Upregulation of an Epithelial miRNA Is Associated with Immune Evasion in Progressive Bronchial Premalignant Lesions.

Squamous cell carcinoma (SCC) of the lung is a leading cause of cancer mortality in the U.S. due to late-stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular alterations involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs progress to carcinoma. We hypothesize that molecular characterization of PMLs and nonlesion areas will allow us to identify alterations associated with histology and lesion progression. We used mRNA sequencing to profile biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n=49), a brushing of the airway field (normal fluorescing area) and endobronchial biopsies were collected over time in repeat locations with serial bronchoscopies. The discovery cohort, included 29 subjects, 197 biopsies, and 91 brushes, while the validation cohort included 20 subjects, 111 biopsies and 49 brushes. The mRNA-Seq data were aligned to hg19 using STAR, and gene/transcript levels were summarized using RSEM. Immune, stromal, and epithelial cell content were inferred using xCell. Biopsy molecular subtypes were discovered using consensus clustering in the discovery cohort and used to train a nearest centroid subtype predictor to assign subtypes in the validation cohort and the brushes. We identified four distinct molecular subtypes in the discovery cohort bronchial biopsies using genes (n=3936) co-expressed across the the discovery cohort brushes and biopsies and two additional RNA-seq lung SCC-related datasets. One of the four molecular subtypes is enriched (p<0.001) for samples with dysplasia histology, high basal cell content, and the classic SCC tumor subtype. These associations are replicated in the validation cohort. There is also significant concordance between the molecular subtypes defined in the biopsies and their corresponding brush from the normal-appearing airway. Genes associated with IFN-gamma signaling and T cell-mediated immunity were observed to be downregulated among lesions that remained stable or progressed to more severe histology within the high-grade subtype. Staining adjacent biopsies to those profiled revealed that decreased expression of genes related to T cell-mediated immunity is associated with decreased numbers of CD4+ and CD8+T cells within the lesions and the surrounding tissue. We have identified four molecular subclasses of premalignant lung SCC lesions that may associate with prognosis. Molecular classification of PMLs may lead to biomarkers of future disease progression that could be used to stratify patients into prevention trials and to monitor efficacy of the treatment. Additionally, the results suggest that personalized lung cancer chemoprevention that targets specific cancer-related pathways or the immune system may have potential therapeutic benefits. Citation Format: Jennifer E. Beane, Sarah Mazzilli, Ania Tassinari, Joshua Campbell, Christopher Moy, Michael Schaffer, Catalina Perdomo, David Jenkins, Mary Beth Pine, Gang Liu, Sherry Zhang, Hangqio Lin, Jessica Vick, Evan Johnson, Suso Platero, Christopher Stevenson, Marc Lenburg, Mary Reid, Samjot Dhillon, Avrum Spira. Bronchial premalignant lesions have distinct molecular subtypes associated with future histologic progression [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A05.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.

Objective: B cells and plasma cells (PCs) play a vital role in the pathogenesis of lung cancer and provide prognostic predictive value. However, the role of B cells in the bronchial premalignant lesions (PMLs), the precursor of lung squamous cell carcinoma, is poorly understood. Prior work has shown that progression of proliferative subtype, a PML molecular subtype enriched with bronchial dysplasia, to a higher histology grade is associated with downregulation of antigen processing and presentation. In this study, we seek to understand the role of B cells, PCs, and the B-cell receptor (BCR) repertoire in progression of the bronchial PML. Methods: We performed bulk targeted BCR sequencing on 69 endobronchial biopsies obtained from 29 subjects at high-risk for developing lung cancer. The Immcantation pipeline were applied to obtain the V(D)J germline segment assignment using the human reference from IMGT, clonal cluster assignment, and mutational load quantification for each BCR sequences by pooling all the BCR sequences from the same subjects. The clones with frequency less than 10-4 were filtered out. We then performed the repertoire analysis, such as clonality diversity, isotype switching frequency, and somatic hypermutation rate (SHM) rate at sample levels and investigated the association with transcriptional signature derived from bulk RNA-seq and the clinical progression of PMLs. Results: The B cell and PC transcriptional signatures and B cell chemoattractants gene expression are associated with progression in proliferative subtype PMLs. The number of BCR clones, the class switch from IgG3 to IgG1, and the mutational rates of total and CDR3 regions were positively correlated with the B cells and PCs transcriptional signatures. Among the 31 PMLs of proliferative subtype, regressive lesions showed higher proportions of IgG heavy chain usage, higher SHM rate in CDR3 and total BCR regions, and higher frequency of class switch from IgD/M to IgG and from IgG3 to IgG1. Biopsies within the same patient had a higher proportion of shared BCR clones when they were sampled at the same timepoint than when they were sampled from the same anatomic location at different timepoints. The preserved clones of BCRs at the same anatomic location over different times had higher clonality, more class switch from IgD/M to IgG or IgA and from IgG3 to IgG1, and increased SHM rates than the non-preserved clones. Conclusions: These results suggest B and plasma cells exert anti-tumor effects that prevent bronchial PMLs from progressing to higher grade lesions by switching to IgG1 BCRs and increasing SHM rates. Citation Format: Darren J. Chiu, Carter Merenstein, Marc Lenburg, Sarah Mazzilli, Jennifer Beane. The role of B-cell receptor repertoire in lung squamous premalignant lesions [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 6097.

Lung cancer is the leading cause of cancer deaths in developed countries. Recently, autofluorescence bronchoscopy has been reported to improve the early detection of lung cancer in high-risk individuals. In the present study, we evaluated the efficacy of autofluorescence bronchoscopy for the early detection of bronchial premalignant and malignant lesions. From November 2000 through March 2004, 123 high-risk individuals (114 men and 9 women with a mean age of 68 years) were enrolled. Among 282 biopsy specimens, 93 (33.0%) were premalignant or malignant lesions. The sensitivity and negative predictive value for the detection of bronchial premalignant and malignant lesions were significantly higher with the addition of autofluorescence bronchoscopy than white light bronchoscopy alone. Moreover, the sensitivity for the detection of bronchial premalignant lesions was extremely higher with the addition of autofluorescence bronchoscopy than white light bronchoscopy alone, whereas there was no significant difference between autofluorescence bronchoscopy and white light bronchoscopy alone for the detection of non-malignant and malignant lesions. Autofluorescence bronchoscopy is a novel modality for the early detection of bronchial abnormality, especially for bronchial premalignant lesions.

Introduction: Bronchial premalignant lesions (PMLs), precursors to lung squamous cell carcinoma, represent a relevant system for studying early tumor development. Some PMLs will progress through increasing grades of dysplasia to become squamous cell carcinoma, while others will regress back to normal airway without intervention. Previous work has identified molecular subtypes of PMLs and identified immune alterations associated with lesion progression/persistence and regression. Here, we used gene expression profiling to identify genes that may be responsible for immune suppression or activation in PMLs. Methods: We collected endobronchial biopsies (n=295) longitudinally from 50 high-risk smokers in approximately one-year intervals at Roswell Park Comprehensive Cancer Center. Two biopsies were collected from each lesion, one for standard pathological assessment and one for RNA sequencing. We derived eight immune-related gene expression phenotypes based on immune-oncologic gene coexpression signatures taken from literature sources. Mediation analysis was used to identify genes that may be upstream modulators or downstream effectors of the association between immune phenotypes and lesion progression/persistence. Gene-Set Enrichment Analysis (GSEA) and Virtual Inference of Protein-activity by Enriched Regulon analysis (VIPER) were used to identify specific signaling and transcription pathways that these mediators were acting through. Results: Predicted immune phenotypes related to B cell activation, interferon signaling, and antigen presentation were significantly associated with lesion progression/persistence within the Proliferative molecular subtype (FDR ≤ 0.01). Via mediation analysis, we identified 15 genes as candidate immune regulators or downstream immune effectors essential for activation/suppression of the immune response in PMLs. One of these genes, GSTP1, is up-regulated in progressing lesions and negatively correlated with several immune activation pathways, thus representing a possible mechanism for immune suppression in these lesions. Further in silico analysis suggests that GSTP1 acts as a repressor of STAT3 signaling and NF-κB pathways, and in vitro experiments are underway to determine if suppression of GSTP1 is sufficient to induce expression of these immune pathways. Conclusion: Therapies are needed to intercept lung cancer development and these results suggest a targetable pathway to activate the immune system to stimulate PML regression. Citation Format: Carter Merenstein, Julia Breda, Sarah Mazzilli, Eric Burks, Christopher Stevenson, Mary Reid, Avrum Spira, Marc Lenburg, Jennifer Beane. Mediators of early immune response in bronchial premalignant lesions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1098.

The molecular events involved in the development of bronchial premalignant lesions (PMLs), and their progression to lung squamous cell carcinoma, are not well understood. Prior work characterized lung PML molecular subtypes by identifying co-expressed gene modules associated with histologic severity and progression/persistence. The proliferative subtype was enriched for PMLs with dysplasia. Genes related to interferon signaling and antigen processing/presentation (Module 9) were decreased in progressive/persistent lesions within this subtype, suggesting early immune suppression is related to PML progression. However, the mechanisms that drive these alterations are unclear. We investigated the role of microRNAs (miRNAs) in regulating gene expression associated with PML outcomes. mRNA and miRNA were extracted and sequenced from longitudinally collected endobronchial biopsies from patients with PMLs (148 samples, 30 patients). miRNAs targeting each gene co-expression module were identified based on target gene enrichment and the degree of negative correlation between the miRNA expression and its targets. Expression association with outcome within the proliferative subtype was tested with a mixed effects model adjusting for batch and patient as random effect. Cell type specificity of miRNA expression was tested based on cell type specific sequencing data from FANTOM5 and cell type marker correlation analysis. Genes regulated by NLRC5 were identified with ChIPseq data from Ludigs et al. Target gene suppression was confirmed by transfecting SW900 cells with miR-149-5p. miR-149-5p level in PML biopsies was examined by miRNA in situ hybridization (miR-ISH). miR-149-5p is identified as potential regulator of Module 9 gene expression and is significantly up-regulated in progressive/persistent PMLs. Its expression is highly enriched in epithelial cells in FANTOM5 and positively correlates with basal cell markers within PMLs. Predicted targets of miR-149-5p are down-regulated in the progressive PMLs in both our and data from Merrick et al. MHC-I and related gene expressions are down-regulated in progressing/persistent PMLs. These genes are regulated by the transcriptional coactivator NLRC5 which is a predicted target gene of miR-149-5p. We find that overexpressing miR-149-5p in SW900 cells decreases the expression levels of both NLRC5 and NLRC5 regulated genes. Additionally, miR-ISH targeting miR-149-5p in proliferative biopsy samples confirm its expression in epithelium compartment and association with outcome. Our data suggest epithelial miR-149-5p might be a key regulator of gene expression contributing to PML progression. We hypothesize that by suppressing NLRC5, miR-149-5p inhibits MHC-I gene expression of epithelial cells, promoting early immune depletion and lesion progression. miR-149-5p might therefore be a therapeutic target for preventing PML progression. Citation Format: Boting Ning, Roxana M. Pfefferkorn, Gang Liu, Sherry Zhang, Hanqiao Liu, Christopher Stevenson, Sarah A. Mazzilli, Avrum E. Spira, Marc E. Lenburg, Jennifer E. Beane. The role of epithelial miR-149 in immune modulation and progression of bronchial premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1488.

Background: Sampling the bronchial airway during bronchoscopies in screening populations at high risk for lung cancer has increased detection of squamous bronchial pre-malignant lesions (PML). The majority of these lesions will regress or remain stable without any clinical intervention. However, a subset of lesions will progress to invasive malignancy. In this work, we investigate the gene expression changes in epithelial cell states and immune cell proportion shifts associated with premalignant histologic stage using single-cell RNA sequencing to begin to identify molecular features associated with PML regression or progression. Methods: Cells (n=3,325) were isolated from 11 endobronchial biopsies from 9 patients, where biopsy histology range from normal, dysplasia, carcinoma in situ (CIS) and squamous cell carcinoma. Immune cells (CD45+) and epithelial cells (CD45− EPCAM+) were sorted into separate 96-well plates using fluorescence activated cell sorting (FACS) and sequenced using CEL-Seq2 single cell RNA seq protocol. Results: After filtering low quality cells, we profiled 2,998 cells (1,052 CD45+ and 1,946 CD45−) with an average of 1,190 genes per cell. Within the epithelial cells, we focused on basal cells as they are the airway progenitor cells. We observed distinct differences in expression of KRT5+ (basal) cells between different histologic grades, where KRT5+ cells present in a hyperplasic lesion expressed multiple secretory cell markers suggesting they were transitioning to a secretory cell phenotype and KRT5+ cells from a CIS lesion from a current smoker expressed higher levels of smoking inducible genes, such as GSTM1 and CYP1A1. Lastly, KRT5+ cells from a lung squamous carcinoma tumor expressed higher levels of many genes related to cell cycle progression. Among the immune populations, low-grade PMLs with non-dysplastic histology were enriched for CD8+ T cells, whereas higher-grade PMLs and an invasive tumor were enriched for myeloid cells. Conclusions: To date, we have begun to identify histology-associated cellular and molecular profiles in bronchial premalignancy and early-stage carcinoma. Future resampling of these patients and expansion of cases will allow us to discover biomarkers associated with lesion progression and molecular targets for lung cancer interception. Citation Format: Conor Shea, Lukas Kalinke, Kitty De Jong, Kate Gowers, Diane Ding, Sherry Zhang, Gang Liu, Jack Cunningham, Ipsita Dey-Guha, Mark Hennon, Sai Yendumuri, Christopher Stevenson, Steven Dubinett, Avrum Spira, Marc Lenburg, Samuel Janes, Mary Reid, Jennifer Beane, Sarah Mazzilli, Joshua D. Campbell. Cellular and molecular changes associated with lung squamous premalignant lesion severity identified by single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2212.

102 Background: The immune response to frank malignancy has been the focus of published work, but little is known about the adaptive immune response to bronchial premalignant lesions (PMLs), precursors of lung squamous cell carcinoma. This study was designed to characterize the T cell receptor (TCR) repertoire in PMLs and its association with clinical, pathological and molecular features. Methods: Endobronchial biopsies (n=294) and brushings (n=137) from high-risk subjects (n=50), undergoing lung cancer screening at approximately 1-year intervals via auto fluorescence bronchoscopy and CT, were profiled by RNA-Seq as part of the Pre-Cancer Genome Atlas (PCGA). We applied the TCR Repertoire Utilities for Solid Tissue/Tumor (TRUST) tool to identify TCR CDR3 sequences in the RNA-Seq data. We quantified the ratio of private (i.e., found in one patient only) and public (i.e. found in two or more patients) TCRs that are found in curated databases with known antigen specificities. We measured the correlation of TCR diversity with previously derived PML transcriptional signatures and molecular subtypes; and with mutational burden among a subset of biopsies (n=115) also profiled with whole exome sequencing. Results: We detected 40,421 unique TCR sequences, of which 3,396 (8.4%) were found in more than one sample and 1,057 (2.6%) were found in two or more patients (i.e. Public). TCRs with known antigen specificities were enriched among public TCRs (p < 0.001). In PMLs with a proliferative molecular subtype reflective of dysplasia (n=80), TCR diversity was decreased in PMLs that regressed versus PMLs that progressed (p=0.045). TCR diversity was negatively associated with a transcriptional signature of T-cell mediated immune activation (Spearman’s rho -0.26, p < 0.001) but was not associated with mutational burden. Conclusions: To our knowledge, this is the first characterization of the TCR repertoire associated with bronchial premalignant lesions. TCR diversity may help predict the efficacy of the host immune response to PMLs but it is not associated with mutational burden. Further studies are needed to leverage these findings and explore the potential for immunoprevention of PMLs.

Recently, the National Lung Screening Trial reported a 20% reduction in lung cancer mortality and lung cancer chemoprevention trials targeting the arachidonic acid pathway have demonstrated decreases in lung cancer associated markers. These studies highlight possibilities for future reductions in lung cancer mortality through early detection and chemoprevention. Our group has previously identified smoking- and lung cancer-specific gene expression alterations in cytologically normal airway epithelial cells that can serve as a clinically-relevant biomarker for the early detection of lung cancer. Here, in an effort that could lead to markers of lung cancer risk, we identify changes in these cells associated with regression of premalignant airway lesions. Airway epithelial cells were collected via bronchoscopy from patients with bronchial dysplasia at baseline, on-treatment, and post-treatment with green tea extract (GTE) or placebo ranging from 2 to 6 months (n=27 patients, n=63 samples). RNA from the samples was processed and hybridized to Affymetrix Human Gene 1.0 ST microarrays. A linear mixed effect model was used to identify a gene expression signature predictive of subsequent dysplasia regression. Further a paired t-test was used to identify genes associated with dysplasia regression over time. Using gene set enrichment analysis (GSEA) we identified that the baseline dysplasia regression signature was enriched (FDR<0.05) among genes whose altered expression was associated with: dysplasia regression over time; the presence or future development of lung cancer; and human bronchial biopsies at successive morphological stages of lung squamous carcinogenesis. The genes associated with dysplasia regression were validated small cohorts of independent samples from chemoprevention trials testing Sulindac, Myo-inositol, and GTE. Analysis of the Connectivity Map identified compounds that reverse the dysplasia regression signature in vitro and are therefore candidate chemoprevention agents. Our studies suggest that the airway “field of injury” is modulated by bronchial premalignant lesions. The molecular signatures identified may be important tools for stratifying high-risk smokers for chemoprevention trials, as surrogate endpoints of efficacy in these trials, and for identification of novel molecular targets for chemoprevention. In addition, the molecular signature of regression of airway dysplasia may have additional utility as a biomarker predictive of the presence of or future lung cancer development. Citation Format: Jennifer Ebel Beane, Kahkeshan Hijazi, Katrina Steiling, Gang Liu, Sherry Zhang, Stephen Lam, Marc Lenburg. Airway molecular alterations associated with premalignant lesion progression and lung cancer development. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A27.

Continuous carcinogen exposure through smoking creates a field of injury along the airway and resulting in mutations which promote remodeling of the bronchial epithelium. Over time lesions can form and exhibit increasing abnormalities progressing from goblet cell hyperplasia (GCH) which is the earliest detectable premalignant state through hyperplasia and squamous metaplasia to incremental stages of dysplasia, carcinoma in situ (CIS) and eventually to frank carcinoma. Unfortunately, more than half of all lung cancer patients are diagnosed with metastatic disease with a dismal 5-year survival rate of only 5.2%. However, when diagnosed at an early stage, the 5-year survival rate is more than 10 times as high. Our group has identified NOTCH1 as the most frequently mutated gene in progressive lung premalignant lesions (PMLs). However, these mutations are significantly less common in advanced lung squamous cell carcinoma (LUSC), indicating a role for NOTCH1 in the pre-tumorigenic environment rather than contributing to the actual tumor mass. Transcriptomic profiling of NOTCH1 mutated lesions identified up regulation of peri-goblet cells (PGC), a newly identified bronchial epithelial cell type that is associated with smoking, and a down regulation of immune components. Using highly multiplex Imaging Mass Cytometry (IMC) to analyze the epithelium of NOTCH1 mutated PMLs and its interplay with the immune system in situ. We identified a shift in epithelial composition with an increase in un- and de-differentiated cells. Interestingly, PGC appear to be an intermediate cell type on a previously undescribed path to goblet cell differentiation. In a parallel approach we are modeling NOTCH1 driven dysplasia in vitro to gain insight into the molecular mechanisms of lesion progression. Primary human bronchial epithelial cells (hBECs) grown at the air-liquid interface (ALI) into mature epithelia are exposed to cigarette smoke condensate (CSC) or IL-13 to model the early stages of bronchial airway dysplasia. Continuous exposure of these differentiating cultures to either stimulus increases the abundance of goblet cells and PGCs in a dose dependent manner with IL-13 additionally leading to the loss of baso-apical organization of the epithelium. After successfully modeling the first stage of bronchial epithelial dysplasia we are now advancing our model by co-culturing primary hBECs with NOTCH1 mutated cells and to disentangle the pathway of basal-PGC-goblet cell differentiation. Understanding driving forces in early lesion progression will further our ability to discern progressive lesions from those that regress naturally and facilitate interventions that force lesions into regression. Citation Format: Roxana Michaela Pfefferkorn, Darren Jianjhih Chiu, Divya Lakshmi Venkatraman, Jennifer Ellen Beane, Sarah Anne Mazzilli, Joshua David Campbell. The role of NOTCH1 in bronchial pre-malignant lesions. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR004.

Molecular and cellular alterations to the normal pseudostratified columnar bronchial epithelium results in the development of bronchial premalignant lesions representing a spectrum of histology from normal to hyperplasia, metaplasia, dysplasia (mild, moderate, and severe), carcinoma in situ and invasive carcinoma. Several studies have identified molecular alterations associated with lesion histology and progression. The broad and continuous spectrum of histologic and molecular changes makes reproducible stratification of lesions across multiple studies challenging. Here we propose a transformer-based framework that flexibly utilizes transcriptomic and histologic patterns to distinguish lesions with bronchial dysplasia or worse from normal, hyperplasia, and metaplasia. We leveraged H&E whole slide images (WSIs) of endobronchial biopsies and bulk gene expression data (GE) from previously published studies and on-going lung precancer atlas efforts obtained from patients as high-risk for lung cancer. Models trained using both WSIs and GE compared to a single data modality had higher performance. On an external testing dataset of WSIs, the area under the ROC curve (AUROC) of the model trained on WSIs plus GE was 0.761±0.015 compared to 0.690±0.027 for model trained on WSIs. On external testing datasets of GE, the AUROC of the model trained on WSIs plus GE was 0.890±0.023 versus 0.816±0.032 for a model trained on GE. Based on these results, we leveraged data across 4 studies to train a flexible fusion model that allows one or both data modalities to be used in training. The model achieved an AUROC of 0.809±0.036 on external testing WSIs data and 0.903±0.022 on external testing GE data. Despite model training on a binary label, model probabilities are associated with histologic grade and the model identifies gene expression alterations associated with bronchial dysplasia across multiple studies. This framework maps bronchial premalignant lesions that contain at least one data modality into a spectrum of disease. In the future, a framework trained on multiple data modalities may be useful in predicting premalignant disease severity, progression, and interception agent efficacy.

Endoscopic follow-up of low-grade precancerous bronchial lesions in high-risk patients: long-term results of the SELEPREBB randomised multicentre trial.

Background: Bronchial pre-malignant lesions (PMLs) are the putative precursors for bronchial squamous cell carcinoma. PMLs represent a spectrum of histologies, from low-grade lesions (hyperplasia, metaplasia) to high-grade lesions (dysplasia, carcinoma in situ). The majority of these lesions will regress or remain stable without clinical intervention while a subset of lesions will progress to invasive carcinoma. We performed single-cell RNA sequencing (scRNAseq) of these lesions to elucidate the cross-talk between epithelial, stromal, and immune populations in lesions of increasing histological grade. Methods: Thirty lesions from seventeen participants were biopsied via bronchoscopy. Cells were sorted by CD45+/- FACS gating and sequenced with the Cel-Seq2 protocol. Celda was used to bi-cluster genes into modules and cells into clusters. Cells were filtered by mitochondrial percentage (%mito < 50%), minimum UMI counts (nUMI > 300), and doublet detection. Cell types were labeled by marker gene expression. Results: After filtering low quality cells, we analyzed 4,382 cells. We observed expected smoking related shifts in epithelial cell type proportions, including an increase in secretory cells (χ2 = 31.39, p = 2.11 X 10-8) and a decrease in ciliated cells (χ2 = 4.83, p = 0.028) among current smokers. Distinct differences in expression of transcriptional modules were observed between KRT5+ (basal) cells from different histologic grades. Basal cells from high grade lesions expressed smoking detoxification and cell cycle gene programs, while low grade lesion basal cells expressed differentiation gene programs. We also identified a group of cells from CIS lesions involved in an epithelial-to-mesenchymal transition, marked by an increase in SPARC and COL4A1 expression and a decrease in CDH1 expression. Subpopulations of immune cells identified include macrophages, CD4/8+ T, B, dendritic cells, and natural killer cells. Several clusters of CD4+ and CD8+ T cells displayed an exhausted phenotype, marked by the expression of PD-1, CTLA4, LAG3, and TIGIT. Samples with high grade histology (dysplasia, carcinoma in situ) were enriched in CD4+ Tregs and myeloid cells compared to low grade histology samples (hyperplasia, metaplasia), which were enriched in Natural Killer and cytotoxic CD8+ T cells (χ2 = 298.95, p = 0.001). Discussion: Our results suggest that changes in specific transcriptional programs are associated with the transition of epithelial cells to more invasive states and that changes in immune populations are associated with increasing histological grade. These signatures can suggest novel avenues for chemoprevention and cancer interception. Citation Format: Conor Shea, Lukas Kalinke, Kitty De Jong, Kate Gowers, Diane Ding, Sherry Zhang, Gang Liu, Jack Cunningham, Ipsita Dey-Guha, Mark Hennon, Sai Yendamuri, Christopher Stevenson, Avrum Spira, Mary E. Reid, Marc E. Lenburg, Sam M. Janes, Jennifer E. Beane, Sarah A. Mazzilli, Joshua D. Campbell. Epithelial, stromal, and immune changes associated with lung squamous premalignant lesion severity identified by single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2201.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma. Prior work has shown that among proliferative subtype PMLs, progression to or persistence of a high-grade histology is associated with downregulation of an immune-related gene module. Furthermore, microRNA-149 (miR-149) was identified as a candidate negative regulator of this expression program. In this study, we aim to investigate the spatial distribution of miR-149 and its association with the immune environment and PML progression/persistence in PMLs. We identified 19 paraffin embedded bronchial biopsies transcriptionally defined as belonging to the proliferative subtype obtained from 9 subjects. Three consecutive sections were stained with H&E for histology annotation, chromogenic miRNA in situ hybridization (miRNA-ISH) for miR-149 abundance and localization, and with 30 antibodies targeted to epithelial and immune populations using imaging mass cytometry (IMC) to perform phenotyping. We trained a robust pixel classifier to detect foci of miR-149 signal in miRNA-ISH and quantified the density of miR-149 signal. For the IMC data, we used Mesmer for cell segmentation. PhenoGraph clustering based on quantification of each antibody in each cell was used for cell population identification. Ten recurrent cellular neighborhoods were identified by K-means clustering. miR-149 density was greater in the epithelium compared with stromal tissue. The miR-149 density in dysplastic epithelium normalized to miR-149 in normal epithelium is higher in the progressive/persistent PMLs than regressive PMLs. Additionally, in progressive/persistent PMLs, we find lower average epithelial staining for the miR-149 target NLRC5 associated with a cellular neighborhood that is enriched for goblet cells and PDL1+MUC5B+ secretory cells. Cell composition analysis showed that progressive/persistent PMLs have lower CD8 T cell infiltrate in both the stroma and epithelium. These results suggest that increased miR-149 expression in progressive/persistent PMLs contributes to an immune suppressive environment characterized by a shift in secretory cell populations and T cells within the bronchial epithelium. Interventions targeting miR-149 expression may be a potential interception strategy for high-grade PMLs. Citation Format: Darren J. Chiu, Roxana Pfefferkorn, Emily Green, Boting Ning, Myrtha Constant, Marc Lenburg, Eric Burks, Sarah Mazzilli, Jennifer Beane. Multiplexed immune phenotyping and miRNA-ISH in bronchial premalignant lesions reveals a mechanism of immune suppression. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR003.

Lung cancer is the leading cause of cancer death in the United States. Squamous cell lung cancers (SCC) comprise approximately 30% of lung cancer cases, and 48,000 people are projected to die this year in the U.S. from SCC. Bronchial dysplasia (BD) is a precursor of SCC, however not all BDs progress to carcinoma. Persistent or progressive BD is associated with a higher risk for the development of invasive lung cancer. We hypothesize that characterization of the difference between persistent and regressive BD will identify high risk patients and suggest novel therapeutic targets for prevention of progression to lung SCC. Gene expression analysis was used to identify differences between persistent and regressive BD. Subjects recruited to Colorado SPORE in Lung Cancer screening studies that met sputum atypia criteria and underwent multiple bronchoscopies were evaluated. Sixty-three frozen baseline biopsies showing BD by histology were classified as persistent/progressive (group 1) or regressive (group 2) according the presence or absence of BD on follow-up biopsies at the same sites. H&E staining was performed on frozen sections to confirm histology, and RNA was harvested and used to create gene expression arrays. PLK1 was selected as a gene of interest based on significantly increased levels of expression and pathway analyses implicating increased activity of cellular pathways associated with PLK-1 expression. Additionally, bronchial epithelial cells were cultured from fresh biopsies taken immediately adjacent to persistent or regressive bronchial sites, and RNA, DNA, and protein were harvested. qRT-PCR was performed on RNA extracted from these persistent and regressive biopsy epithelial cultures. PLK1 expression in epithelial cultures was shown to be increased in persistent as compared to regressive cell lines suggesting that this cell cycle control enzyme may represent a possible chemoprevention target in persistent/progressive dysplasias. RNA was also extracted from a variety of immortalized cancer cell lines of both squamous and adenocarcinoma subtypes, and qRT-PCR was used to establish baseline expression of these genes in cancer cells. Eleven of 20 lung cancer cell lines showed at least 20 fold higher PKL-1 expression compared to non-tumorigenic cell lines while 4 out of 4 lines of SCC origin showed 75 fold or higher expression further supporting a role for this enzyme in SCC progression. Citation Format: Micah Friedman, Meredith Tennis, Lori Dwyer-Nield, Robert Keith, Raphael Nemenoff, Wilbur Franklin, Mark Geraci, Michael Edwards, Sugita Michio, Dan Merrick. Biomarkers of progressive squamous lung dyplasia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2356. doi:10.1158/1538-7445.AM2013-2356