and exploration. Therefore, this study goes a step further on the basis of the previous research: to investigate the value of dual-layer spectral CT-based multimodal radiomics in accessing the invasiveness of lung adenocarcinoma manifesting as GGNs. In this study, 125 GGNs with pathologically confirmed preinvasive adenocarcinoma (PIA) and lung adenocarcinoma were divided into a training set (n=87) and a test set (n=38). Each lesion was automatically detected and segmented by the pre-trained neural networks, and 63 multimodal radiomic features were extracted. The least absolute shrinkage and selection operator (LASSO) was used to select target features, and a rad-score was constructed in the training set. Logistic regression analysis was conducted to establish a joint model which combined age, gender, and the rad-score. The diagnostic performance of the two models was compared by the receiver operating characteristic (ROC) curve and precision-recall curve. The difference between the two models was compared by the ROC analysis. The test set was used to evaluate the predictive performance and calibrate the model. Five radiomic features were selected. In the training and test sets, the area under the curve (AUC) of the radiomics model was 0.896 (95% CI: 0.830-0.962) and 0.881 (95% CI: 0.777-0.985) respectively, and the AUC of the joint model was 0.932 (95% CI: 0.882-0.982) and 0.887 (95% CI: 0.786-0.988) respectively. There was no significant difference in AUC between the radiomics model and joint model in the training and test sets (0.896 vs. 0.932, P=0.088; 0.881 vs. 0.887, P=0.480). Multimodal radiomics based on dual-layer spectral CT showed good predictive performance in differentiating the invasiveness of GGNs, which could assist in the decision of clinical treatment strategies.

Transcriptional factor MAZ promotes cisplatin-induced DNA damage repair in lung adenocarcinoma by regulating NEIL3

BackgroundCisplatin remains a common chemotherapy drug for lung adenocarcinoma (LUAD) in clinical treatment. Long-term use of cisplatin in patients may lead to acquired drug resistance, resulting in poor prognoses of patients. NEIL3 was a glycosylase-encoding gene highly expressed in LUAD. NEIL3 can repair telomerase DNA damage in the S phase. Nevertheless, there are few reports on whether NEIL3 is involved in cisplatin resistance and its related mechanisms in LUAD. MethodsThe expression of NEIL3 in LUAD patients was analyzed by bioinformatics. The regulator upstream of NEIL3 was predicted via hTFtarget. The possibly involved pathways of NEIL3 were obtained by performing Gene Set Enrichment Analysis. qRT-PCR and western blot were applied to test the expression level of genes and protein LUAD cells. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay were conducted to validate the binding relationship between MAZ and NEIL3. Cell function assays were performed to test the DNA damage, cell viability, cell migration and invasion, and cell cycle of LUAD cells in the treatment group. ResultsNEIL3 and its upstream regulatory factor MAZ were highly expressed in LUAD tissue, and NEIL3 was enriched in cell cycle and mismatch repair pathways. Dual-luciferase assay and ChIP assay proved that MAZ could target NEIL3. Cell experiments identified that MAZ/NEIL3 axis could repress DNA damage to advance cisplatin resistance of cancer cells, and foster cell migration and invasion in LUAD. ConclusionMAZ-activated NEIL3 could propel the cisplatin resistance in LUAD by repressing DNA damage.

Knockdown of ERO1L attenuates tumor growth, migration and invasion in lung adenocarcinoma through Wnt/β‑catenin pathway

ABSTRACT Recent studies confirm the critical roles of endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) in malignant behavior of various cancers. Nevertheless, what function ERO1L plays in lung adenocarcinoma (LUAD) remains uncovered. The expressions and clinical significance of ERO1L in LUAD were investigated using the TCGA dataset. The ERO1L levels were examined by RT-qPCR. The LUAD cell proliferation was valued using colony formation as well as CCK-8 assays. The invasion and migration abilities of LUAD cells were detected through Transwell in addition to wound healing assays. The effects of ERO1L on LUAD cell apoptosis were determined by flow cytometric analysis. Moreover, we also established mouse xenograft models of LUAD cells to confirm the functions of ERO1L in vivo. The ERO1L levels in tumors were identified by immunohistochemistry. Western blot was used for the detection of the levels of Wnt/βcatenin signaling-related proteins. The TCGA database revealed that ERO1L expressions were higher in LUAD tissues than those in non-cancerous tissues. ERO1L overexpression was related to poorer overall survival of LUAD patients. In addition, ERO1L silence suppresses LUAD cell clone formation, proliferation, migration as well as invasion but induces apoptosis. Moreover, we also verified that ERO1L silence could promote LUAD growth in vivo. Based on the mechanism analysis, ERO1L was confirmed to regulate LUAD development via Wnt/βcatenin cascade signal. ERO1L, the expression of which was increased in LUAD tissues, functioned as an oncogene. ERO1L silence significantly attenuated LUAD tumorigenesis, likely via inhibition of Wnt/βcatenin signaling, indicating that ERO1L could be exploited as a promising biomarker in LUAD treatment.

Retraction Note: Long noncoding RNA LINC00968 inhibits proliferation, migration and invasion of lung adenocarcinoma through targeting miR-22-5p/CDC14A axis.

[This retracts the article DOI: 10.1007/s13205-021-02981-8.].

Abstract 3621: Identification of podocalyxin-positive tumor buds in pancreatic ductal adenocarcinoma: Implications for solid tumor collective invasion

Abstract High expression of the single-pass transmembrane sialomucin, podocalyxin, has been shown to predict poor disease outcome in a number of solid tumor types, including pancreatic ductal adenocarcinoma (PDAC) (Taniuchi et al., 2016). In addition to the classical TMN staging measures of disease progression (tumor size, lymph node involvement, and distant metastasis) local invasion at the primary tumor site is a valuable indicator of solid tumor progression. Local invasion has been observed histologically as tumor buds, defined by the presence of small cohesive clusters of tumor cells in the invasive tumor-stromal interface. To date, tumor budding has been characterized as an indicator of poor prognostic outcome in colorectal carcinoma (CRC), pancreatic ductal adenocarcinoma (PDAC), invasive ductal breast carcinoma (IDC), lung adenocarcinoma (LUA), stomach and esophageal carcinomas, oral squamous cell carcinoma, periampullary adenocarcinoma, and head and neck squamous cell carcinoma (HNSCC). Our group has previously modelled tumor budding experimentally using the MCF7 human breast cancer cell line (Graves et al., 2016). We showed that forced over-expression of podocalyxin was a driver of local collective invasion and tumor budding in vitro and in vivo. Because podocalyxin is highly expressed in PDAC we sought to determine if its expression was involved in the process of tumor budding in this tumor context as well. We assessed large format histological sections from a cohort of patient PDAC tumors and identified high podocalyxin expression in tumor buds. Further, we generated podocalyxin-null MiaPaCa2 PDAC cell lines and demonstrated that podocalyxin-expression is required for collective invasion in 3D culture conditions. We are currently evaluating in vivo xenograft tumor models with these podocalyxin-expressing and podocalyxin-null MiaPaCa2 cells to determine whether changes in 3D culture invasion are representative of tumor budding in this model. Expression of podocalyxin by tumor buds provides rationale to support investigation into the impact of therapeutically targeting these invasive cells. Citation Format: Erin M. Bell, Steve E. Kalloger, Pamela M. Dean, Janel L. Kopp, Kelly M. McNagny, Calvin D. Roskelley. Identification of podocalyxin-positive tumor buds in pancreatic ductal adenocarcinoma: Implications for solid tumor collective invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3621.

Abstract 6513: Single cell and spatial profiling reveal molecular and immune evolution from precancers to invasive lung adenocarcinomas in genetic and carcinogen-induced mouse models

Abstract Introduction: An improved understanding of early lung carcinogenesis may facilitate development of novel diagnostic, screening, and prevention strategies to decrease lung cancer mortality. We have previously led a series of studies on the molecular and immune landscape of lung adenocarcinoma (LUAD) precursors. However, resected human specimens only provide molecular snapshots. Here, we sought to establish and characterize human-relevant murine lung precancer models to depict molecular evolution during early lung carcinogenesis and to provide novel insights for lung cancer interception. Methods: We have established 3 genetically engineered mouse models (GEMMs) (K: KrasLSL-G12D/+, KP: KrasLSL-G12D/+/Trp53R172H/+, KL: KrasLSL-G12D/+/Lkb1fl/fl) and 2 carcinogen-induced models (CITMs) (UWT: Urethane in wild type mice, URG: Urethane in Rag2−/− mice). Lung tissues were serially collected at multiple time points after induction and subjected to whole exome sequencing (WES), single cell RNA sequencing (scRNA-seq), spatial transcriptomics (Visium), and imaging mass cytometry (IMC). Results: Pathological review of specimens from 252 mice revealed normal lung, precancers and LUAD at different timepoints mirroring the evolution of human LUAD. Overall, the tumor burden was significantly higher in GEMMs than CITMs (p&amp;lt;0.05). Tumor mutation burden progressively increased with progression from precancers to LUADs across all models. scRNA-seq demonstrated a progressive decrease of CD4+ T cell, CD8+ T cell, NK cell, and B cell infiltration in all models except the B/T cell-deficient WRG model; and a progressive increase of neutrophils (in KL model) and macrophages (in K, KP, UWT and URG models) along with progression of precancers. These findings were confirmed by spatial Visium and IMC profiling. Infiltration of T cells, B cells and NK cells inside tumors was not different between GEMMs (K, KP, KL) and CITMs (UWT). However, UWT showed significantly higher density of immune cells at the peritumor regions (P&amp;lt;0.05). Compared to the immune-competent UWT model, the B/T-cell deficient URG model showed similar progression and tumor burden at early phases, but rapid progression and larger tumor burden in the later phases. URG had no mature B/T cells but significantly higher NK cell infiltration than UWT (p&amp;lt;0.05). Conclusion: We have established 5 murine carcinogen-dependent and -independent precancer models, morphologically resembling human LUAD and its precursors. Although progressive immunosuppression along with progression of lung precancers is universal across all models, the evolution patterns and the molecular/immune features underlying immunosuppression vary in different models, particularly between CITMs and GEMMs. These models may be valuable assets for studying early lung carcinogenesis and lung cancer interception. Citation Format: Bo Zhu, Jian-Rong Li, Hong Chen, Pingjun Chen, Junya Fujimoto, Yanhua Tian, Muhammad Aminu, Chenyang Li, Lingzhi Hong, Alexandre Reuben, Edwin Roger Parra Cuentas, Ou Shi, Monique Nilsson, Alissa Poteete, Shawna Hubert, Khaja Khan, Wei Lu, Daniel Kraushaar, Xingzhi Song, Jianhua Zhang, Don Gibbons, Luisa Solis Soto, Ignacio Wistuba, Jia Wu, John Heymach, Chao Cheng, Jianjun Zhang. Single cell and spatial profiling reveal molecular and immune evolution from precancers to invasive lung adenocarcinomas in genetic and carcinogen-induced mouse models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6513.

Abstract 5884: Targeting T regulatory cells to intercept progression from preinvasive to invasive lung adenocarcinoma using a novel KRAS-driven murine model

Abstract Non-small cell lung cancer is the leading cause of cancer-related death in the US and worldwide, with a 5-year survival of ~18-20%. Understanding of the biology of early lung cancer is necessary to intercept early steps of progression. Indeed, increased utility of low-dose CT guided lung cancer screens in populations at risk have begun to detect early premalignant disease; however, little is known about the underlying biology that drives progression of preinvasive lesions to invasive adenocarcinoma. Moreover, there exists a lack of reliable animal models to assess interception strategies to target disease progression. We have developed and characterized a lung cancer cell-of-origin-specific KRAS-driven mouse model which accurately mirrors progression of human precursor lesions to invasive disease. Transcriptional analysis of preinvasive and invasive stages identified cellular and molecular alternations observed in human lesions, including upregulation of cell proliferation, extracellular matrix remodeling, and an increasingly complex immune microenvironment. Interrogation with immunofluorescence microscopy and Hyperion mass cytometry imaging showed marked reprogramming of the immune microenvironment including increased T cell infiltration and abundance of T regulatory cells (Tregs). Surprisingly, depletion of T cells impaired progression of preinvasive lesions, implicating the critical immunosuppressive roles of Tregs. Spatial transcriptomics (10X Genomics Visium) revealed unique spatial transcriptional changes in the progression from preinvasive to invasive stage disease, including specific ligand-receptor interactions highly expressed in Tregs. Genetic and pharmacological approaches using FoxP3-DTR-GFP mice and low dose cyclophosphamide have begun to define the functional role of Tregs in early premalignant disease. This study demonstrates critical roles of immunosuppressive Tregs in driving progression of preinvasive to invasive lung cancer, and advocates for Treg targeting as a potent immunoprevention approach for patients at risk of developing lung cancer. Citation Format: Mitchell Martin, Michael J. Crowley, Geoffrey Markowitz, Alain Borczuk, Chen Zhang, Liron Yoffe, Arshdeep Singh, Nasser K. Altorki, Vivek Mittal. Targeting T regulatory cells to intercept progression from preinvasive to invasive lung adenocarcinoma using a novel KRAS-driven murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5884.

In-depth proteomics reveals the characteristic developmental profiles of early lung adenocarcinoma with epidermal growth factor receptor mutation.

Lung adenocarcinoma progresses stepwise from atypical adenomatous hyperplasia to adenocarcinoma in situ (AIS), followed by minimally invasive adenocarcinoma (MIA), and then obvious invasive adenocarcinoma. In this study, we examined the protein expression profiles of early and epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinomas. Fifteen cases of small and EGFR mutation-positive adenocarcinomas were collected, including AIS, MIA, and small invasive adenocarcinoma (SIA). We examined their protein expression profiles by tandem mass tag (TMT)-labeling liquid chromatography-mass spectrometry (LC-MS/MS) and compared the results between AIS and MIA versus SIA. The differentially expressed proteins were then verified by Western blot analysis and immunohistochemistry (IHC). The clinicopathological implications of the proteins were also examined by IHC. A total of 4220 proteins were identified by LC-MS/MS analysis. Pathway analysis of the differentially expressed proteins revealed that pathways related to interferon α/β signaling, glutamate and glutamine metabolism, and gluconeogenesis were upregulated in SIA relative to AIS. Among the 13 differentially expressed proteins, cellular retinoic acid binding protein 2 (CRABP2), delta(24)-sterol reductase (DHCR24), and adenylate kinase 4 (AK4) were expressed significantly more strongly in SIA than in AIS. Patients with high expression of CRABP2, DHCR24, and AK4 showed a significantly poorer outcome than those with low expression. In comparison with AIS, SIA shows differences in several different protein expression pathways. Furthermore, CRABP2, DHCR24, and AK4 are useful IHC markers for diagnosis of lung adenocarcinoma invasiveness and may be associated with malignant progression of AIS.

Establishment and validation of nomograms for predicting survival of lung invasive adenocarcinoma based on the level of pathological differentiation: a SEER cohort-based analysis.

The pathological differentiation of invasive adenocarcinoma (IAC) has been linked closely with epidemiological characteristics and clinical prognosis. However, the current models cannot accurately predict IAC outcomes and the role of pathological differentiation is confused. This study aimed to establish differentiation-specific nomograms to explore the effect of IAC pathological differentiation on overall survival (OS) and cancer-specific survival (CSS). The data of eligible IAC patients between 1975 and 2019 were collected from the Surveillance, Epidemiology, and End Results (SEER) database, and randomly divided in a ratio of 7:3 into a training cohort and a validation cohort. The associations between pathological differentiation and other clinical characteristics were evaluated using chi-squared test. The OS and CSS analyses were performed using the Kaplan-Meier estimator, and the log-rank test was used for nonparametric group comparisons. Multivariate survival analysis was performed using a Cox proportional hazards regression model. The discrimination, calibration, and clinical performance of nomograms were assessed by area under receiver operating characteristic curve (AUC), calibration plots, and decision curve analysis (DCA). A total of 4,418 IAC patients (1,001 high-differentiation, 1,866 moderate-differentiation, and 1,551 low-differentiation) were identified. Seven risk factors [age, sex, race, tumor-node-metastasis (TNM) stage, tumor size, marital status, and surgery] were screened to construct differentiation-specific nomograms. Subgroup analyses showed that disparate pathological differentiation played distinct roles in prognosis, especially in patients with older age, white race, and higher TNM stage. The AUC of nomograms for OS and CSS in the training cohort were 0.817 and 0.835, while in the validation cohort were 0.784 and 0.813. The calibration curves showed good conformity between the prediction of the nomograms and the actual observations. DCA results indicated that these nomogram models could be used as a supplement to the prediction of the TNM stage. Pathological differentiation should be considered as an independent risk factor for OS and CSS of IAC. Differentiation-specific nomogram models with good discrimination and calibration capacity were developed in the study to predict the OS and CSS in 1-, 3- and 5-year, which could be used predict prognosis and select appropriate treatment options.

Prognostic effect of ground-glass opacity in subcentimeter invasive lung adenocarcinoma.

Although subcentimeter nodules represent precursor or minimally invasive lung cancer in most cases, there are still a few that are subcentimeter invasive adenocarcinoma (IAC). The aim of this study was to investigate the prognostic effect of ground-glass opacity (GGO) and the optimal surgical procedure in this special group. Patients with subcentimeter IAC were enrolled and were categorized into pure GGO, part-solid, and solid nodules based on the radiological appearance. Cox proportional hazards model and the Kaplan-Meier method were used for survival analyses. A total of 247 patients were enrolled. Among them, 66 (26.7%) were in the pure-GGO group, 107 (43.3%) were in the part-solid group, and 74 (30.0%) were in the solid group. Survival analysis demonstrated a significantly worse survival in the solid group. Cox multivariate analyses confirmed that the absence of GGO component was an independent risk factor for worse recurrence-free survival (RFS) and overall survival (OS). As for surgical procedures, lobectomy did not provide a significant better RFS or OS than sublobar resection in the whole cohort or in a subgroup of patients with solid nodules. The radiological appearance stratified the prognosis of IAC with size of smaller than or equal to 1 cm. Sublobar resection may be feasible for subcentimeter IAC, even for those appearing as solid nodules; however, caution should be taken when applying wedge resection.