Premalignant Tissues Research Articles

Abstract 2712: Establishing the early genetic progression of cancers with unobtainable premalignant disease

Abstract Premalignant tissue samples collected at different timepoints along the path to invasive tumors have been used to establish the typical order of somatic genetic events in several cancer types. For many other cancer types, however, precancer tissue is unobtainable, leaving the genetic progression unknown. Here, we demonstrate an approach that can reconstruct progression histories for such cancers from exome sequencing of primary tumors. We validated our recent computational method, PhylogicNDT, on classic, HPV-negative head and neck squamous cell carcinoma (HNSCC), recapitulating the previous experimentally determined sequence of genetic events that occur during progression. We uncovered progression of HPV-positive HNSCC tumors, which lack premalignant tissue, identifying subtype specific early drivers. We associated the timing of major changes in ploidy with the development of intra-tumor genetic heterogeneity and shorter overall survival. In cancers with unobtainable premalignant tissue, our approach can fill the knowledge gap about early progression, providing the basis for development of experimental models and methods for early detection, interception, and prognostication. Citation Format: Ignaty Leshchiner, Edmund A. Mroz, Justin Cha, Daniel Rosebrock, Oliver Spiro, Juliana Bonilla-Velez, William Faquin, Armida Lefranc-Torres, Derrick Lin, William Michaud, Gad Getz, James W. Rocco. Establishing the early genetic progression of cancers with unobtainable premalignant disease [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 2712.

Abstract 3055: Field cancerization and microbiome in lung cancer

Abstract Lung Cancer carcinogenesis is the outcome of a field of premalignant changes that occur in the bronchial tree leading to the overt emergence of the malignant lung cancer. We sought to study the proteome of the microbiome associated with the premalignant bronchial mucosa epithelia. The overall goal is the identify functional relationships between the bronchial mucosa microbiome and the risk of developing lung cancer. We evaluated the proteome of the microbiome using mass spectrometry and specialized microbiome authentication software. This method was applied to a case-control study of plasma samples donors of physician health study who donated plasma at different times prior to the onset of Non-Small Cell Lung Cancer. We also examined bronchial mucosa tissue samples near to, and distant from, the tumor harvested from surgical lobectomy specimens. Highly significant protein biomarker differences were found in the plasma and in the tissue samples that were blindly associated with cancer. These included specific proteins from the mucosal microbiome that may have functional role in the carcinogenic process. These key diagnostic risk markers were shared between the premalignant bronchial mucosa tissue and the plasma, supporting the concept that risk markers of bronchial mucosa lung cancer carcinogenesis can be detected in the peripheral circulation. Citation Format: Rayan I. Alhammad, Ngoc B. Vuong, Weidong Zhou, Claudius Mueller, Donald J Johann Jr, Amith R. Katta, Monique V. Hoek, Fahad M. Alsaab, Alessandra Luchini, Lance Liotta. Field cancerization and microbiome in lung cancer [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 3055.

Single Cell Meta-Analysis of Endothelial to Mesenchymal Transition (EndMT) in Glucose Metabolism of the Digestive Diseases.

Background: Endothelial-to-mesenchymal transition (EndMT) is poorly understood in digestive diseases, and the function of metabolism in EndMT is uncertain. Objective: The goal of this study is to elucidate the role of EndMT in digestive diseases and to describe its metabolic state. Method: The GEO database was used to extract single-cell data in order to discover EndMT subpopulations in digestive organs such as premalignant lesions and cancer of the stomach, intestine, and pancreas. Results: By single-cell RNA sequencing in digestive diseases, we generated a single-cell atlas from tissues of patients spanning a cascade of premalignant lesions and cancer. We next established a single-cell network elucidating the cellular and molecular characteristics of endothelial cells (ECs) across many lesions and identified key genes linked with EndMT in premalignant lesions and cancer lesions. The EndMT activation of a wide variety of metabolic signaling pathways was discovered in ECs, and further study of premalignant lesions and cancer tissue indicated that glucose metabolism increased in premalignant lesions and reached a maximum in cancer tissue. Finally, it was shown that INSR and LDHA might be used as prognostic markers for developing premalignant lesions to cancer involving glucose metabolism in digestive diseases. Conclusion: For the first time, we discovered EndMT’s role in digestive diseases and described its metabolism, underscoring its crucial role in glucose metabolism in the disease. We found several targets via gene screening that are beneficial for predicting premalignant lesions that progress to cancer.

Adipocytokines and Insulin Resistance: Their Role as Benign Breast Disease and Breast Cancer Risk Factors in a High-Prevalence Overweight-Obesity Group of Women over 40 Years Old.

Insulin levels, adipocytokines, and inflammatory mediators trigger benign breast disease (BBD) and breast cancer (BC). The relationship between serum adipocytokines levels, overweight-obesity, metabolic disturbs, and BC is unclear. Methods: To analyze the serum levels of the adipocytokines, insulin, and the HOMA IR in women without breast disease, with BBD or BC, and the role of these as risk factors for benign breast disease or breast cancer. Results: Adipsin values > 0.91 and visfatin levels > 1.18 ng/mL represent a risk factor to develop BBD in NBD lean women (OR = 18; and OR = 12). Data in overweight-obese women groups confirm the observation due to insulin levels > 2.6 mU/mL and HOMA IR > 0.78, with OR = 60.2 and 18, respectively; adipsin OR = 26.4, visfatin OR = 12. Breast cancer risk showed a similar behavior: Adipsin risk, adjusted by insulin and visfatin OR = 56 or HOMA IR and visfatin OR = 22.7. Conclusion: Adipose tissue is crucial for premalignant and malignant tissue transformation in women with overweight-obesity. The adipocyte–breast epithelium interaction could trigger a malignant transformation in a continuum, starting with BBD as premalignant disease, especially in overweight-obese women.

Somatic whole genome dynamics of precancer in Barrett\u2019s esophagus reveals features associated with disease progression

While the genomes of normal tissues undergo dynamic changes over time, little is understood about the temporal-spatial dynamics of genomes in premalignant tissues that progress to cancer compared to those that remain cancer-free. Here we use whole genome sequencing to contrast genomic alterations in 427 longitudinal samples from 40 patients with stable Barrett’s esophagus compared to 40 Barrett’s patients who progressed to esophageal adenocarcinoma (ESAD). We show the same somatic mutational processes are active in Barrett’s tissue regardless of outcome, with high levels of mutation, ESAD gene and focal chromosomal alterations, and similar mutational signatures. The critical distinction between stable Barrett’s versus those who progress to cancer is acquisition and expansion of TP53−/− cell populations having complex structural variants and high-level amplifications, which are detectable up to six years prior to a cancer diagnosis. These findings reveal the timing of common somatic genome dynamics in stable Barrett’s esophagus and define key genomic features specific to progression to esophageal adenocarcinoma, both of which are critical for cancer prevention and early detection strategies.

Presence of Skin Tissue-Resident Memory T Cells in Human Nonmalignant and Premalignant Melanocytic Skin Lesions and in Melanoma.

The infiltration of tissue-resident memory (TRM) cells in melanoma correlates with improved survival, suggesting an important role for TRM cells in immunity against melanoma. However, little is known about the presence of TRM cells in nonmalignant and premalignant melanocytic lesions. This study aimed to evaluate the presence of TRM cells in human skin melanocytic lesions, representing the spectrum from healthy skin to metastatic melanoma. FFPE sections from healthy skin, sun-exposed skin, benign nevi, lentigo maligna (LM), primary LM melanoma, and primary cutaneous and metastatic melanoma were analyzed by immunohistochemistry. The number of infiltrating cells expressing TRM-associated markers, CD3, CD4, CD8, CD69, CD103, and CD49a, was quantified by digital analyses. Multiplex immunofluorescence was performed to analyze coexpression of TRM cell markers. More T cells and CD69+ cells were found in melanoma lesions, as compared with healthy skin and nevi. CD103+ and CD49a+ cell numbers did not significantly differ. More importantly, no differences were seen in expression of all markers between healthy skin and benign nevi. Similar results, except for CD69, were observed in LM melanoma, as compared with LM and sun-exposed skin. Interestingly, multiplex immunofluorescence showed that nevi tissues have comparable CD103+ T cell numbers with healthy skin but comprise more CD103+ CD8+ cells. Expression of TRM cell markers is significantly increased in melanoma, as compared with nonmalignant skin. Our data also show that TRM cells are not abundantly present already in premalignant tissues. Further studies on the specificity of TRM cells for melanocyte/melanoma antigens may reveal their significance in cancer immunosurveillance.

Comparison of tumor and two types of paratumoral tissues highlighted epigenetic regulation of transcription during field cancerization in non-small cell lung cancer

BackgroundField cancerization is the process in which a population of normal or pre-malignant cells is affected by oncogenic alterations leading to progressive molecular changes that drive malignant transformation. Aberrant DNA methylation has been implicated in early cancer development in non-small cell lung cancer (NSCLC); however, studies on its role in field cancerization (FC) are limited. This study aims to identify FC-specific methylation patterns that could distinguish between pre-malignant lesions and tumor tissues in NSCLC.MethodsWe enrolled 52 patients with resectable NSCLC and collected resected tumor (TUM), tumor-adjacent (ADJ) and tumor-distant normal (DIS) tissue samples, among whom 36 qualified for subsequent analyses. Methylation levels were profiled by bisulfite sequencing using a custom lung-cancer methylation panel.ResultsADJ and DIS samples demonstrated similar methylation profiles, which were distinct from distinct from that of TUM. Comparison of TUM and DIS profiles led to identification of 1740 tumor-specific differential methylated regions (DMRs), including 1675 hypermethylated and 65 hypomethylated (adjusted P < 0.05). Six of the top 10 tumor-specific hypermethylated regions were associated with cancer development. We then compared the TUM, ADJ, and DIS to further identify the progressively aggravating aberrant methylations during cancer initiation and early development. A total of 332 DMRs were identified, including a predominant proportion of 312 regions showing stepwise increase in methylation levels as the sample drew nearer to the tumor (i.e. DIS < ADJ < TUM) and 20 regions showing a stepwise decrease pattern. Gene set enrichment analysis (GSEA) for KEGG and GO terms consistently suggested enrichment of DMRs located in transcription factor genes, suggesting a central role of epigenetic regulation of transcription factors in FC and tumorigenesis.ConclusionWe revealed distinct methylation patterns between pre-malignant lesions and malignant tumors, suggesting the essential role of DNA methylation as an early step in pre-malignant field defects. Moreover, our study also identified differentially methylated genes, especially transcription factors, that could potentially be used as markers for lung cancer screening and for mechanistic studies of FC and early cancer development.

A murine MODEL of e-cigarettes exposure to evaluate the carcinogenic effects of vaping on the bladder urothelium.

566 Background: Data evaluating the carcinogenic potential of e-cigarette (ECS) by-products on the bladder urothelium is scarce, and to date, no group has studied the direct impact of ECS on the urothelium at the genomic and epigenetic level. While it has been published that ECS exposure causes bladder hyperplasia in mice, no bladder tumors formed and the field is therefore lacking appropriate preclinical models to be able to study ECS related bladder cancer. Importantly, whole organ mapping studies of tumor bearing human bladders have identified DNA methylation field changes in “adjacent normal” urothelium as early events in bladder carcinogenesis. Whether ECS exposure results in early field alterations of DNA methylation in the urothelium is unknown. Methods: Mice (4 females, 4 males; 35 weeks old) were exposed to 12% nicotine (120mg/ml) in 1:1 polyethylene glycol: vegetable glycerol (PG/VG) or vehicle (PG/VG) daily: 3 hr sessions, 3 sec vape duration, 10 min interval between vapes. Nicotine and cotinine serum concentrations were measured at 3 hrs, and then at 5 days using LC-MS. Bladders were harvested after 4 weeks. DNA was extracted from microdissected urothelial layer. Reduced representation bisulfite sequencing (RRBS) was performed (Zymo Research) using 100ng of DNA input and paired-end chemistry. Alignment was performed using Bismark. Number of loci covered by each sample was determined and data was filtered to only include loci with &gt; 10 reads. Differentially methylated loci were determined. Adjusted p-value &lt; 0.01 was used as threshold to generate heatmaps. Gene Set Enrichment Analysis was performed to contextualize the DNA methylation changes. Results: Acute and repeated exposure resulted in significantly higher levels of nicotine and cotinine serum concentration in the nicotine group vs. the vehicle group without significant adverse events. No tumors were observed on gross visualization at the time of urothelium harvest. The number of loci in the RRBS libraries with &gt; 10 reads ranged from 130,000 to 250,000. 53 loci were differentially methylated between nicotine and vehicle. These loci did not correspond to the top hyper/hypo methylated genes in premalignant bladder tissue from recently reported whole-cystectomy mapping. Conclusions: We established an effective and safe murine model of ECS exposure closely resembling the human experience of vaping. Significant changes of DNA methylation were observed in mice exposed to ECS vapor vs. a vehicle control after four weeks of daily exposure. Ongoing experiments include longer vaping exposures as well as comparison of RRBS to a recently developed commercial mouse methylarray. Understanding epigenetic and genomic changes as a result of ECS exposure in preclinical models can serve as a foundation for studies developing assays to identify ECS users whose bladders contain early field defects that may place them at higher risk for bladder cancer.

STLA101 assay for the detection of cancerous progression in Barrett’s esophagus: A multi-institutional study.

249 Background: Since 1975, Esophageal adenocarcinoma (EAC) incidence has increased more than any other cancer (̃700%). Pre-malignant metaplastic tissue, or Barrett's Esophagus (BE), is the only known precursor for EAC. Periodic histological assessment of endoscopic biopsy along dysplasia-carcinoma sequence is used to assess progression risk. Current surveillance protocols have clinical limitations as 80% of patients still present with stage II-IV EAC. The aim of this study is to compare novel proteomic expression patterns to distinguish stable BE patients from those who developed EAC using the STLA101 assay. Methods: After Institutional Review Board approval patients with BE/GERD were identified. Two cohorts were selected: progressors (progressed to EAC at subsequent interval of 0.5-3 years) and non–progressors (̃10 yrs of f/u surveillance without progression along dysplasia/carcinoma path). 24 normal esophageal mucosa samples with no h/o of BE were also retrieved for baseline expression levels. Tissue sections were microdissected to isolate pure BE cells from surrounding stromal cells. BE cells underwent a multi-step process where tissue was heated and digested to produce a liquid biopsy for mass spec. Isotopically labeled control peptides representing the STLA101 panel were spiked in with digested tissue samples to quantify all of the markers simultaneously using a Triple-quad mass spectrometer. Results: A total of 95 tissues were analyzed. 29 BE biopsies progressed to EAC within a mean of 380 days while the remaining 28 BE and 14 GERD specimens did not progress over a 10-yr period. All 8 biomarkers were expressed at a higher level in BE tissue that progressed to cancer when compared to BE/GERD tissue that remained stable over 10 years. The assay had a 100% technical success rate as no samples yielded “non-detectable” data points. Conclusions: The differential overexpression of the STLA101 panel on progressive and non-progressive BE tissues demonstrates the clinical utility of STLA101 as a predictive biomarker panel for early detection of esophageal carcinogenesis. Non-progressive BE tissues were similar to normal mucosa, therefore, patients whose BE tissue samples resemble this profile may receive a reduced screening cadence. On the other hand, patients with overexpression of the STLA101 panel may be eligible to receive more frequent screening or therapeutic intervention.

Role of dental practitioner in smoking cessation interventional programs

Oral cancer, periodontitis, reduced response to periodontal therapy, implant failure, tooth loss, dental caries, delayed wound healing, chronic candidiasis, staining, halitosis and premalignant tissue alteration are all caused or linked to smoking and smokeless tobacco use. Importantly, smoking cessation therapies (both brief and extensive) have been proved to be a very cost-effective method of prolonging life and lowering disease. Health care providers, notably dentists and dental hygienists, are responsible for providing smoking cessation care to individuals with oral difficulties. The purpose of this research was to review the available information about the effectiveness of dental practitioners in smoking cessation interventional programs. According to the existing literature, dental practitioners can play an essential role in the control and spread of tobacco-related oral disorders by including smoking cessation interventional programs. The negative health impacts of tobacco use as well as the benefits of quitting tobacco use and tobacco cessation counselling, should all be part of the dentist office's arsenal. Dental professionals are in a unique position to assist tobacco users seeking dental care by assisting them in quitting smoking.

Tracing and Targeting the Origins of Childhood Cancer

Despite the success of treating childhood cancers with cytotoxic agents, novel therapeutic strategies are required to achieve the next leap in cure rates. A promising avenue may be to target the origin of childhood cancers. Here, we review recent advances in tracing the origins of pediatric tumors. Cancer-to-normal cell comparisons by single-cell mRNA sequencing reveal the fetal state of cancer cells, as well as their cell of origin. Recent phylogenetic analyses have uncovered large tissue-resident precursor clones to childhood cancers, which already possess key genomic alterations leading to tumor formation. Both the transcriptional fetalness and genomic status of the premalignant tissue bed provide further avenues for targeted therapy. Overall, these advances begin to describe the precise origins of pediatric tumors and pave the way for novel methods in detecting, treating, and perhaps even preventing childhood cancers.

The membrane-linked adaptor FRS2β fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis

Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2β, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2β deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2β-deficient premalignant tissues. We found that colocalization of FRS2β and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2β contain more stroma. The elucidation of the FRS2β-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.

S431 Proteomic Assay for Barrett’s Esophagus Progression: A Multi-Institutional Retrospective Study

Introduction: Incidence of Esophageal adenocarcinoma (EAC) in the US has increased at a greater rate than any other cancer. EAC arises from pre-malignant metaplastic tissue - Barrett’s Esophagus (BE). Periodic histological assessment of an endoscopic biopsy is used to assess progression along dysplasia-carcinoma sequence. Current surveillance protocols have clinical limitations as 80% of EAC patients present in advanced cancer stages. The aim of this study is to compare 8 proteomic expression patterns to distinguish stable BE patients from those who developed EAC using the STLA101 assay. Methods: After Institutional Review Board approval (Mayo Clinic and KUMC) patients with BE were identified. 2 cohorts were selected: progressors (progressed to EAC at subsequent interval of 0.5-3 years) and non–progressors (who had 10 yrs of f/u surveillance without progression along dysplasia/carcinoma path). 11 normal esophageal mucosa samples were also retrieved for analysis. Serial tissue sections were microdissected to reduce proteomic noise from surrounding stromal cells. Isolated cells underwent a multi-step process where cross-links were reversed, and proteins were digested and reduced into a mass spec-compatible lysate. Isotopically labeled control peptides of the STLA101 panel were spiked in with digested tissue samples to quantify all of the markers simultaneously using a Q-Exactive HFX mass spectrometer in a CAP/CLIA setting. Results: A total of 42 tissues were analyzed. Cohort tissue descriptions can be found in Table 1. 13 BEs progressed to EAC within a mean of 334 days while the remaining 18 BEs did not progress over a 10-yr period. All 8 biomarkers were expressed at a higher level when comparing dysplastic BE tissue that progressed to cancer to BE tissue that remained stable over 10 yrs and normal esophageal mucosa. There was not a significant difference in age, sex and length of BE between the groups. The assay had a 100% technical success rate as no samples yielded “non-detectable” data points. Conclusion: The overexpression of the STLA101 panel in dysplastic BE tissue that progressed to cancer demonstrates the potential predictability of the assay when assessing molecular hallmarks of carcinogenesis. The expression quantities of the non-progressive BE tissues were closer to normal mucosa and may warrant patients with this profile type to be screened less. Patients with high expression quantities of the STLA101 panel may be candidates for a more frequent screening cadence or therapeutic intervention.Figure 1.: Mass spectrometry-based expression patterns of 2 markers in the STLA101 panel demonstrating clear overexpression of proteins involved in the malignant transformation of Barrett’s precancerous tissue into esophageal adenocarcinoma. The expression mean of non-diseased esophageal squamous mucosa, labeled as “normal”, are represented by blue bars. The green bars represent non-progressive BE tissue that remained stable for over 10-years according to collaborating institutional databases. The red bars represent progressive BE tissue retrieved at a mean of 334 days before adenocarcinoma diagnosis.Table 1.: Specimen types analyzed in this study

Renalase is a novel tissue and serological biomarker in pancreatic ductal adenocarcinoma.

Dysregulated expression of the secretory protein renalase can promote pancreatic ductal adenocarcinoma (PDAC) growth in animal models. We characterized renalase expression in premalignant and malignant PDAC tissue and investigated whether plasma renalase levels corresponded to clinical PDAC characteristics. Renalase immunohistochemistry was used to determine the presence and distribution of renalase in normal pancreas, chronic pancreatitis, PDAC precursor lesions, and PDAC tissues. Associations between pretreatment plasma renalase and PDAC clinical status were assessed in patients with varied clinical stages of PDAC and included tumor characteristics, surgical resection in locally advanced/borderline resectable PDAC, and overall survival. Data were retrospectively obtained and correlated using non-parametric analysis. Little to no renalase was detected by histochemistry in the normal pancreatic head in the absence of abdominal trauma. In chronic pancreatitis, renalase immunoreactivity localized to peri-acinar spindle-shaped cells in some samples. It was also widely present in PDAC precursor lesions and PDAC tissue. Among 240 patients with PDAC, elevated plasma renalase levels were associated with worse tumor characteristics, including greater angiolymphatic invasion (80.0% vs. 58.1%, p = 0.012) and greater node positive disease (76.5% vs. 56.5%, p = 0.024). Overall survival was worse in patients with high plasma renalase levels with median follow-up of 27.70 months vs. 65.03 months (p < 0.001). Renalase levels also predicted whether patients with locally advanced/borderline resectable PDAC underwent resection (AUC 0.674; 95%CI 0.42–0.82, p = 0.04). Overall tissue renalase was increased in both premalignant and malignant PDAC tissues compared to normal pancreas. Elevated plasma renalase levels were associated with advanced tumor characteristics, decreased overall survival, and reduced resectability in patients with locally advanced/borderline resectable PDAC. These studies show that renalase levels are increased in premalignant pancreatic tissues and that its levels in plasma correspond to the clinical behavior of PDAC.

850 OBJECTIVE IDENTIFICATION OF OESOPHAGEAL ADENOCARCINOMA USING MASS SPECTROMETRY IMAGING

Abstract Outcomes for oesophageal adenocarcinoma continue to be poor, as patients are often diagnosed too late for curative treatment. New tools to assist early and rapid diagnosis are needed. Tissue phospholipids quantified by mass spectrometry imaging (MSI) can objectively diagnose non-oesophageal cancers from control tissue in minutes. However, whether MSI can objectively identify oesophageal adenocarcinoma is unknown and represents a significant challenge, as this microenvironment is complex with phenotypically similar tissue-types. Methods Desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) and bespoke chemometrics were used to assess the phospholipidomes of oesophageal adenocarcinoma and relevant control tissues, in surgical (discovery) and endoscopic (validation) cohorts. Structural annotations verified by tandem MS fragmentation patterns were performed to understand the characteristics of the key differential phospholipids. Bioinformatics, quantitative polymerase chain reaction, and immunohistochemistry were performed to describe the reprogramming of phospholipogenesis in oesophageal adenocarcinoma. Finally, genetic silencing experiments were performed to understand whether this genetic reprogramming impacts the oesophageal adenocarcinoma phospholipid signature. Results Multivariable models based on tissue phospholipids from 117 patients were highly discriminant for oesophageal adenocarcinoma both in discovery (area-under-curve = 0.97) and validation cohorts (AUC = 1). Cancer samples were enriched for polyunsaturated phosphatidylglycerols with longer acyl chains, with stepwise enrichment in pre-malignant tissues. Expression of fatty acid and glycerophospholipid synthesis genes was significantly upregulated, and characteristics of fatty acid acyls matched phospholipid acyls. Mechanistically, reducing ACLY expression in oesophageal adenocarcinoma cells shortened GPL chains, therefore linking de novo lipogenesis to the oesophageal adenocarcinoma phospholipidome. Conclusion DESI-MSI (i) objectively identified invasive oesophageal adenocarcinoma from a number of pre-malignant tissues (ii) described the characteristics of cancer phospholipids, which have unknown function (iii) unveiled a new mechanism of phospholipidomic reprogramming. These results call for accelerated diagnosis studies using DESI-MSI in the upper gastrointestinal endoscopy suite, as well as functional studies to determine how polyunsaturated phosphatidylglycerols are generated, and how they contribute to oesophageal carcinogenesis.

Somatic mutations in benign breast disease tissues and association with breast cancer risk

BackgroundBenign breast disease (BBD) is a risk factor for breast cancer (BC); however, little is known about the genetic alterations present at the time of BBD diagnosis and how these relate to risk of incident BC.MethodsA subset of a long-term BBD cohort was selected to examine DNA variation across three BBD groups (42 future estrogen receptor-positive (ER+) BC, 36 future estrogen receptor-negative (ER−) BC, and 42 controls cancer-free for at least 16 years post-BBD). DNA extracted from archival formalin fixed, paraffin-embedded (FFPE) tissue blocks was analyzed for presence of DNA alterations using a targeted panel of 93 BC-associated genes. To address artifacts frequently observed in FFPE tissues (e.g., C>T changes), we applied three filtering strategies based on alternative allele frequencies and nucleotide substitution context. Gene-level associations were performed using two types of burden tests and adjusted for clinical and technical covariates.ResultsAfter filtering, the variant frequency of SNPs in our sample was highly consistent with population allele frequencies reported in 1 KG/ExAC (0.986, p < 1e−16). The top ten genes found to be nominally associated with later cancer status by four of 12 association methods(p < 0.05) were MED12, MSH2, BRIP1, PMS1, GATA3, MUC16, FAM175A, EXT2, MLH1 and TGFB1, although these were not statistically significant in permutation testing. However, all 10 gene-level associations had OR < 1 with lower mutation burden in controls compared to cases, which was marginally statistically significant in permutation testing (p = 0.04). Comparing between the three case groups, BBD ER+ cases were closer to controls in mutation profile, while BBD ER− cases were distinct. Notably, the variant burden was significantly higher in controls than in either ER+ or ER− cases. CD45 expression was associated with mutational burden (p < 0.001).ConclusionsSomatic mutations were more frequent in benign breast tissue from women who did not develop cancer, opening questions of clonal diversity or immune-mediated restraint on future cancer development. CD45 expression was positively associated with mutational burden, most strongly in controls. Further studies in both normal and premalignant tissues are needed to better understand the role of somatic gene mutations and their contribution to future cancer development.

Abstract 946: A novel monoclonal antibody targeting cell-surface plectin has potent antitumor activity in ovarian cancer models

Abstract Background: Cell surface plectin (CSP) is an abundant bioavailable target exclusively found on the surface of ovarian epithelial cancers while remaining entirely intracellular in pre-malignant tissue. Under current treatment options, 80% of women, even if remission is achieved, will experience recurrence of disease demonstrating an urgent need for more effective therapeutics. Herein, we investigate the anticancer effects of a CSP-targeting monoclonal antibody (ZB131) in in-vitro and in-vivo models of ovarian cancer (OC) as well as elucidate key signaling pathways it modulates. We demonstrate that treatment with ZB131 is efficacious both as a monotherapy and in combination with chemotherapy. Methods: A first-in-class CSP-targeting monoclonal antibody, ZB131, was developed and characterized for specificity and affinity via ELISA and kinetic binding assays. In-vitro efficacy of ZB131 was determined by Cell Titer-Glo assay and flow cytometry, impact on cell-cycle by flow cytometry, and on migration by scratch assay using multiple OC cell lines. ROS accumulation was evaluated by DCFDA assay. Phosphorylation changes of signaling kinases were measured by LiCor. In-vivo validation was performed in an OC xenograft model. The expression of proliferative markers was evaluated by IHC staining of tumor sections. Treatment toxicity was assessed from serum chemistry tests and histological evaluation of major organs. Similar in-vivo and in-vitro techniques were used to characterize ZB131's effect in combination with cisplatin. Results: ZB131 demonstrates high affinity (0.4±0.1nM) and specificity for both murine and human CSP. Treatment with ZB131 decreased cell proliferation, migration, induced G0/G1 growth arrest, and increased ROS accumulation followed by cell death in OC cell lines. ZB131 induced two coordinated events, decreased phosphorylation of cell proliferation cascades (AKT, PRAS40, ERK1/2, and GSK3alpha/beta) and increased phosphorylation of stress signaling pathways (JNK, c-Jun, p38, MSK1/2, and AMPK). In vivo, ZB131 therapy caused a 50% tumor growth reduction as well as decreased expression of cyclin D and Ki67 with no toxic side effects. Impressively, ZB131 synergizes with cisplatin resulting in a greater than 50-fold decrease in IC50 in-vitro and showed sustained tumor growth reduction with greater than 85% tumor necrosis over the course of treatment in-vivo. Conclusion: CSP is a novel highly abundant therapeutic target exclusively localized to the cell surface of ovarian, colorectal, lung, and pancreatic cancers which collectively account for more than half of all estimated cancer deaths. Our findings illustrate that CSP-targeting with ZB131 induces cytotoxicity by activating stress pathways and dampening proliferation and survival pathways. This study serves as the basis for informing a novel treatment strategy for CSP-positive tumors. Citation Format: Samantha Melinda Perez, Julien Dimastromatteo, Kimberly A. Kelly. A novel monoclonal antibody targeting cell-surface plectin has potent antitumor activity in ovarian cancer models [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 946.

Abstract 750: Race differences in telomere length in benign prostate and subsequent risk of prostate cancer

Abstract Telomere shortening is linked to aging and thought to be associated with increased risk for cancer. Most cancer studies have used leukocyte telomere length as a surrogate measure for telomere length with few focused upon telomeres in the target tissue of cancer origin. To determine whether telomere length in pre-malignant prostate tissue is associated with subsequent prostate cancer risk, we conducted a case-control study of 530 case-control pairs matched on date, age at cohort entry, and race nested within a historical cohort of 10,478 men with a benign prostate biopsy. Overall mean telomere length assessed using modified quantitative real-time PCR was not different in benign prostate biopsies of prostate cancer cases vs. matched controls (1.65 ± 0.35 vs. 1.64 ± 0.39; p=0.41). However, race was significantly associated with telomere length with African American (AA) men having shorter telomeres in benign prostate biopsy compared with white men (1.59 ± 0.37 vs. 1.70 ± 0.37; p&amp;lt;0.0001). In race-stratified analyses, telomere length only showed a suggestive association with prostate cancer risk in white men, wherein those with telomere length in the highest quartile had 1.5-fold greater risk of prostate cancer compared to men with prostate telomere lengths in the lowest quartile (OR=1.56; 95% CI = 0.89 - 2.73 ). In addition, white men in the highest prostate telomere length quartile had almost three-fold greater risk of aggressive prostate cancer, defined as Gleason group 3 and above, tumor stage 3 or above or PSA 20 ng/ml or higher , compared with men with telomere lengths in the lowest quartile (OR=2.73; 95% CI =1.05 - 7.15). In summary, white men have longer telomeres in benign prostate tissue compared with AA men, and those with the longest telomeres may be at increased risk for prostate cancer, particularly the more aggressive form of the disease. Citation Format: Benjamin A. Rybicki, Sudha M. Sadasivan, Yalei Chen, Ian Loveless, Nilesh S. Gupta, Dhananjay A. Chitale, Sean R. Williamson, Andrew G. Rundle, Deliang Tang. Race differences in telomere length in benign prostate and subsequent risk of prostate cancer [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 750.

METHYLATION PROFILING OF DAPK1, LRPPRC, RAB6C, AND ZNF471 IN SALIVA AND TISSUES AS NOVEL EPIGENETIC MARKERS OF ORAL SQUAMOUS CELL CARCINOMA

<h3>Background</h3> Despite rapid progress in the diagnosis and treatment of oral squamous cell carcinoma (OSCC), the survival rates remain poor. Early identification of premalignant lesions is critical in reducing mortality. Epigenetic changes, such as aberrant DNA methylation resulting in altered gene expression, contribute to oral carcinogenesis. <h3>Objective</h3> To identify aberrantly methylated genes in saliva and tissue of OSCC patients as novel biomarkers of patients' prognosis. <h3>Methods</h3> Genome-wide methylation changes were identified by differential methylation hybridization microarray. The results were compared against datasets from TCGA-HNSCC. Promoter sequences of <i>DAPK1, LRPPRC, RAB6C</i>, and <i>ZNF471</i> were validated by bisulfite genome sequencing. The methylation status of these genes was tested in saliva and tissue specimens by targeted next-generation sequence. <h3>Results</h3> Promoter sequences of <i>DAPK1, LRPPRC, RAB6C,</i> and <i>ZNF471</i> were significantly hypermethylated in tumors compared with matched normal tissue (<i>P</i> < .0001). Sensitivity and specificity of methylation markers for detection of OSCC were in the range of 70% to 100%, with AUC 0.83 and above. Salivary DNA methylation levels were higher in premalignant lesions and OSCC in comparison to healthy controls (<i>P</i> < .05). The r-value between premalignant tissue versus saliva and OSCC versus saliva were in the range of 0.6297 to 0.8023 and 0.7823 to 0.9419, respectively. <h3>Conclusions</h3> Our data confirms that the methylation pattern of these candidate genes are significantly higher in premalignant and OSCC tissues and saliva. Thus, the methylation profiling of these candidate genes has the potential to serve as novel non-invasive markers of OSCC.

Accelerated aging in normal breast tissue of women with breast cancer

BackgroundDNA methylation alterations have similar patterns in normal aging tissue and in cancer. In this study, we investigated breast tissue-specific age-related DNA methylation alterations and used those methylation sites to identify individuals with outlier phenotypes. Outlier phenotype is identified by unsupervised anomaly detection algorithms and is defined by individuals who have normal tissue age-dependent DNA methylation levels that vary dramatically from the population mean.MethodsWe generated whole-genome DNA methylation profiles (GSE160233) on purified epithelial cells and used publicly available Infinium HumanMethylation 450K array datasets (TCGA, GSE88883, GSE69914, GSE101961, and GSE74214) for discovery and validation.ResultsWe found that hypermethylation in normal breast tissue is the best predictor of hypermethylation in cancer. Using unsupervised anomaly detection approaches, we found that about 10% of the individuals (39/427) were outliers for DNA methylation from 6 DNA methylation datasets. We also found that there were significantly more outlier samples in normal-adjacent to cancer (24/139, 17.3%) than in normal samples (15/228, 5.2%). Additionally, we found significant differences between the predicted ages based on DNA methylation and the chronological ages among outliers and not-outliers. Additionally, we found that accelerated outliers (older predicted age) were more frequent in normal-adjacent to cancer (14/17, 82%) compared to normal samples from individuals without cancer (3/17, 18%). Furthermore, in matched samples, we found that the epigenome of the outliers in the pre-malignant tissue was as severely altered as in cancer.ConclusionsA subset of patients with breast cancer has severely altered epigenomes which are characterized by accelerated aging in their normal-appearing tissue. In the future, these DNA methylation sites should be studied further such as in cell-free DNA to determine their potential use as biomarkers for early detection of malignant transformation and preventive intervention in breast cancer.