DEGs between subsolid nodule and nLung C20 NKT cells

Over the past decade, substantial progress has been made in the development of targeted and immune-based therapies for patients with advanced non-small-cell lung cancer. To further improve outcomes for patients with lung cancer, identifying and intercepting disease at the earliest and most curable stages are crucial next steps. With the recent implementation of low-dose computed tomography scan screening in populations at high risk, there is an emerging unmet need for new diagnostic, prognostic and therapeutic tools to help treat patients suspected of harbouring premalignant lesions and minimally invasive non-small-cell lung cancer. Continued advances in the identification of the earliest drivers of lung carcinogenesis are poised to address these unmet needs. Employing multimodal approaches to chart the temporal and spatial maps of the molecular events driving lung premalignant lesion progression will refine our understanding of early carcinogenesis. Elucidating the molecular drivers of premalignancy is critical to the development of biomarkers to detect those incubating a premalignant lesion, to stratify risk for progression to invasive cancer and to identify novel therapeutic targets to intercept that process. In this Review, we summarize emerging insights into the earliest cellular and molecular events associated with lung squamous and adenocarcinoma carcinogenesis and highlight the growing opportunity for translating these insights into clinical tools for early detection and disease interception to transform the outcomes for those at risk for lung cancer. While progress has been made in treating advanced non-small-cell lung cancer, patients would further benefit from methods that detect progressive premalignant lesions and strategies that effectively intercept progression. In this Review, Mazzilli et al. explore recent advances in identifying premalignant lesions, highlighting their disease aetiology and devising approaches for stratification and interventions aimed at halting the progression to cancer.

Lung adenocarcinoma (LUAD) is one of the most prevalent and lethal cancer types worldwide. However, our understanding of the transition from normal-appearing tissue (NAT) to adenomatous lung premalignant lesions (aPMLs) and LUADs, particularly within a spatial context, remains limited. This study aims to address this gap by systemically analyzing the pathologic continuum of NAT > aPML > LUAD using spatial transcriptomics (ST). High-resolution spatial profiling was conducted on 56 samples from 25 patients with paired aPMLs and LUADs using the Visium ST platform. Non-negative matrix factorization was conducted to identify transcriptional programs for each sample, following clustering analysis to define consensus metaprograms across the cohort. Additionally, spatial molecular imaging was performed on an expanded cohort of 188 cores arranged into eight tissue microarrays using the Xenium in situ platform with a customized lung cancer gene panel to establish a single-cell spatial atlas of the disease continuum and systemically investigate spatial organization, cellular neighborhoods and interactions. Eight distinct metaprograms (MP1∼8) were identified, distinguishing stromal (MP2), myeloid (MP4), lymphoid (MP6), and epithelial (MP3, MP5) compartments. Additionally, metaprograms were identified for the lung capillary bed (MP7), stressed cellular state (MP8), and mosaic cellular patterns (MP1). These metaprograms correlated strongly with pathological annotations and captured fine tissue structures like lymphoid aggregates. LUADs and aPMLs showed distinct MP profiles, with MP3 and MP6 being highly abundant in LUADs, while MP1, MP4, MP5 and MP7 were more abundant in aPMLs. Specifically, we observed positive correlations between MP3 and MP6, as well as between MP4 and MP5 in sample distributions, indicating co-evolution of tumor and immune microenvironments during disease progression. Xenium data complemented these findings by revealing distinct compositions of microenvironmental cellular states and tumor neighborhood structures across aPML and LUAD lesions. A total of 44 cell states were defined. Specifically, NK cells were depleted in aPMLs and LUADs compared with NAT, accompanied by an enrichment of regulatory T cells and myofibroblastic cancer-associated fibroblasts (CAFs) in the latter. Additionally, pro-inflammatory cellular subsets, including IL1B+ macrophages and IL6+ inflammatory CAFs were more abundant in tumor neighborhoods of aPMLs compared to LUADs, potentially contributing to the pre-malignant to malignant transition. This study offers a comprehensive molecular and cellular landscape of aPML and LUAD, laying a crucial foundation for future mechanistic studies aimed at early disease interception. Yibo Dai, Fuduan Peng, Ansam Sinjab, Sujuan Yang, Minyue Chen, Warapen Treekitkarnmongkol, Lorena I. Gomez Bolanos, Tieling Zhou, Alejandra G. Serrano, Jianlong Liao, Guangsheng Pei, Yunhe Liu, Yang Liu, Jiahui Jiang, Kyung Serk Cho, Yanshuo Chu, Kai Yu, Ruiping Wang, Jiping Feng, Zahraa Rahal, Guangchun Han, Naoe Jimbo, Takuo Hayashi, Satsuki Kishikawa, Kazuya Takamochi, Akshay Basi, Avrum Spira, Steven Dubinett, Tomokazu Itoh, Takashi Yao, Kenji Suzuki, Luisa M. Solis, Stephen Swisher, Mingyao Li, Junya Fujimoto, Ignacio I. Wistuba, Jared Burks, Kadir Akdemir, Hind Refai, Katy Rezvani, Jeffrey Myers, Humam Kadara, Linghua Wang. Spatial transcriptomics identifies unique tumor and microenvironment pathomic programs that are associated with the lung premalignancy and adenocarcinoma continuum [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 759.

Lung cancer is the leading cause of cancer-related mortality worldwide, and there is a critical need for early detection strategies to reduce its burden. In high-risk individuals, low-dose computed tomography (LDCT) reduces lung cancer mortality by detecting cancer at earlier stages. Gene expression obtained from bronchial and nasal brushings effectively discriminates benign from malignant lung nodules detected via LDCT. Lung premalignant lesions (PMLs), precursors to cancer, represent an opportunity for further mortality reduction, but detection methods remain underexplored. This study uses single-cell RNA sequencing (scRNA-seq) to investigate molecular and cellular alterations in the airway field of patients with PMLs. Endobronchial biopsies from lung sites with suspect PMLs and bronchial and nasal brushes from normal-appearing epithelium were collected from high-risk patients (n=46). Epithelial and immune cells were profiled using scRNA-seq via the CEL-Seq2 protocol (n=9405 biopsy, n=3416 bronchial brush, and n=2002 nasal brush cells). Cell and gene clustering were performed via Celda. Co-expressed gene modules and cell-type abundance differences by sample type, smoking status, and lesion histology were identified with differential composition analysis and linear mixed models. Gene module associations with phenotypes were validated in independent bronchial brushes profiled by bulk mRNA-seq (n=89 samples, n=30 patients). Clustering of epithelial cells (40 cell clusters, 70 gene modules) showed nasal brushes are enriched in cells from 9 cell clusters (p<0.001) with high expression of detoxification and mucous production gene modules. Nine gene modules associated with smoking status (p<0.001) across sample types were enriched for xenobiotic metabolism genes. The effect of smoking on these gene modules was strongest in bronchial biopsy samples, decreased in bronchial brushes and weakest in nasal brushes. This stepwise decrease was significant for each pairwise comparison (p <= 0.001). Five basal cell clusters enriched with carcinoma in situ lesion or tumor cells (termed high-grade basal cells) expressed 7 gene modules linked to keratinization and inflammation (p<0.01). Cells from a bronchial brush sample clustered with high-grade basal cells. The expression of a gene module upregulated in high-grade basal cells was associated with the worst lesion histology sampled during the procedure (p=0.006) in bronchial brushes profiled by bulk RNA-seq. This study identifies smoking and lesion severity associated molecular and cellular alterations in the airway fields of patients with PMLs. These findings suggest that airway field brushings may be useful in detecting the presence of high-grade PMLs. Regan D. Conrad, Conor Shea, Lukas Kalinke, Kitty De Jong, Sherry Zhang, Zhihan Li, Gang Liu, Erin Kane, Emily Siedlecki, Kelsey Simon, Kate E. Otter, Kate Gowers, Mark Hennon, Sai Yendamuri, Steven Dubinett, Avrum Spira, Mary Reid, Sam Janes, Marc Lenburg, Sarah Mazzilli, Joshua Campbell, Jennifer Beane. Single-cell characterization of airway field alterations in patients with lung squamous premalignant lesions [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 5066.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with varying degrees of airway wall thickening, chronic bronchitis, and emphysema. A better understanding of the underlying pathology is needed to improve the personalized treatment of the disease and identify new therapeutic targets. Available data from 56 COPD patients included in the GLUCOLD study were used (61.1±7.7 years, 89% male, and FEV1 of 62.5±8.9% predicted). Clinical characterization was performed including bronchoscopy and collection of bronchial biopsies at baseline. RNA from bronchial biopsies was sequenced and used for unsupervised clustering, using a 98 COPD gene signature previously identified in bronchial brushes comparing patients with COPD to non-COPD controls. Next, we assessed differences in the clinical expression of COPD, lung function decline, inflammatory cell counts, and gene expression between clusters. Validation was performed in an independent dataset. We identified two clusters: CAGE1 (n=39) and CAGE2 (n=17). CAGE2 patients had higher percentage of sputum lymphocytes, and more CD4+ and CD8+ T-cell counts in their bronchial biopsies. In addition, their FEV1 improved less in response to 30-months treatment with inhaled corticosteroids (ICS) (change in FEV1- CAGE1: +24.4mL; CAGE2: -29.1mL; p-value=0.048), and they experienced a faster decline in their lung function follow-up (CAGE1: -44.0mL/year; CAGE2: -69.9mL/year; p-value=0.002). Gene expression analysis showed more activation of T- and B-cell immune responses in CAGE2. We identified a new COPD endotype, CAGE2, characterized by ICS unresponsiveness and faster lung function decline. Additionally, we show a different pathobiology in CAGE2 with more activation of T- and B-cell immune responses. ### Competing Interest Statement A. Spira is an employee of Johnson and Johnson. ### Clinical Trial NCT00158847 ### Funding Statement The study was sponsored by the Dutch Ministry of Health via the Public Private Funding Program (PPP), GlaxoSmithKline (The Netherlands), the University Medical Centre Groningen and the Leiden University Medical Centre. Rui Marcalo is supported by Fundacao para a Ciencia e Tecnologia through the grant 10.54499/UI/BD/151337/2021. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of Groningen University Medical Center gave ethical approval for this work. Ethics committee of Leiden University Medical Center gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

The molecular drivers bronchial premalignant lesion progression to invasive lung squamous cell carcinoma are not well defined. Prior work profiling longitudinally collected bronchial premalignant lesion biopsies by RNA sequencing defined a proliferative subtype, enriched with bronchial dysplasia. We found that a gene co-expression module associated with interferon gamma signaling and antigen processing/presentation was down-regulated in progressive/persistent versus regressive lesions within the proliferative subtype, suggesting a functional impact of these genes on immune evasion. RNA from these same premalignant lesions was profiled by microRNA (miRNA) sequencing and a miRNA-gene network analysis identified hsa-miR-149-5p as a potential regulator of this antigen presentation gene co-expression module associated with lesion progression. hsa-miR-149-5p was found to be predominantly expressed in the epithelium and up-regulated in progressive/persistent versus regressive proliferative lesions while targets of this miRNA, the transcriptional coactivator of MHC-I gene expression, NLRC5 , and the genes it regulates were down-regulated. MicroRNA in situ hybridization of hsa-miR-149-5p in tissue from adjacent fixed biopsies showed that hsa-miR-149-5p was increased in areas of bronchial dysplasia in progressive/persistent versus regressive lesions. Imaging mass cytometry showed that NLRC5 protein expression was decreased in progressive/persistent versus regressive lesions within areas of hyperplasia, metaplasia, and dysplasia. Additionally, basal cells with high versus low levels of NLRC5 were found to be in close spatial proximity to CD8 T cells, suggesting that these cells exhibit increased functional MHC-I gene expression in lesions with low hsa-miR-149-5p expression. Collectively, our data suggests a functional role for hsa-miR-149-5p in bronchial premalignant lesions and may serve as a therapeutic target for PML immunomodulation. STATEMENT OF SIGNIFICANCE:Integrative analysis across bronchial premalignant lesions has identified and localized a potential regulator of immune evasion in progressive/persistent lesions that could be a novel therapeutic target.

Abstract Background: The goal of the Early Detection Research Network (EDRN) Lung Team Project #2 (LTP-2) is to establish a cohort (n= 300) of high-risk people with indeterminate pulmonary nodules (6mm-30mm) to clinically validate the diagnostic accuracy of existing molecular and imaging biomarkers discovered and analytically validated through the EDRN program. These biomarkers comprise signals measured in serum (glycan or cytokine), plasma (protein, miRNA, RNA, or methylated circulating tumor DNA), airway epithelial cells (RNA or DNA), nasal brush cells (RNA or DNA), and/or chest CT images. Methods: Eligible participants had an indeterminate nodule discovered incidentally or through low-dose CT screening within 14 months prior to enrollment and were aged 45-90 years, with 20 pack-years or more cigarette smoking history, free of lung cancer, and willing to provide blood, nasal brushing, and optionally bronchoscopic brush biopsy specimens. Baseline medical and demographic data were collected from each subject. Baseline and years 1 and 2 follow-up CT images were obtained. Each specimen at each respective site was labeled with a 2-D barcode provided by the DMCC and specimen information was entered into the Validation Study Information Management System (VSIMS) developed by the EDRN Data Management and Coordinating Center (DMCC). Aliquots of blinded specimens from selected cases and controls will be dispensed to each EDRN site for biomarker analysis. Analysis plan: Primary endpoint: The positive diagnostic likelihood ratio (DLR+) will be used to measure biomarker performance within values of DLR+ >= 2 or DLR- <= 0.5, a range considered clinically useful for any patients with indeterminate lung nodules. The sample size calculation targeted >80% power to test the null hypotheses that a) a positive biomarker does not alter risk across a range of specificities, and b) a negative biomarker does not alter risk across a range of sensitivities. The EDRN Data Management and Coordinating Center (DMCC) will unblind and complete analysis and report results. Exploratory endpoint: The diagnostic accuracy of combined biomarkers will be calculated using a logistic regression model with flexible functional forms. Results and Discussion: More than 300 subjects were enrolled into the LTP2 cohort including a sufficient number of cases and controls (cohort size n>300) to reach power according to calculated estimates. Chest CT images were collected and are in the process of transfer to a central database. Plasma specimens have been distributed to test sites. RNA and DNA extraction from NBC and AEC is in process and based on results thus far the specimen quality and quantity is more than sufficient from the number of subjects needed to reach power for the primary endpoint and to conduct the planned exploratory analyses. Citation Format: James C. Willey, Eric L. Grogan, Stephen A. Deppen, James G. Herman, Michael N. Kammer, Matthew B. Schabath, Lynn Sorbara, Harvey I. Pass, Jun-Chieh J. Tsay, Ziding Feng, Avrum Spira, Erin L. Crawford, Jackie Dahlgren, Steven M. Dubinett, Kimberly R. Rieger-Christ, Marc E. Lenburg. EDRN Lung Team Project 2 validation of molecular biomarkers for the early detection of lung cancer in the setting of indeterminate pulmonary nodules [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 6104.

Abstract Understanding the earliest changes during lung adenocarcinoma (LUAD) development can set the stage for discovery of fertile targets for disease interception, thereby mitigating the dire public health burden of LUAD. We previously identified bulk-level molecular and immunological changes that are enriched in normal-appearing tissue (NAT) in the local niche of human LUAD, as well as those that commence in adenomatous premalignant lesions (aPMLs, LUAD precursors) and that are further enriched in LUADs. Yet, in-depth understanding of the identities, states, and properties of specific cell subsets in NAT and precancer that trigger LUAD remain largely elusive due to inherent roadblocks to sampling and characterizing aPMLs that are at the center of this trajectory. Here, we aimed to map molecular profiles, states, and interactions of cell subsets that underlie initiation in lesion-adjacent NAT, and to determine how features of these cells evolve along the aPML-LUAD spectrum. We analyzed an expanding cohort of archived matching NATs, aPMLs and LUADs, including challenging small samples, from up to 17 patients. Samples were analyzed using combined high-resolution, multi-modal Visium spatial transcriptomics (ST, n=17) and proteomics (n=12), as well as single-cell RNA-sequencing of fixed cells (scFFPE-seq) from consecutive sections of the same tissues from 15 patients. We also studied a subset of samples by high-plex spatial proteomics (COMET) or subcellular spatial gene expression analysis (Xenium). In total, we sequenced more than half a million single cells comprising diverse epithelial, immune, and stromal subsets, which were concordant with lineage clusters identified by ST analysis of the same samples. Loss of alveolar differentiation was a hallmark of tumor cell-enriched areas in aPMLs and LUADs, with a more pronounced effect in the LUADs. Alveolar intermediate cells (AICs), which we have previously shown to be involved in LUAD initiation, were found in lesion-adjacent NAT, and their signature was enriched in matching aPMLs and further in LUADs. In line with these findings, trajectory analysis showed that AICs are derived from normal alveolar type 2 cells and likely progress towards tumor cells. Spatial neighborhood analysis pointed towards strong crosstalk between AICs and macrophages. We also noted heterogeneity in immune infiltration including patterns indicative of progressive features along the NAT-aPML-LUAD continuum. For example, B cell lineages were not only increased in abundance in LUADs relative to aPMLs, but they also mobilized into relatively more mature tertiary lymphoid aggregates/structures. While T regulatory cells aggregated close to LUADs, they were rather scattered in matching aPMLs. Our multimodal and spatial atlas of NAT, nearby aPML and LUAD elucidates the earliest cellular events underlying transition of NAT to LUAD and that could inform of targets for interception of this trajectory. Citation Format: Ansam Sinjab, Fuduan Peng, Yunhe Liu, Sujuan Yang, Tieling Zhou, Alejandra G. Serrano, Jiping Feng, Lorena Gomez Bolanos, Guangchun Han, Daniel Gustavo Rosen, Stephen G. Swisher, Avrum Spira, Steven M. Dubinett, Luisa M. Solis Soto, Mingyao Li, Junya Fujimoto, Jared Burks, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A multimodal spatial-omics atlas of lung precancer and progression to adenocarcinoma [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 3893.

Studying lung adenocarcinoma (LUAD) early carcinogenesis is challenging, primarily due to the lack of LUAD precursors specimens. We amassed multi-omics data from 213 LUAD and LUAD precursors to identify molecular features underlying LUAD precancer evolution. We observed progressively increasing mutations, chromosomal aberrations, whole genome doubling and genomic instability from precancer to invasive LUAD, indicating aggravating chromosomal instability (CIN). Telomere shortening, a crucial genomic alteration linked to CIN, emerged at precancer stage. Moreover, later-stage lesions demonstrated increasing cancer stemness and decreasing alveolar identity, suggesting epithelial de-differentiation during early LUAD carcinogenesis. The innate immune cells progressively diminished from precancer to invasive LUAD, concomitant with a gradual recruitment of adaptive immune cells (except CD8+ and gamma-delta T cells that decreased in later stages) and upregulation of numerous immune checkpoints, suggesting LUAD precancer evolution is associated with a shift from innate to adaptive immune response and immune evasion mediated by various mechanisms.

Abstract Lung cancer is the leading cause of cancer-related death. The five year survival rate had remained stagnant for decades, but more effective screening protocols using low-dose computed tomographic screening for high-risk individuals, along with progress in treatment for advanced-stage lung cancer with targeted therapy and immunotherapy, have helped decrease lung cancer mortality. However, improved approaches to lung cancer prevention and early detection are still urgently needed. Adenocarcinoma (LUAD) is the most common form of lung cancer. Following surgical resection of pre-invasive adenocarcinoma in situ, the five-year survival rate approaches 100%. This emphasizes the need for improved understanding of characteristics of aggressive premalignant lesions that would benefit from early interventions. In contrast to the well-characterized histopathologic sequence of lung squamous cell carcinoma, atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS) are the only known precursors in the sequence of LUAD pathogenesis. The features of AAH and AIS that lead to invasive LUAD are poorly characterized. The relationship of AIS and AAH to the histologically diverse subtypes of LUAD, ranging from the typically slow growing lepidic to aggressive solid subtypes, are also unclear. We hypothesized that transcriptomic changes in subsets of premalignant lesions are linked to distinct genomic and clinicopathologic features of malignant disease. To investigate this hypothesis, we performed exome sequencing and bulk RNA sequencing of laser capture microdissected tissue from tumor margins that included premalignant lesions, invasive tumor, and adjacent normal tissues. With our analysis, we discovered de novo subtypes of LUAD premalignant lesions based on gene expression. One of these subtypes had gene expression alterations similar to aggressive invasive LUAD solid tumors. This molecular adenomatous premalignant lesion subtype was also associated with an increased accumulation of cancer driver mutations. This subtype was further characterized by altered expression of immune-related pathways. Molecular signatures measured in adenomatous PML may thus enhance our understanding of pathway dysregulation and mutational heterogeneity occurring during LUAD carcinogenesis, and implicate immunotherapeutic strategies to prevent their progression to cancer. Citation Format: Kelley Anderson, Linh Tran, Kostyantyn Krysan, William Wallace, Gregory Fishbein, Emily Green, Gang Liu, Hanqiao Liu, Erin Kane, Sarah Mazzilli, Steven Dubinett, Avrum Spira, Marc Lenburg, Jennifer Beane. Molecular subtyping of lung adenocarcinoma premalignant lesions identifies features associated with aggressive disease [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR007.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies 1 . Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8 + alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8 + cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Abstract Background: Epidemiological studies showed higher intake of cruciferous vegetables or their active compounds, isothiocyanates (ITCs), with lower risk of lung cancer. ITCs inhibited tobacco-carcinogen induced lung adenocarcinoma in animal models, along with reduced cellular proliferative marker Ki-67 and increased apoptotic markers Caspase-3 and TUNEL. However, human data lack. The aim of our study was to assess if oral intake of sulforaphane (SFN) for 12 months would stop/reverse the progression of bronchial histopathology, reduce Ki-67 index and/or increase Caspase-3 and TUNEL indices. Methods: A randomized clinical trial (NCT03232138) was conducted in former smokers with ≥ 30 pack-years and quitting smoking for 1-10 years in USA. Forty-three subjects were randomly assigned to the SFN (n = 21) or placebo group (n = 22) in 2018-2022. Each participant was instructed to take 4 Avmacol® or identical placebo capsules BID for 12 months. Each Avmacol® capsule contained 15 mg of glucoraphanin, which yielded a daily internal dose of 120 μmol SFN. Biopsies were collected from 6 predetermined bronchial sites at baseline and the end of treatment, respectively. The primary outcomes were the average changes of histopathology scores, Ki-67, Caspase-3 and TUNEL indices in post- vs. pre-treatment biopsies. Results: Thirty-seven participants completed the study. In the SFN group (n = 17) at baseline, 13 were men, all white, mean age 64.1 (SD 5.3) years, body mass index (BMI) 30.6 (6.1) kg/m2, years of smoking 39.5 (8.2), cigarettes per day (CPD) 23.8 (10.7), and years of quitting smoking 4.3 (2.9). The corresponding figures in the placebo group (n=20) were 9 men, 19 white, age 68.0 (3.2), BMI 28.6 (4.2), years of smoking 42.2 (4.5), CPD 25.5 (8.4), and years of quitting smoking 6.8 (3.2). The baseline medians of total Ki-67 positive nuclei were 22.3 nuclei/mm2 in the SFN group and 22.9/mm2 in the placebo group (p = 0.96). After treatment, the number of positive Ki-67 nuclei decreased by 5.3/mm2 (-24%) in the SFN group and increased by 18.0/mm2 (+79%) in the placebo group (p = 0.014) with adjustment for baseline Ki-67 and other covariates. The post- and pre-treatment difference was even greater in high-density (3+) positive Ki-67, with -46% decrease in the SFN group vs. +99% increase in the placebo group (p = 0.004). There was a dose-response effect of higher compliance with SFN intake on reducing Ki-67 index (ptrend = 0.01). SFN treatment had no impact on Caspase-3, TUNEL, or bronchial histopathology. No severe adverse event was observed in the SFN group. Conclusion: Daily intake of 120 μmol SFN for 12 months significantly reduced Ki-67 index in bronchial tissue. The findings support the potential chemopreventive effect of SFN against lung cancer development in high-risk former smokers (Grant # R01CA213123). Citation Format: Jian-Min Yuan, Thomas W. Kensler, Sanja Dacic, Douglas J. Harman, Renwei Wang, Paula Balogh, Lindsey Seigh, Yen T-H Pham, Jennifer Adams-Haduch, Shivendra Singh, James G. Herman, Avrum Spira, David O. Wilson. Randomized placebo-controlled phase II clinical trial on oral supplementation of sulforaphane for 12 months that reduced bronchial Ki-67 index in former smokers at high risk for lung cancer [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 749.

Abstract Introduction: Higher consumption of dietary sulforaphane (SFN), a phytochemical found in many cruciferous vegetables, has been shown to be associated with lower risk of lung cancer (LC). SF activates the NRF2-pathway, which initiates cellular detoxification, antioxidant, and anti-inflammatory responses. To date, no chemoprevention trial for LC has examined the impact of SFN on LC-related gene expression (GE) as an endpoint in nasal and bronchial tissue. The objective of our study was to examine whether oral intake of SF for 12 months impacts the GE of previously identified LC- and premalignant lesions (PML)-related genes in bronchial and nasal brushing samples. Methods: A randomized, placebo-controlled clinical trial (NCT03232138) was conducted in former smokers with ≥ 30 pack-years and quitting smoking for 1-10 years in Western Pennsylvania, USA. Forty-three subjects were randomly assigned to the SFN treatment (n = 21) or placebo group (n = 22) in 2018-2022. Each participant was instructed to take 4 Avmacol® or identical placebo capsules BID for 12 months. Each Avmacol® capsule contained 15 mg of glucoraphanin, which yielded a daily internal dose of 120 μmol SFN. Nasal swabs and bronchial brushings were obtained at baseline and after 12 months. Among the 37 participants who completed the study, RNA was extracted from their nasal swabs and bronchial brushings. We constructed single sample gene signature scores (GSS) using the GSVA algorithm for the following: 1) the NRF2-pathway, 2) genes associated with LC risk in bronchial biopsies, and 3) genes associated with PML. The genes in these sets were stratified by their up- or down-regulation in LC or PML. We evaluated the association between the interaction of SFN supplementation over time (pre-/post-treatment) on GSS using linear mixed models, adjusted for the fixed effects of age at baseline, RNA integrity number (RIN), packyears of smoking, and random effect of participant. Results: GE data was available for 31 participant pairs with bronchial and/or nasal samples. Within nasal tissue, SFN treatment increased both the GSSs of the NRF2-pathway (p=0.0089) and the downregulated LC-risk genes (p=0.0047), and decreased the upregulated LC-risk genes (p=0.012) over time, whereas SFN had no significant impact on GSSs of the NRF2-pathway and LC-risk genes in the bronchial tissue. No association between SFN treatment over time and the PML-related GSS was observed in nasal or bronchial tissue. Conclusion: Our initial analysis indicates that SFN treatment over 12 months impacted LC-associated GE in nasal tissue but not in bronchial tissue from former smokers. The discrepancy results may be due to small sample size and additional studies are warranted to confirm if SFN has direct impact on LC-risk related genes in bronchial tissue (NIH grant No. R01CA213123). Citation Format: Kathryn Demanelis, Avrum Spira, Yohana Kefella, Renwei Wang, Jennifer Adams-Haduch, Thomas W. Kensler, Jennifer E. Beane, David O. Wilson, Jian-Min Yuan. Impact of oral supplementation of sulforaphane for 12 months on lung cancer-related gene expression in former smokers at high risk for lung cancer: Results from a randomized placebo-controlled phase II clinical trial [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 748.

Abstract Microscopic vascular invasion (VI) is predictive of recurrence in stage I lung adenocarcinoma (LUAD) but is difficult to assess in resection specimens and cannot be accurately predicted prior to surgery. Thus, new biomarkers are needed to identify this aggressive subset of stage I LUAD tumors. To assess molecular and tumor microenvironment (TME) features associated with angioinvasive LUAD we profiled 171 resected stage I tumors with and without VI by RNA-seq, including 24 tumors by high-resolution spatial transcriptomics (stRNA-seq, 10x Genomics Visium). Visium capture areas were selected by an experienced thoracic pathologist to include invasive foci, tumor regions distal to foci, and tumors without invasive foci. We identified a molecular signature from the bulk RNA-seq discovery cohort (n=103) containing three gene expression clusters increased in VI+ stage I LUAD including genomic instability (C1), tissue remodeling (C2), and hypoxia (C3), and one increased in VI- (C4, immune surveillance). Analysis of the stRNA-seq revealed high inter-tumor patient heterogeneity, with most spots clustering by tumor identity, suggesting tumor-intrinsic properties. Scoring the stRNA-seq for VI signature clusters revealed that C2 was highly expressed outside the VI focus and declined as a function of distance toward it, while C1 and C3 increased. C2 was most strongly enriched in regions of desmoplastic but not normal stroma while C4 was enriched in normal-appearing adjacent lung and low-grade histologic patterns including lepidic and papillary. Given increased signature expression in regions of invasive tumors at a distance from VI foci, we leveraged the discovery cohort to develop a transcriptomic predictor of VI. We applied a nested cross-validation approach to select a machine learning model, which demonstrated an AUC of 0.86 for VI+ LUAD in our independent test set (n=60). In contrast, the predictor was unable to detect lymphatic invasion (LI) in VI- LUAD, suggesting a different molecular process may differentiate VI from LI. The VI-associated predictor increased across the spectrum of indolent to aggressive stage I LUAD histopathology and was predictive of recurrence-free survival, even in VI- LUAD, implying detection of pre-angioinvasive LUAD or VI foci that were missed during pathology review. Finally, we used RNA-seq data from multi-region sampling of stage I LUAD cases in TRACERx, a longitudinal study of NSCLC evolution, to evaluate the VI predictor’s robustness to intra-tumor heterogeneity (ITH). Across all tumors, we observed strong correlation between scores from randomly sampled tumor-matched regions (n=136; R = 0.91, p < 2.2e-16). The difference of scores between unmatched regions (inter-tumor heterogeneity) was significantly higher than matched regions (ITH) (p < 2.2e-16). Our study suggests that VI-associated gene expression extends from the site of intravasation and can be used to predict the presence of VI. This may enable the prediction of angioinvasive LUAD from biopsy specimens, allowing for more tailored treatment prior to surgery. Citation Format: Dylan Steiner, Lila Sultan, Jason Weis, Travis Sullivan, Emily Green, Hanqiao Liu, Sherry Zhang, Gang Liu, Avrum Spira, Sarah Mazzilli, Kimberly Christ, Eric Burks, Jennifer Beane, Marc Lenburg. Spatially informed profiling of stage I lung adenocarcinoma reveals an extensive gene expression signature of vascular invasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B025.

BACKGROUND: Accurate assessment of the probability of lung cancer (pCA) is critical in patients with pulmonary nodules (PNs) to help guide decision-making. We sought to validate a clinical-genomic classifier developed using whole-transcriptome sequencing of nasal epithelial cells from patients with a PN <= 30 mm who smoke or have previously smoked. RESEARCH QUESTION: Can the pCA in individuals with a PN and a history of smoking be predicted by a classifier that uses clinical factors and genomic data from nasal epithelial cells obtained by cytologic brushing? STUDY DESIGN AND METHODS: Machine learning was used to train a classifier using genomic and clinical features on 1,120 patients with PNs labeled as benign or malignant established by a final diagnosis or a minimum of 12 months of radiographic surveillance. The classifier was designed to yield low-, intermediate-, and high-risk categories. The classifier was validated in an independent set of 312 patients, including 63 patients with a prior history of cancer (other than lung cancer), comparing the classifier prediction with the known clinical outcome. RESULTS: In the primary validation set, sensitivity and specificity for low-risk classification were 96% and 42%, whereas sensitivity and specificity for high-risk classification was 58% and 90%, respectively. Sensitivity was similar across stages of non-small cell lung cancer, independent of subtype. Performance compared favorably with clinical-only risk models. Analysis of 63 patients with prior cancer showed similar performance as did subanalyses of patients with light vs heavy smoking burden and those eligible for lung cancer screening vs those who were not. INTERPRETATION: The nasal classifier provides an accurate assessment of pCA in individuals with a PN <= 30 mm who smoke or have previously smoked. Classifier-guided decision-making could lead to fewer diagnostic procedures in patients without cancer and more timely treatment in patients with lung cancer.

Results of pathway enrichment using ROAST (FDR<0.05). The column "Direction" refers to pathway enrichment among genes up-regulated (Up) or down-regulated (Down) in the presence of PMLs.

PDF file - 690KB, Supplementary Figure S1. Laser capture microdissection. Supplementary Figure S2. Experimental validation of PTBP3 expression. Supplementary Table S1. Patient demographics. List of SCC patients with their age, gender, grade of premalignant lesion, and assays performed. Supplementary Table S2. Alignment statistics. The number and percentage of reads aligning uniquely to the nuclear (autosomal and sex) or mitochondrial chromosomes, or not uniquely aligned, are shown for each sample.

Supplementary Table 4 from Characterizing the Impact of Smoking and Lung Cancer on the Airway Transcriptome Using RNA-Seq