Development Of Premalignant Lesions Research Articles

Prevalence of oral premalignant lesions in the area with the highest prevalence of gastrointestinal cancer

Objective: According to research conducted in Iran, from 141450 cases of reported cancer between 2003-2006, 1896 cases of oral and lip cancer have been reported. The timely and prompt diagnosis of this disease reduces mortality, increases longevity, reduces damage to adjacent structures, reduces financial costs, and unwanted side effects. The current study aimed to investigate the prevalence of oral premalignant lesions in the oral cavity and the factors related to it in Golestan province. Methods: In this descriptive-analytic cross-sectional study, 1900 Iranian subjects living in Golestan province, regardless of gender, aged 40 and above, participated voluntarily and randomly. Initially, a questionnaire containing demographic information, assessment of habits and risk factors was completed for each patient in an interview form, followed by oral examination on all patients. Statistical analysis of data was done by SPSS 22. Results: The mean age of these individuals was 49.5 ± 10.3 years and the highest premalignant lesions were observed in the age range of 70-80 years. Of the 1900 participants, 69 (3.6%) had pre-malignant lesions. Among the pre-malignant lesions, the prevalence of lichen planus was 49.28%, leukoplakia was 14.49%, and the suspected malignancy was 18.8%. In addition, there was a significant relationship between the use of tobacco with the development of pre-malignant lesions (p <0.01). Cigarette alone showed the highest association with the development of pre-malignant lesions. Conclusions: In this study, there was a significant relationship between risk factors such as age, ethnicity and smoking with the development of premalignant lesions in people. However, risk factors such as alcohol consumption and family history did not show a significant relationship.

Cellular Senescence in Normal Mammary Gland and Breast Cancer. Implications for Cancer Therapy.

Cellular senescence (CS) is a major homeostatic biological process, which plays a key role in normal tissue development and provides protection from stressful cell insults. The role of CS in mammary-gland development and breast cancer is not well understood. While there is a lack of experimental data on the role of CS in the development of the pre-pubertal mammary gland, there is evidence for a biphasic senescence response in adult normal-mammary-epithelial cells, where the bypass of the first senescence barrier (M0) seems to be a key step in the development of premalignant lesions, with genetic abnormalities that resemble in situ breast carcinoma. Further, there is accumulating evidence for the role of cellular senescence in breast-cancer response, regarding treatment and patient outcome. Here, we review the current literature on cellular senescence, in epithelial-mammary cells, breast-cancer cells, and breast-tumor-microenvironment-resident cells. Furthermore, we discuss its putative role in breast-cancer response, regarding treatment and disease progression. In addition, we provide preliminary evidence of CS in breast-cancer-microenvironment cells, such as tumor-associated fibroblasts and tumor-infiltrating lymphocytes, by employing the novel GL13 lipofuscin stain, as a marker of cellular senescence.

Diagnostic Value of Cytokeratin 17 during Oral Carcinogenesis: An Immunohistochemical Study.

Background Little is known about the role of cytokeratin 17 (CK17) during oral carcinogenesis. CK17 expression in oral leukoplakia (OL), the most encountered oral potentially malignant disorders and oral squamous cell carcinoma (OSCC), remains very limited. To determine the role of CK17 during oral carcinogenesis and its potential diagnostic marker in oral premalignant and malignant lesions, this study evaluated CK17 expression in OL without dysplasia, OL with dysplasia, and OSCC. CK17 expression in these tissues was compared with those of normal oral mucosa (NOM). Additionally, the relationship between CK17 expression and clinicopathologic factors of OSCC was investigated. Methods CK17 expression was evaluated in 186 samples consisting of 12 NOM, 33 OL without dysplasia, 58 OL with dysplasia, and 83 OSCC using immunohistochemistry. The proportion of positively immunostained cells was evaluated and scored. Results CK17 was expressed in 8.3%, 54.5%, 74.1%, and 90.4% of NOM, OL without dysplasia, OL with dysplasia, and OSCC, respectively. NOM had a significantly lower CK17 score than OL with dysplasia (p=0.0003) and OSCC (p < 0.0001). A significant association between CK17 expression and histopathologic differentiation of OSCC was found. Tumors with well differentiation had high CK17 expression compared with those of moderate and poor differentiation. Conclusion CK17 was overexpressed in OL with dysplasia and OSCC, suggesting that CK17 plays a pivotal role in the development of premalignant lesions and OSCC. Of clinical significance, CK17 may be a good diagnostic marker for oral premalignant lesions and OSCC. Additionally, CK17 could be used as an objective tool to classify histopathologic grade in OSCC. The findings that CK17 expression is high in OSCC but low in NOM imply that CK17 may serve as a potential therapeutic target for OSCC.

Abstract A09: Pregnancy reprograms the epigenome of mammary epithelial cells and blocks the development of premalignant mammary lesions in response to cMYC-overexpression

Abstract Pregnancy instates a series of cellular and molecular modifications in the mammary epithelial cells (MECs) of the adult female mammary gland. Pregnancy is also known for decreasing the predisposition of rodent and human MECs to oncogenesis. In order to understand the molecular basis for this protective effect, we analyzed the epigenetic changes in the enhancer landscape of murine, post-pregnancy MECs, and their effect on gene regulation, tissue development, and oncogenesis. Using in vivo and in vitro models of normal and malignant mammary development, we found that completion of a pregnancy cycle changed the dynamics of cellular proliferation and gene expression in response to a second wave of pregnancy hormones. Our results also demonstrated that post-pregnancy MECs are resistant to the molecular programs driven by cMYC overexpression, a response that blocked the development of premalignant lesions but did not perturb the pregnancy-induced epigenomic landscape. Citation Format: Mary J. Feigman, Matthew A. Moss, Chen Chen, Samantha L. Cyrill, Michael Ciccone, Marygrace Trousdell, Shih Ting Yang, Wesley D. Frey, John E. Wilkinson, Camila O. dos Santos. Pregnancy reprograms the epigenome of mammary epithelial cells and blocks the development of premalignant mammary lesions in response to cMYC-overexpression [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A09.

IA06 Intercepting Lung Cancer by Understanding Premalignant Changes in the Airway Field

Lung cancer is the leading cause of cancer death. In order to decrease mortality, we need innovative strategies to intercept cancer development by diagnosing the disease at its earliest and potentially most curable stage. Development of lung cancer risk biomarkers and interception strategies requires a detailed understanding of the earliest molecular alterations involved in lung carcinogenesis that occur in the respiratory epithelium. Exposure to cigarette smoke creates a field of injury throughout the entire respiratory tract by inducing a variety of genomic alterations that can lead to an at-risk airway where lung squamous premalignant lesions develop. Lung squamous cell carcinoma arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions through a stepwise histologic progression from normal epithelium to hyperplasia, squamous metaplasia, dysplasia (mild, moderate, and severe), carcinoma in situ, and finally to invasive cancer. The presence of high-grade persistent or progressive dysplasia is a marker of increased lung cancer risk, although many lesions have varied outcomes. Recent molecular profiling of endobronchial biopsies representing a range of histologic scores revealed an upregulation of cell cycle, proliferation, and DNA repair pathways and downregulation of inflammation and immune-associated pathways in high-grade progressive/persistent lesions. This work provided a foundation with which to further our understanding of the mechanisms that drive these early alterations and to develop robust biomarkers to detect their presence and future behavior. Recent projects that build upon this work to elucidate the pathways responsible for histologic progression will be presented. These projects will be put into the context of the collaborative effort to create a lung precancer atlas that will include large-scale genomic, immunogenomic, and clinical multidimensional data. This larger effort will serve to both validate existing observations and biomarkers and further enhance lung cancer interception efforts.

Inhibitory Effect of Nelumbo nucifera Leaf Extract on 2-Acetylaminofluorene-induced Hepatocarcinogenesis Through Enhancing Antioxidative Potential and Alleviating Inflammation in Rats.

Leaf extract of Nelumbo nucifera (NLE) has been demonstrated to possess anti-atherosclerosis, improve alcohol-induced steatohepatitis, prevent high-fat diet-induced obesity, and inhibit the proliferation and metastasis of human breast cancer cells. This study determines the chemopreventive role of NLE against 2-acetylaminofluorene (AAF)-induced hepatocellular carcinoma (HCC) in rats. AAF was used to induce hepatocarcinogenesis in rats through genetic and nongenetic effects. After administration for 12 weeks, NLE (0.5–2%) supplementation orally inhibited AAF (0.03%)-induced hepatic fibrosis which appears during the development of premalignant lesions in rats. After the 6-month experiment, NLE supplementation resulted in decreasing AAF-induced serum parameters of hepatic injury, including the level of triglycerides, total cholesterol, alpha-fetoprotein (AFP), and inflammatory mediator interleukin (IL)-6 and tumor necrosis factor (TNF)-α as well as the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γGT). NLE supplementation also reduced AAF-induced lipid peroxidation and 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation in the rat liver. Hepatic histopathological investigation revealed that NLE supplementation attenuated the AAF-induced HCC and glutathione S-transferase-Pi (GST-Pi) expression. Furthermore, NLE supplementation increased the expression of transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream targets, including catalase, glutathion peroxidase (GPx), and superoxide dismutase 1 (SOD-1) in the rat liver. Our findings indicate that NLE supplementation inhibited AAF-induced hepatocarcinogenesis by enhancing antioxidative potential and alleviating inflammation in rats.

Helicobacter pylori: qué debemos saber en el año 2018. Revisión de la evidencia y nuevas recomendaciones de tratamiento

Helicobacter pylori (HP) is the most widely chronic human infection around the world, and&#x0D; the main risk factor for the development of gastric cancer. Our country has high rates of&#x0D; this neoplasia and a high prevalence of HP infection. Even both have fallen in the last year,&#x0D; is a major concern to diagnose the population infected with HP in early stages, before the&#x0D; development of premalignant lesions and properly eradicate this infection. In this review, we&#x0D; discussed the different methods for the diagnosis of HP and factors that change positivity&#x0D; as the use of proton pump inhibitors. Also, we discussed the factors to be considered in the&#x0D; choice of the treatment, like local resistance to antibiotics, specially clarithromycin. In the last&#x0D; years has been documented in Chile a significant increase in resistance to clarithromycin, from&#x0D; 20 to 46%, which predicts inadequate effectiveness for the classic triple therapy. As the result&#x0D; of the previous analysis we discussed new possible therapies, including bismuth quadruple&#x0D; therapy and concomitant therapy.

Abstract A05: Bronchial premalignant lesions have distinct molecular subtypes associated with future histologic progression

Abstract 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&amp;lt;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.

Abstract 3248: Genomic characterization of premalignant lung squamous cell carcinoma lesions

Abstract Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airway and is often preceded by the development of premalignant lesions. However, not all premalignant lesions progress to lung SqCC and many will regress spontaneously. Understanding the somatic alterations and molecular subtypes associated with progression will allow us to identify biomarkers for early detection and develop therapeutic strategies for disease prevention and interception. Methods: Biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute. For each subject, multiple sites were sampled repeatedly over time. One biopsy from each region was sent for pathological review while another biopsy was taken for molecular studies. Whole-exome sequencing (WES) was performed at Uniform Services University to 120x coverage and RNA-seq was performed at Boston University School of Medicine. Results: The median number of somatic mutations across all premalignant lesions that underwent DNA-seq (150 biopsies from 20 subjects) was 0.45 per megabase and displayed a modest association with histological grade (p=0.05). The most frequently mutated known lung cancer genes included NOTCH1 (14%), TP53 (6%), FAT1 (3%), PIK3CA (2%), KRAS (&amp;lt;1%), and CDKN2A (&amp;lt;1%). One patient had a moderate dysplastic lesion without any detectable arm-level copy number changes or known cancer mutations. Six months later, this lesion had progressed to severe dysplasia and obtained many genomic alterations commonly observed in squamous cell carcinoma including 3q gain, 3p loss, and mutations in TP53, NOTCH1, and CDKN2A. Using RNA-seq, we identified 4 distinct molecular subtypes using 197 biopsies from 29 subjects. One subtype was enriched for samples with dysplasia histology, high basal cell content, and the “Classical” SqCC tumor gene expression subtype (p&amp;lt;0.001). These associations replicated in an independent set of 111 biopsies from 20 subjects. Genes associated with IFN-gamma signaling and T cell mediated immunity were down-regulated among lesions that persisted or progressed vs. those that regressed within the high-grade subtype. Staining of adjacent biopsies revealed that decreased expression of these immune pathways was associated with decreased numbers of CD4+ and CD8+ T cells within the lesions and surrounding tissue. Conclusions: The somatic alterations observed in known cancer genes may be among the earliest events in lung SqCC development and may be useful as biomarkers for early detection. Molecular classification of these lesions into molecular subtypes may lead to biomarkers of disease progression that could be used to identify at-risk patients for aggressive surveillance or for prevention trials. Citation Format: Joshua D. Campbell, Xijun Zhang, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Samjot S. Dhillon, Gang Liu, Sherry Zhang, Hanqiao Liu, Jessica Vick, Christopher Moy, Stefano Monti, Evan Johnson, Matthew Meyerson, Matthew Wilkerson, Clifton Dalgard, Suso Platero, Chris Stevenson, Marc Lenburg, Mary Reid, Jennifer Beane, Avrum Spira. Genomic characterization of premalignant lung squamous cell carcinoma lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3248.

Immunohistochemical Evaluation of Bcl-2 and Ki-67 in Buccal Mucosa of Induced- Immunosuppressed Albino Rats

Introduction: Organ transplantation provides life-saving therapy for patients with end-organ disease. Tacrolimus is an immunosuppressive drug used to lower the risk of organ rejection. This study was performed to investigate the immunohistochemical expressions of Ki-67 (a proliferation associated antigen) and Bcl-2 (antiapoptotic protein) and their correlation with the increased risk of development of neoplasms in the buccal mucosa of tacrolimus-induced immunosuppressed albino rats. Material & methods: Twenty adult male albino rats were divided into two equal groups. Group one treated with tacrolimus in a daily oral dose 0.5 mg/kg for 3 months. Group two served as negative control, received distilled water in comparable volume. Tissue samples were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin to evaluate histopathological finding. Other sections were used to reveal the immunohistochemical expression of Ki-67 and Bcl-2 antibodies through computerized image j analysis software. The expression of both markers was analyzed statistically through using Mann-Whitney test. Results: Histopathological finding showed moderate dysplasia in tacrolimus-treated group. There was marked increase in normal mitotic figures, basal cell hyperplasia, loss of basal cell polarity, swirling of the spinous layer, prominent nucleoli, hyperchromatism, and altered N/C ratio. Immunohistochemical finding revealed marked increase in the nuclear expression of Ki-67 in basal cells and cytoplasmic expression of Bcl-2 in all layers of keratinocytes in tacrolimus-treated group. Statistical finding in tacrolimus – treated group showed significant difference in the expression of Ki-67 and highly significant difference in the expression of Bcl-2. Conclusion: There is an obvious relation between prolonged systemic use of tacrolimus and development of premalignant lesions. The marked increase in Ki-67 and Bcl-2 expression may play a role in tumorgenesis.

A Large Set of miRNAs Is Dysregulated from the Earliest Steps of Human Hepatocellular Carcinoma Development

Hepatocellular carcinoma (HCC) typically results from a stepwise process characterized by the development of premalignant lesions, such as low- or high-grade dysplastic nodules (LGDNs and HGDNs, respectively), in a cirrhotic setting. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional regulation of gene expression that can act as oncogenes or tumor suppressors. Whether and which miRNAs are involved in the early stages of HCC development remains elusive. Here, small-RNA sequencing was applied to profile miRNA expression in 55 samples (cirrhotic nodules; CNs), LGDNs, HGDNs, early HCCs, and small progressed HCCs, obtained from 17 patients bearing HCCs of different etiologies. An miRNA expression signature of 62 miRNAs distinguishing small progressed HCCs from matched CNs was identified. Interestingly, 52 of these miRNAs discriminated CNs from LGDNs/HGDNs, regardless of etiology, and remained modified along the tumorigenic process. Functional analysis of the predicted mRNA targets of deregulated miRNAs identified common modifications between the early and late stages of HCC development likely involved in the stepwise process of HCC development. Our results demonstrate that miRNA deregulation happens very early in HCC in humans, implying their crucial role in the tumorigenic process. The identification of miRNAs discriminating CNs from neoplastic nodules may have relevant translational implications in early diagnosis.

Abstract 5002: Premalignant squamous cell lung carcinoma lesions have distinct molecular subtypes associated with histologic progression

Abstract Background: Squamous cell carcinoma (SCC) of the lung is a leading cause of cancer mortality in the US 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 events involved in the progression of PMLs to lung SCC are not clearly understood, and not all PMLs go on to form carcinoma. By molecularly characterizing PMLs and non-lesion areas in the airway of individuals with PMLs, we hypothesize that we will be able to identify subgroups of PMLs that are more likely to progress. Methods: 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=30), we sampled bronchial biopsies repeatedly over time (394 +/- 170 days) with serial bronchoscopies (6 +/- 5 biopsies/subject) as the biopsied area progressed towards or regressed away from frank malignancy. mRNA-Seq (n=197 biopsies) data was aligned to hg19 using STAR, and gene/transcript levels were summarized using RNA-Seq using RSEM and Ensembl 74 annotation. Immune, stromal, and epithelial cells content were inferred using the ESTIMATE algorithm and pathway activity of in vitro derived oncogenic signatures was estimated using GSVA for each sample. Molecular subtypes were derived using non-negative matrix factorization (NMF) and consensus clustering. Linear modeling was used to associate PML outcome metrics and pathway activity scores with subtype membership. Results: We identified six distinct molecular subtypes of bronchial biopsies using NMF across the 10% most variable genes (n=2,322 gene). One subtype contained samples with stable high-grade histology (p&amp;lt;0.01). This subtype was also enriched for basal epithelial cell content and oncogenic pathway activity including AKT and EGFR (p&amp;lt;0.01). Another subtype was enriched for immune cell content and had elevated expression of immune-related pathways including chemokine, cytokine, and T-cell receptor signaling. Increased immune infiltrate was also observed in H&amp;E stains from biopsies taken adjacent to the sequenced biopsy. Conclusions: Molecular classification of premalignant lesions may lead to biomarkers of 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 interventions targeting specific cancer-related pathways or the immune system may be have potential therapeutic benefits. Citation Format: Jennifer E. Beane, Sarah Mazzilli, Joshua Campbell, Christopher Moy, Michael Schaffer, Catalina Perdomo, David Jenkins, Gang Liu, Sherry Zhang, Hanqiao Liu, Jessica Vick, Evan Johnson, Suso Platero, Marc Lenburg, Mary Reid, Samjot S. Dhillon, Avrum Spira. Premalignant squamous cell lung carcinoma lesions have distinct molecular subtypes associated with histologic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5002. doi:10.1158/1538-7445.AM2017-5002

Abstract 3259: The genomic landscape of premalignant lung squamous cell carcinoma lesions

Abstract Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airway and is often preceded by the development of premalignant lesions. However, not all premalignant lesions progress to lung SqCC and many regress without therapeutic intervention. Understanding the somatic alterations that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention. Methods: Airway biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and chest CT at the Roswell Park Cancer Institute. For each subject (n=30), multiple premalignant lesions were sampled repeatedly over time (n=144 samples). One biopsy from each region was sent for pathological review while another biopsy was taken for molecular studies. DNA was also isolated from the blood or cytologically normal bronchial brushings to serve as a matched normal control. Exome capture was performed using the Illumina TruSeq Rapid Exome kit and sequenced to a mean depth of coverage of 120x at Uniform Services University and Walter Reed National Military Medical Center. Results: The median number of somatic mutations across all premalignant lesions was 0.73 per megabase (range: 0.10 - 9.8 per Mb) and displayed a modest association with histological grade (p=0.07). The most frequently mutated lung cancer genes included KMT2C (12%), NOTCH1 (11%), FAT1 (6%), TP53 (5%), and CDKN2A (&amp;lt;1%). Known oncogenic hotspot mutations were observed in PIK3CA (1%) and KRAS (&amp;lt;1%). The majority of lesions did not have overlapping sets of mutations with other samples from the same patient, indicating that most of these lesions arose from clonally distinct populations. The two lesions with the relatively high mutation rates (&amp;gt;7/Mb) were taken from adjacent sites over two time points in the same individual with a prior history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations including FAT1 indicating a common evolutionary ancestor. Conclusions: The somatic alterations observed in known cancer genes such as TP53, KMT2C, NOTCH1, and FAT1 may be among the earliest driver events in lung SqCC development and may be useful as biomarkers for early detection as well as targets for lung cancer interception. Citation Format: Joshua Campbell, Xijun Zhang, Samjot S. Dhillon, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Gang Liu, Sherry Zhang, Hanqiao Lin, Jessica Vick, Christopher Moy, Stefano Monti, Evan Johnson, Matthew Meyerson, Steven Dubinett, Suso Platero, Matthew Wilkerson, Clifton Dalgard, Marc Lenburg, Mary Reid, Jennifer Beane, Avrum Spira. The genomic landscape of premalignant lung squamous cell carcinoma lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3259. doi:10.1158/1538-7445.AM2017-3259

Abstract 1467: A large set of miRNAs is deregulated since the earliest steps of human HCC

Abstract Introduction: Hepatocellular carcinoma (HCC) is the result of a stepwise process preceded by the development of premalignant lesions. To assess the involvement of microRNAs (miRNAs) in hepatocarcinogenesis it is critical to understand whether and which miRNAs are involved in the early stages of tumor development. To this end, changes in expression of miRNAs were investigated in low and high grade dysplastic nodules (LGDNs, HGDNs), early HCCs (eHCCs) and progressed HCCs (pHCCs). Materials and Methods: Small RNA sequencing (smallRNA-Seq) was applied to search for liver miRNAs and to profile their expression patterns in 14 cirrhotic nodules (CNs), 9 LGDNs, 6 HGDNs, 6 eHCCs and 20 pHCCs) from 17 patients with liver lesions of different etiology (HCV, HBV and alcohol). To investigate miRNAs differentially expressed in pHCCs towards their matched cirrhotic tissue, we applied the Random-Variance Model (F-test) and Multivariate Permutation Test (Fold Change, FC ≥ 2.0). QRT-PCR analysis was also performed on 14 additional patients with the same etiology of the first cohort for validation of the smallRNA-Seq results. Results: We identified a 62 miRNA expression signature that differentiates pHCCs from matched CNs. Notably, 52/62 miRNAs dysregulated in pHCCs were likewise altered in LGDN and HGDN, suggesting their possible role in HCC onset. Indeed, 28 miRNAs showed lower expression in CN that was significantly increased in LGDNs and remained high up to pHCCs. In contrast, 24 miRNAs displayed an opposite behavior, as their expression was very low or absent all throughout the tumorigenic process, compared to CNs. Interestingly, 2 miRNAs (let-7a-5p, miR-101-3p) showed a progressive decrease, suggesting that they are involved in later stages of the process. To further support the finding that dysregulation of miRNAs is a very early event in HCC development, we performed qRT-PCR in a series of LGDN, HGDN, eHCC and pHCCs from 14 additional patients. We selected for this analysis a handful of miRNAs found dysregulated by NGS analysis. The results demonstrate that all the 6 miRNAs examined (miR-429, miR-141-3p, miR-200a-3p and miR-200b-3p, miR-375 and miR-21-5p), were dysregulated at all stages of the tumorigenic process as compared to CNs. Conclusion: The results demonstrate that miRNAs deregulation may be a critical player in human hepatocarcinogenesis, from early stages to pHCC. They also suggest that some miRNAs might represent biomarkers useful for differential diagnosis of dysplastic and neoplastic liver lesions. Note: This abstract was not presented at the meeting. Citation Format: Francesca Rizzo, Luca Di Tommaso, Pia Sulas, Elena Coviello, Chiara Novello, Alessandro Weisz, Massimo Roncalli, Amedeo Columbano. A large set of miRNAs is deregulated since the earliest steps of human HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1467. doi:10.1158/1538-7445.AM2017-1467

Abstract 895: Genomic characterization of premalignant lung squamous cell carcinoma lesions

Abstract Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions. However, not all premalignant lesions will progress to lung SqCC and many of these lesions will regress without therapeutic intervention. Understanding the molecular events that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention. Methods: Bronchial brushings and biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute. For each subject (n = 30), both premalignant lesions (PMLs) and the cytologically normal mainstem bronchus were sampled repeatedly over time (n = 288 samples). DNA and RNA were isolated from a total of 197 bronchial biopsies of PML (average of 5 per subject) and 91 bronchial brushings. DNA was also isolated from the blood to serve as a matched normal. Exome capture was performed using the Agilent SureSelect Human All Exon+UTR 70MB kit and sequenced to a mean depth of coverage of 75x (n = 85 samples from 22 subjects). RNA libraries were prepared with Illumina TruSeq (mRNA-Seq: n = 288 samples from 30 subjects and miRNA-Seq: n = 183 samples from 26 subjects). Results: We identified gene and miRNA expression changes associated with histological grade as well as progressive/stable disease. The Hippo pathway, Wnt signaling, p53 signaling, and immune-related pathways are modulated with histological grade and disease progression. Genes associated with histological grade in the cytologically normal airway and in the biopsies were significantly concordantly enriched (FDR&amp;lt;0.05) demonstrating a strong relationship between the PMLs and the field of injury. The somatic mutation rate of PMLs displayed no significant association with histological grade (p = 0.65). Mutations in previously characterized lung cancer genes included TP53 (3%), CREBBP (3%), FAT1 (3%), and NOTCH1 (9%). Examining copy number alterations revealed a single metaplastic lesion with an arm-level amplification on chr5p containing TERT. The two lesions with the highest mutation rates (&amp;gt;3/Mb) were taken from adjacent sites over two time points in the same individual with a history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations (p = 2.2 × 10−17) indicating a common evolutionary ancestor, and contained mutations in CREBBP and FAT1, suggesting they are at increased risk for progressing to frank malignancy. Conclusions: We performed genomic profiling of PMLs in the airways of high-risk smokers. The gene expression and somatic alterations that were observed in known cancer genes may be among the earliest events in cancer development. Citation Format: Joshua D. Campbell, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Samjot S. Dhillon, Gang Liu, Sherry Zhang, Hangqio Lin, Jessica Vick, Christopher Moy, Evan Johnson, Matthew Meyerson, Suso Platero, Marc Lenburg, Mary Reid, Avrum Spira, Jennifer Beane. Genomic characterization of premalignant lung squamous cell carcinoma lesions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 895.

Abstract 896: The airway field of injury reflects gene expression changes associated with the presence of lung squamous premalignant lesions

Abstract Lung squamous cell carcinoma (SCC) arises in the epithelial layer of the bronchial airways and is preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs that develop go on to form carcinoma. In addition, the majority of lung cancer chemoprevention agents tested to date are ineffective. Molecular characterization of the airway field of injury in individuals with PMLs could provide novel insights into the earliest molecular events associated with carcinogenesis and identify biomarkers to guide lung cancer detection and chemoprevention. RNA-sequencing was conducted on cytologically normal airway brushings from current and former smokers with (n = 50) and without (n = 25) PMLs as part of the British Columbia Lung Health Study. Linear modeling strategies were used to identify 280 differentialy expressed genes at FDR&amp;lt;0.002 between subjects with and without PMLs. Pathway analysis using GSEA and ROAST revealed enrichment of genes involved in the electron transport chain and oxidative phosphorylation pathways in subjects with PMLs. These findings were validated by measuring the cellular bioenergetics of cultured epithelial cells from biopsies of PMLs and non-lesion areas. Baseline oxygen consumption rates were 2.5 fold higher (p&amp;lt;0.001) and the spare respiratory capacity was 1.5 fold higher (p&amp;lt;0.001) in PML cultures. These data suggest that metabolism-associated gene expression observed in the field of injury of PMLs is correlated with PMLs. In addition, there is a significant concordant enrichment (FDR&amp;lt;0.05) between the signature and gene expression in PMLs adjacent to SCC tumors, in SCC tumors, and in the field of individuals with lung cancer. This concordance led to the development of a 200-gene biomarker that accurately predicts the presence of PMLs (AUC = 0.90 n = 17 independent samples). Importantly, this biomarker was also predictive (AUC -.72) of progression/stability vs. regression of these premalignant lesions in an independent cohort of cytologically normal airway brushings collected as part of the RPCI screening clinic (n = 18). This is the first study to comprehensively profile gene expression changes in airway epithelial cells in the presence of PMLs. A subset of these changes reflects the earliest changes in the process of lung squamous cell carcinogenesis including the genes involved in cellular metabolism. However, the molecular alterations in the field of injury are dynamic as bronchial lesions either progress or regress these changes may be leveraged to monitor efficacy in chemoprevention trials. In addition monitoring molecular changes in high-risk smokers may identify smokers with PMLs that should receive lung cancer screening as well as lay the foundation for personalized lung cancer chemoprevention. Citation Format: Sarah A. Mazzilli, Ania Tassinari, Yaron Gethalter, Gang Lui, Mary Pine, Stephen Lam, Mary Reid, Suso Platero, Marc Lenburg, Avrum Spira, Jennifer Beane. The airway field of injury reflects gene expression changes associated with the presence of lung squamous premalignant lesions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 896.

Abstract A11: Airway gene expression indicates an increase in oxidative phosphorylation in the field of injury of individuals with premalignant lesions

Abstract Lung squamous cell carcinoma (SCC) claims the lives of approximately 50,000 individuals in the United States annually due to late stage diagnosis and lack of effective treatment options. Lung SCC arises in the epithelial layer of the bronchial airways and is preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs that develop go on to form carcinoma. Our group is now using high-throughput genomic techniques to characterize the process of premalignant progression by examining PMLs and non-lesion areas in the airway of individuals with lesions (i.e. the “field of injury”,) to identify events that lead to the development of SCC. mRNA-Seq was conducted on cytologically-normal proximal airway epithelium collected by brushing the mainstem bronchus from current and former smokers with (n=43) and without (n=32) PMLs (metaplatic lesions excluded). Linear modeling strategies were used to identify genes altered between subjects with and without PMLs (n=801 out of 13,916 genes at FDR&amp;lt;0.01). Genes were significantly concordantly enriched (FDR&amp;lt;0.01) in an overlapping sample set profiled by microarray (n=57 out of the 75 samples, 33/57 with PMLs) and enriched among up-regulated genes in the independent sample set (n=158, 38/158 with PMLs). Pathway analysis by GSEA revealed enrichment (FDR&amp;lt;0.05) of oxidative phosphorylation (OXPHOS) /respiratory electron transport among genes up-regulated in the airways of subjects with PMLs. OXPHOS is the most efficient metabolic pathway that generates energy in the form of ATP by utilizing the structures and enzymes of the mitochondria and OXPHOS is often elevated during repair epithelial tissue. However, OXPHOS metabolism is often superseded in the development of cancer by glycolysis. To validate these computational findings related to the OXPHOS pathway, we examined oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) in lung cell lines. These cell lines have either high OXPHOS and low glycolytic gene expression (H1299) or low OXPHOS and high glycolytic gene expression (HCC4006). Consistent with the gene expression pattern in these cell lines, The OCR for H1299s and HBECs were 10% higher than HCC4006 cells reflecting higher OXPHOS activity (p = 0.06). Whereas the ECAR was 39% higher in HCC4006 than H1299 cells and 46 % higher than HBECs (p&amp;lt;0.001and p&amp;lt;0.001 respectively), reflecting higher glycolytic metabolism corresponding to the gene expression. Next, we measured protein expression of genes that were elevated in the field of injury of PMLs, which included: mitochondrial membrane protein, translocase of the outer membrane (TOMM 22) and a subunit of complex IV of the electron transport chain, cytochrome C oxidase (COX-IV), in FFPE sections of lung tissue from the N-nitroso-tris-chloroethylurea (NTCU) mouse model of lung SCC. The NTCU model develops PMLs in the bronchial epithelium that progress to lung SCC in a histologically similar pattern to that observed in humans; it is important to note the carcinogen is administered cutaneously and not inhaled thus the field of injury may differ from inhaled carcinogens such as tobacco smoke. Protein levels of TOMM 22, and COX-IV were found to be elevated in dysplastic lesions compared to untreated controls. Together these data suggest that metabolism-associated gene expression is correlated with cellular metabolism and furthermore there is an increase in OXPHOS associated with the development of PMLs. While we are currently in the process of validating these findings in human normal and PMLs, our results suggest that there is potential that therapeutically increasing or maintaining OXPHOS in premalignant lesions or the field of injury may be a mechanism of prevention for lung cancer. Citation Format: Sarah A. Mazzilli, Gang Liu, Xiaohui Zhang, Huiqing Si, Stephen Lam, Marc Lenburg, Avrum Spira, Jennifer E. Beane. Airway gene expression indicates an increase in oxidative phosphorylation in the field of injury of individuals with premalignant lesions. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A11.

Abstract 2878: Development of the pre-cancer genome atlas (PCGA) for squamous cell lung carcinoma

Abstract Squamous cell cancer (SCC) of the lung is a leading cause of cancer mortality in the US, 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 events involved in the progression of PMLs to lung SCC are not clearly understood and not all PMLs go on to form carcinoma. By molecularly characterizing PMLs and non-lesion areas in the airway of individuals with PMLs we hypothesize that we will be able to identify early events in the process of lung carcinogenesis that lead to SCC. We used next-generation sequencing to profile bronchial brushings and 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 = 26), we sampled the PML(s) and the mainstem bronchus repeatedly over time (394 +/- 170 days) with serial bronchoscopies (5 +/- 3 biopsies/subject) as the PML progressed towards or regressed away from frank malignancy. mRNA-Seq (n = 192) and miRNA-Seq (n = 183) were performed on the endobronchial biopsies and brushings and exome-Seq was performed on blood DNA from these subjects. RNA-seq data was aligned to the hg19 and gene/transcript levels were summarized using RSEM/Ensembl 74 or Bedtools/ mirBase 18. Single nucleotide variants were quantified using a modified PRADA pipeline and GATK. We identified gene and miRNA expression changes as well as pathways that are associated with biopsy histological grade as well as progressive/stable disease. H&amp;E stains of PMLs clustering with lesions of dissimilar grades were examined to show that molecular classification provides complementary information to pathological grading. Genes altered in the normal airway in the presence of a PML were significantly concordantly enriched with genes identified in the biopsies demonstrating a strong relationship between the PMLs and the field of injury. In addition, cancer-associated missense and loss-of-function mutations were detected in PMLs. One individual had cancer-associated mutations at the initial time point in a single moderate dysplastic PML. Upon resampling ∼40 months later, the PML had progressed to an in situ carcinoma while gaining additional mutations. Furthermore, an adjacent PML at this second time point, also in situ carcinoma, had a significantly overlapping set of mutations with the original lesion suggesting that clonal expansion and invasion occurred between temporal samplings. The multiomic characterization of PMLs reveals gene and miRNA expression changes and somatic mutations that are associated with the lesion severity and progressive disease. These studies provide insights into the early molecular events in the process of lung carcinogenesis and may be predictors of lung cancer risk and lesion progression as well as novel candidates for targeted or preventive therapy. Citation Format: Jennifer E. Beane, Joshua Campbell, Christopher Moy, Catalina Perdomo, Michael Schaffer, Sarah Mazzilli, Yaron Geshalter, Jacob Kantrowitz, Liye Zhang, David Jenkins, Mary Beth Pine, Samjot Dhillon, Gang Liu, Hanqiao Liu, Sherry Zhang, Jessica Vick, Stefano Monti, Evan Johnson, Suso Platero, Marc Lenburg, Mary Reid, Avrum Spira. Development of the pre-cancer genome atlas (PCGA) for squamous cell lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2878. doi:10.1158/1538-7445.AM2015-2878

Su1993 Somatic Mutations in the Mismatch Repair System Are Responsible for a Majority of Unexplained Lynch Syndrome Cases: Time to Revise Lynch Syndrome Screening?

G A A b st ra ct s gastric tissue samples harvested from an independent population ofH. pylori-infected persons in New Orleans to assess expression and methylation status of HIF-1α in vivo. Gastric tissue specimens from African-American subjects harboring an increased risk for gastric cancer exhibited marked decreases in methylation of HIF-1α with increasing disease severity. The highest levels of HIF-1α methylation were found in patients with non-atrophic gastritis and this decreased as disease progressed to atrophic gastritis (0.5-fold) and intestinal metaplasia (0.2-fold), versus gastritis alone. Consistent with decreasing HIF-1α methylation status, gastric tissue from African-American patients harbored increased levels of HIF-1α expression with increasing disease progression and this was not observed in Caucasian patients. Collectively, these data indicate that H. pylori induces HIF-1α in gastric epithelial cells and this is augmented under conditions of iron deficiency. HIF-1α expression in vivo increases in conjunction with decreased HIF-1α methylation and the development of premalignant lesions, which may provide a mechanism underpinning the link between high altitude, iron depletion, and increased gastric cancer rates within the context of H. pylori infection.

Matrix metalloproteinase 7 restrains Helicobacter pylori-induced gastric inflammation and premalignant lesions in the stomach by altering macrophage polarization.

Helicobacter pylori is the strongest risk factor for the development of gastric cancer. Although the specific mechanisms by which this pathogen induces carcinogenesis have not been fully elucidated, high-expression interleukin (IL)-1β alleles are associated with increased gastric cancer risk among H. pylori-infected persons. In addition, loss of matrix metalloproteinase 7 (MMP7) increases mucosal inflammation in mouse models of epithelial injury, and we have shown that gastric inflammation is increased in H. pylori-infected MMP7(-/-) C57BL/6 mice. In this report, we define mechanisms that underpin such responses and extend these results into a genetic model of MMP7 deficiency and gastric cancer. Wild-type (WT) or MMP7(-/-) C57BL/6 mice were challenged with broth alone as an uninfected control or the H. pylori strain PMSS1. All H. pylori-challenged mice were successfully colonized. As expected, H. pylori-infected MMP7(-/-) C57BL/6 mice exhibited a significant increase in gastric inflammation compared with uninfected or infected WT C57BL/6 animals. Loss of MMP7 resulted in M1 macrophage polarization within H. pylori-infected stomachs, as assessed by Luminex technology and immunohistochemistry, and macrophages isolated from infected MMP7-deficient mice expressed significantly higher levels of the M1 macrophage marker IL-1β compared with macrophages isolated from WT mice. To extend these findings into a model of gastric cancer, hypergastrinemic WT INS-GAS or MMP7(-/-) INS-GAS mice were challenged with H. pylori strain PMSS1. Consistent with findings in the C57BL/6 model, H. pylori-infected MMP7-deficient INS-GAS mice exhibited a significant increase in gastric inflammation compared with either uninfected or infected WT INS-GAS mice. In addition, the incidence of gastric hyperplasia and dysplasia was significantly increased in H. pylori-infected MMP7(-/-) INS-GAS mice compared with infected WT INS-GAS mice, and loss of MMP7 promoted M1 macrophage polarization. These results suggest that MMP7 exerts a restrictive role on H. pylori-induced gastric injury and the development of premalignant lesions by suppressing M1 macrophage polarization.