Preneoplastic Lesions Research Articles

Randomized Phase II Trial of Imiquimod with or without 9-Valent HPV Vaccine versus Observation in Patients with High-grade Pre-neoplastic Cervical Lesions (NCT02864147).

We report the results of a randomized phase II trial of imiquimod, a topical immune-response modulator versus imiquimod plus a 9-valent human papillomavirus (HPV) vaccine (9vHPV) versus clinical surveillance in cervical intraepithelial neoplasia (CIN2/3) patients. We randomly allocated 133 patients with untreated CIN2/3 in equal proportions to a 4-month treatment with self-applied vaginal suppositories containing imiquimod (Arm B) or imiquimod plus a 9vHPV (Arm C) versus clinical surveillance (Arm A). The main outcome was efficacy, defined as histologic regression to CIN1 or less. Secondary outcomes were HPV clearance and tolerability. Exploratory objectives included the comparison of cervical CD4/CD8 T-cell infiltration at baseline, mid-study, and posttreatment by flow cytometry among study arms. Of the 114 evaluable patients 77% and 23% harbored CIN2 and CIN3, respectively. Regression to CIN1 or less was observed in 95% of patients in the imiquimod group (Arm B) compared with 79% in the control/surveillance (Arm A); P = 0.043 and 84% in the imiquimod+9vHPV group (Arm C; P = 0.384 vs. Arm A). Neither of the treatment-arm differences from Arm A reached the prespecified α = 0.025 significance level. No significant differences were noted in the secondary outcome of rate of HPV clearance. The number of tissue-resident memory CD4/CD8 T cells in cytobrush samples demonstrated a >5-fold increase in Arm B/imiquimod when compared with Arm A/surveillance (P < 0.01). In contrast, there was no significant difference in T-cell responses among participants in Arm C when compared with Arm A. Imiquimod treatment was well tolerated. Although imiquimod induced a higher regression to CIN1 or less and significant increases in CD4/CD8 T cells infiltrating the cervix, it did not meet its prespecified statistical outcome for efficacy. A higher regression rate than expected was observed in the surveillance arm of this prospective trial. Future clinical trials with imiquimod targeting CIN3 patients are warranted.

Abstract LB139: Disparities in oral precancerous lesion surveillance in the United States upper Midwest

Abstract Objective: To determine whether geographic representation and race of patients in a regional oral cancer surveillance program (University of Minnesota Cancer Active Surveillance Population (UMN CASP)) mirror characteristics in the corresponding National Cancer Institute (NCI) designated comprehensive cancer center catchment. Introduction: Oral cancer poses a significant public health concern that may be mitigated through lesion surveillance. Representation of patients for clinical trials throughout an NCI cancer center catchment area is a crucial goal to promote improved health outcomes. This study investigates the demographic landscape of patients with preneoplastic oral lesions seen at the CASP. Methods: Through the UMN Clinical and Translational Sciences Institute, we queried 30 International Classification of Disease (ICD) 9 and 10 codes corresponding to 120 unique mucosal oral precancerous lesion descriptors of CASP patients. Automated extraction of electronic medical record (EMR) data for 4,496 patients treated by the senior author between 2011 and 2023 identified 1,375 patients with preneoplastic lesions. The proportion of patients residing in three-digit zip code regions of Minnesota were compared to 2021 American Community Survey state population data. Additionally, race data were compared between CASP patients and the UMN Masonic Cancer Center (MCC) patient catchment data. Results: CASP patients were over-represented relative to state population distributions within the Minneapolis-St. Paul metropolitan area and surrounding suburbs (region 554 p&amp;lt;0.0001 and region 551 p&amp;lt;0.0001). Under-representation of CASP patients was observed in all western and southern border regions (regions 559 p&amp;lt;0.0001, 560 p&amp;lt;0.0001, 561 p&amp;lt;0.0001, 562 p=0.0037, 565 p&amp;lt;0.0001, and 567 p=0.0006) as well as portions of central (563 p&amp;lt;0.0001), northern (566 p=0.0015), and north-eastern (558 p&amp;lt;0.0001) Minnesota. Proportional representation was observed in several regions of central (553 and 564), eastern (550), and north-eastern (557 and 556) Minnesota. Comparisons of CASP race data revealed a higher proportion of black patients (CASP 8.59% [95% CI 7.17-10.27%] vs. MCC 7.16% [95% CI 7.14-7.17%]) and a lower proportion of Asian/Pacific Islander patients (CASP 3.74% [95% CI 2.82-4.94%] vs. MCC 5.32% [95% CI 5.30-5.34%]) relative to MCC catchment patients. White, American Indian/Alaskan Native, and mixed-race groups were proportionally represented in both data sets. Conclusions: These data show that system wide EMR searches can be implemented to identify patients in an NCI cancer center catchment area with precancerous or other at-risk conditions for cancer. Importantly, Native patients were proportionally represented in the screening program while several rural locations were under-represented. This information can instruct the expenditure of additional resources for under-represented groups and permit streamlined enrollment of patients for cancer prevention clinical trials. Citation Format: Alexander J. Dwyer, Lindsey Mortensen, Beverly R. Wuertz, Frank G. Ondrey. Disparities in oral precancerous lesion surveillance in the United States upper Midwest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB139.

Abstract LB219: DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays

Abstract Gastrointestinal cancers, including gastric cancer (GC) and colorectal cancer (CRC), continue to be devastating diseases despite advances in screening, early detection, and removal of pre-neoplastic and early cancerous lesions. Even with advancements in targeted chemotherapy and immunotherapies, advanced and metastatic diseases continue to be responsible for the high mortality associated with GC and CRC. Newer, safer, and more effective therapies to prevent, inhibit, or reverse advanced disease progression are urgently needed. Doublecortin-like kinase 1 (DCLK1) is a microtubule associated protein kinase that has been reported to drive tumor initiation, progression, metastasis, and drug resistance in many solid tumor cancers including GC and CRC, where it is upregulated in patient tumors and associated with poor overall survival. An anti-DCLK1 monoclonal antibody has demonstrated potential therapeutic activity against CRC. In this study, we established patient-derived organoids (PDOs) to evaluate the effects of inhibiting DCLK1 kinase activity (DCLK1-IN-1) or targeting DCLK1 with a highly specific CAR-T cells on the treated PDOs. Two GC PDOs were treated with various concentrations of DCLK1 kinase inhibitor DCLK1-IN-1 (0-32 μM) for 72 hrs, viability of the PDOs were determined, histochemistry and immunohistochemistry staining were used to image the live cells and DCLK1 expression. For CRC PDOs, five CRC PDOs were cultured in the presence or absence of PBMC, treated with Mock CAR-T cells or DCLK1 CAR-T cells (three doses) for 4 days. Tumor size, tumor total area, and viability of these PDOs were determined. Here, we demonstrate that treating GC PDOs with DCLK1 kinase inhibitor resulted in a significant dose-dependent inhibition in PDO numbers in both PDOs (two-way ANOVA P&amp;lt;0.0001), a decrease of cell viability, revealing IC50 values of 7.566 and 9.958 μM respectively, and a near total loss of GC PDO viability at 10 μM using CellTiterGlo assay. In CRC PDOs, two of five CRC PDOs showed a significant decrease in total tumor size, tumor areas, and an increase in cell death measured by the DRAQ7 method. Compared to Mock CAR-T cells, treatment with DCLK1 CAR-T cells resulted in a dose dependent decrease of tumor size and total tumor area, with more than a 50% reduction at the 50000 cells dose (p&amp;lt;0.05). Treatment with DCLK1 CAR-T cells also resulted in a dose dependent increase in cell death, with approximately 20% cell death without PBMC, and 50% cell death with PBMC at the 50000 cells dose (p&amp;lt;0.05). These results suggest that targeting DCLK1 represents a potential novel therapeutic strategy for GI cancers, especially in combination with FDA approved drugs: 5-fluorouracil (5-FU) or Gefitinib. Citation Format: Dongfeng Qu, Jinsen Shi, Zhiyun Cao, Eddie BAnnerman-Menson, Nathaniel Weygant, Courtney Houchen. DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB219.

Glyphosate-based herbicide worsens alterations induced by cafeteria diet on rat uterus.

Exposure to glyphosate-based herbicides (GBH) and consumption of cafeteria (CAF) diet, which are widespread in Western society, seem to be associated with endometrial hyperplasia (EH). Here, we aimed to evaluate the effects of a subchronic low dose of a GBH added to CAF diet on the rat uterus. Female Wistar rats were fed from postnatal day (PND)21 until PND240 with chow (Control) or CAF diet. Since PND140, rats also received GBH (2 mg of glyphosate/kg/day) or water through food, yielding four experimental groups: Control, CAF, GBH and CAF+GBH. On PND240, CAF and CAF+GBH animals showed an increased adiposity index. Respect to Control group, no changes on the serum levels of 17β-estradiol and progesterone were found. However, progesterone levels were higher in CAF+GBH group than in CAF and GBH groups. In the uterus, both studied factors alone and in combination induced morphological and molecular changes associated with EH. Furthermore, the addition of GBH provoked an increased thickness of subepithelial stroma in rats fed with CAF diet. As a consequence of GBH exposure, CAF+GBH rats exhibited an increased density of abnormal gland area, considered preneoplastic lesions, as well as a reduced PTEN and p27 expression, both tumor suppressor molecules that inhibit cell proliferation, respect to Control rats. These results indicate that the addition of GBH exacerbates the CAF-effects on uterine lesions and that the PTEN/p27 signaling pathway seems to be involved. Further studies focusing on the interaction between unhealthy diets and environmental chemicals should be encouraged to better understand uterine pathologies.

Abstract 3447: Potential for reduction in invasive cancer incidence by interception of preneoplastic lesions

Abstract Background: Preneoplastic lesions (PNL) are morphologically identifiable lesions lack the ability to invade surrounding tissue but can progress to invasive cancer. Thus, PNL present an opportunity to identify individuals at elevated cancer risk and identify opportunities for early detection and interception of cancer. Objective: We used published data sources to estimate the impact of intercepting progression of PNL to invasive cancer across multiple anatomic sites. We estimated the hypothetical number of cancers that could be avoided in the United States population if interception was successful in preventing a percentage of individuals with PNL from progressing to invasive cancer. Methods: Estimates of prevalence, latency, and probability of percent progression of PNL to invasive cancer were obtained from literature. We considered PNL associated with invasive tumors of the breast, bladder, cervix, colorectum, esophagus, liver, lung, oropharynx, pancreas, prostate, skin (melanocytes), skin (squamous cells), and stomach as well as precursors to leukemia and myeloma. We modeled a cohort of 100,000 individuals from which we estimated the number of cases that could be avoided if interception of a PNL before it progressed to an invasive cancer case was successful. Results were stratified for race and sex; positive- and negative-predictive values were calculated. Results: For PNL with a higher prevalence and percent progression per year, there were more invasive cancers that could be avoided if interception was successful. Detection of PNL associated with pancreas, stomach, oral, and prostate cancers had the greatest potential to avoid invasive cancers in the US population. For example, if 95% of pancreatic intraepithelial neoplasia lesions were detected, 15% progressed to pancreas cancer, and assuming a 43% prevalence over a 5-year latency period, 1,226 potential invasive pancreatic cancer cases could be avoided per year. Similarly, 998 potential invasive stomach cancer cases could be avoided earlier if 95% of gastric intestinal metaplasia lesions were detected assuming 42% progressed to invasive cancer with a 19% prevalence and a 4-year latency period. Additionally, if we could intercept 5% to 95% of all PNL from becoming invasive cancer, we would avoid between 176 to 8,515 number of invasive cases being diagnosed. When stratified by race, symptomatic multiple myeloma can be avoided in individuals with monoclonal gammopathies of undetermined significance if this condition is detected earlier among Black individuals. Conclusion: By detecting PNL and preventing them from advancing to invasive cancer, we can avoid substantial mortality as well as the costs and morbidities associated with treating more advanced cancers. Citation Format: Pranoti Pradhan, Irene Ghobrial, Catherine Marinac, Betsy O’Donnell, Sapna Syngal, Timothy Rebbeck. Potential for reduction in invasive cancer incidence by interception of preneoplastic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3447.

Abstract 3446: Associations of non-steroidal anti-inflammatory drug use and circulating inflammatory proteins with gallbladder dysplasia

Abstract Background: Most gallbladder cancer (GBC) cases arise from the metaplasia-dysplasia-GBC sequence, and chronic inflammation has been associated with its development. The use of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with reduced risk of GBC; however, little is known about these associations with preneoplastic lesions. We thus aimed to study the associations of the use of NSAIDs and inflammatory proteins with gallbladder dysplasia, a precursor to GBC. Methods: Associations of NSAIDs and gallbladder dysplasia were evaluated in 1,925 Chilean cholecystectomy patients, 82 with dysplasia and 1,843 with gallstones but not dysplasia. In addition, we measured 33 circulating inflammatory proteins in a subsample of 68 dysplasia cases diagnosed at the time of sample selection and 136 gallstone controls. Age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated, and associations of inflammatory proteins and gallbladder dysplasia were analyzed. Results: NSAID use was inversely associated with gallbladder dysplasia (OR: 0.48, 95% CI: 0.26-0.83). Comparing the highest versus lowest category of each immune-related protein, eight proteins were inversely associated with dysplasia with ORs ranging from 0.30 (95%CI: 0.12-0.77) for IL-33 to 0.76 (95%CI: 0.59-0.99) for MIP-1B. Of those, GRO remained associated with dysplasia (OR: 0.64, 95%CI: 0.45-0.91) and BCA-1 was borderline-associated (OR: 0.74, 95%CI: 0.54-1.01) in the multivariable logistic regression model additionally adjusted for sex, age and NSAID use. Conclusion: NSAID users were less likely to have gallbladder dysplasia, suggesting that NSAIDs might be beneficial for gallstones patients. Prospective studies are needed to understand the inverse association of immune-related markers and dysplasia, and to evaluate the biological effect of NSAIDs in GBC. Research supported by general funds from the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Division of Cancer Epidemiology and Genetics, and the Office of Research on Women’s Health, National Institutes of Health. Citation Format: Lorena Rosa, Paz Cook, Ruth M. Pfeiffer, Troy J. Kemp, Allan Hildesheim, Burcin Pehlivanoglu, Volkan Adsay, Enrique Bellolio, Juan Carlos Araya, Ligia Pinto, Catterina Ferreccio, Gloria Aguayo, Eduardo Viñuela, Jill Koshiol. Associations of non-steroidal anti-inflammatory drug use and circulating inflammatory proteins with gallbladder dysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3446.

Abstract 3027: p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most common and aggressive gynecological malignancy. It is typically diagnosed in late stages due to lack of diagnostic tests and current therapies available are not efficacious when the tumor is advanced. HGSOC is characterized by ubiquitous loss of functional p53 tumor suppressor protein function, largely due to point mutations that arise very early in carcinogenesis. P53 mutations are detected in pre-neoplastic cells of the fallopian tube mucosa, called p53 signature, and in early precursor lesions called serous tubal intraepithelial carcinoma (STIC). Some mutations promote the misfolding and aggregation of p53, transforming the protein into a cancer-promoting oncogene. Here, we hypothesize that the transition from folded, soluble to aggregated mutant p53 underlies the transformation from a pre-neoplastic lesion (p53 signature, STILs), to STICs and eventually HGSOC. We first set to determine the proportion of cells carrying soluble vs misfolded mutant p53 in fallopian tube tissues collected from n=10 patients undergoing salpingo-oophorectomy. We performed conformation-sensitive staining and quantification of folded and unfolded p53 cases and demonstrated that p53 misfolding, a mandatory precursor to aggregation, is present in STICs and HGSOCs, but notably absent from p53 signatures and surrounding healthy tissue. To better characterize the role of aggregated p53 in the progression of HGSOC, we took advantage of primary human fallopian tube secretory epithelial cells (FTSEC) that mimic early and more advanced stages of carcinogenesis. We utilize the FTSEC cells line FT282, immortalized by introducing hTERT and the aggregation-prone mutant TP53R175H that is found in p53 signature lesions and STILs, as well as the FT282-CCNE1, derived from FT282 cell line with overexpression of CCNE1, representing a more malignant phenotype.We used these models to investigate p53 aggregation, characterize the mutant p53 interactome and test the efficacy of ReACp53, a p53 aggregation-targeting peptide we developed. Our results demonstrated that p53 aggregation is more severe in the FT282-CCNE1 line, with a higher susceptibility to ReACp53 compared to the FT282 cells. Overall, our results demonstrated that aggregation of mutant p53 is a structural defect that can distinguish between pre-malignant and malignant HGSOC lesions and can potentially be targeted, offering a potential window for early intervention in halting ovarian cancer progression. Citation Format: Sara Sartini, Lexi Omholt, Neda A. Moatamed, Alice Soragni. p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3027.

Abstract 6849: Characterizing a discernible stromal fibroblastic gene signature in the shift from high-grade prostatic intraepithelial neoplasia to aggressive prostate cancer

Abstract Background: High-grade prostatic intraepithelial neoplasia (HGPIN) is a preneoplastic lesion that precedes the development of both indolent and aggressive variants of prostate cancer (PCa). However, the specific stromal molecular mechanisms underlying the progression of HGPIN and their contribution to epithelial cancer cell motility and invasiveness remain poorly understood. This study aims to elucidate the driver genomic alterations involved in shaping the perineoplastic stroma of HGPIN, with the goal of unraveling their role in promoting cancer invasion, and metastasis. Methods: We performed gene expression profiling on laser capture microdissected perineoplastic stroma of HGPIN and PCa in tissue samples from 25 PCa cases, including 12 low-grade (Gleason 6) and 13 high-grade (Gleason &amp;gt;/= 8) cases. We used the standard moderated t-test from the Bioconductor limma package to assess the statistical significance of differential gene expression between perineoplastic stroma high grade PCa and perineoplastic stroma HGPIN, as well as between perineoplastic stroma low grade PCa and HGPIN. The -log10 p-values represents the level of statistical significance for each gene, while the log2 fold change represents the magnitude of gene expression difference between the two comparison groups. Additionally, we computed Gene Set Enrichment Analysis (GSEA) scores using the Bioconductor package and discerned the leading-edge genes among the pathways. This study is supported by the NIH-NCI-T32 program for next-generation pathologists at our institution. Results: In the gene expression analysis comparing High Grade PCa and HGPIN, several genes, including POSTN, BGN, GREM1, C1QA, THBS4, COL1A1, COL1A2, SFRP4, RNU6-847P, FCGR2P, and TOMM22, exhibited the highest level of both biological and statistical significance. GSEA revealed that the multi-invasive pathway had the highest enrichment score (&amp;gt;2). Notably, the top overexpressed genes in this pathway exhibited a fibroblastic signature (in bold), including POSTN, BGN, GREM1, FBN1, COL1A2, COL3A1, COL5A2, ASPN, SPARC, VCAN, COMP, INHBA, THBS2, and SFRP4. The presence of this signature indicates the invasiveness of the disease. Importantly, these genes were not associated with the progression to low grade PCa, indicating their exclusive association with the aggressive variant. Conclusion: To our knowledge, this study is the first to explore the stroma's role in HGPIN-to-aggressive PCa progression. It highlights a key stromal pathway enriched with fibroblastic gene signature. These findings offer potential biomarkers for targeted therapies and improved care through HGPIN patient stratification. Citation Format: Mohammad K. Alexanderani, Lucio Queiroz, Mohamed Omar, Claudio Zanettini, Hubert Pakula, Luigi Marchionni, Massimo Loda. Characterizing a discernible stromal fibroblastic gene signature in the shift from high-grade prostatic intraepithelial neoplasia to aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6849.

Abstract 265: Reconstructing the early steps of breast tumor initiation

Abstract Breast cancer screening programs, detect not only the early stages of the disease but also various preneoplastic lesions which are very difficult to predict if and when they may develop into cancer. This uncertainty can lead to over-diagnosis, over-treatment and stress for millions of screened women. As consequence, discovering reliable biomarkers of this evolution could make it possible to monitor these patients appropriately and propose an effective prevention strategy. To meet this challenge, we urgently need to better understand the mechanisms that regulate human mammary homeostasis during early steps of tumor initiation. The hierarchy and the composition of human mammary epithelium, composed of stem, myoepithelial, luminal progenitors and mature luminal cells, are complex. This complexity raises the question of whether a given mammary epithelial cell is equally likely to be the cell-of-origin of breast cancer. Interestingly, increasing evidences show that disruption of lineage integrity can be a precursor of breast tumor initiation, and the basal and the luminal lineages respond differently to a given oncogenic stress and may each follow their own rule towards malignant transformation. These results strongly suggest that each lineage may have its own pliancy to be transformed by a given oncogene, hijacking the inter-tumoral heterogeneity observed in breast cancers. The objectives of this study are to develop a model to reconstruct the early steps of breast tumor initiation and understand the interplay between the initiating oncogenic event and the cell-of-origin. To answer these questions, we used a human mammary epithelial cell lines (HME) that maintains a cellular hierarchy with mammary stem cells (MaSC) and luminal cells (LC). Upon oncogenic expression of HRASG12V or c-Myc, we observed different modifications of cell homeostasis, with altered proportions of the lineages populations in the cell line. Depending on the oncogenic stress induced, we also demonstrated a modification in the morphology and the cellular composition of HME organoids, as well as in the capacity to form tumors. HRASG12V induces increase of MaSC compartment, formation of more branched organoids and generate aggressive basal-like tumors. Conversely, c-MYC leads to an almost loss of MaSC and formation of more proliferative unbranched organoid and generate tumors with limited growth. To address more precisely how cell-of-origin and lineage plasticity influence malignant transformation, we are developing for the first time a lineage tracing model in human mammary epithelium with HME. Moreover, a single cell RNA-seq was performed at different early time points during HME cellular differentiation in homeostasis and upon oncogenic stress to decipher the molecular mechanisms that underpin the very earliest steps of breast tumor initiation. Citation Format: Anaïs Grandon, Shuheng Lin, Martin Castagné, Julien Wicinski, Olivier Rosnet, Christophe Ginestier, Emmanuelle Charafe-Jauffret. Reconstructing the early steps of breast tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 265.

Abstract 7064: BRCA 1/2 haploinsufficiency-associated metabolic dysfunction in benign fallopian tube epithelia

Abstract In 3-17% of women with BRCA1/2 mutations who undergo risk-reducing surgery, pre-neoplastic and neoplastic lesions are found in their fallopian tubes that display differential loss of heterozygosity of BRCA1/2. Precisely how mutations in BRCA1/2 predispose women to develop high grade serous ovarian carcinoma (HGSC) is not completely understood. Although linked to various processes localized in the nucleus, BRCA1/2 may also have essential roles in the cytoplasm, specifically in the regulation of key processes associated with mitochondrial homeostasis and metabolism. Understanding how haploinsufficiency of BRCA1/2 contributes to the potential neoplastic transformation of fallopian tube epithelial cells should reveal new insights into BRCA1/2 function, and ultimately provide novel opportunities for preventative interventions in individuals with this mutation and new treatment opportunities for HGSC associated with BRCA1/2 heterozygosity. We have identified distinct metabolic and mitochondrial deficits in BRCA2 mutation carriers compared to BRCA1 carriers and non-carriers. Fallopian tube epithelial cell lines derived from BRCA2 mutation carriers showed increased levels of mitochondrial respiration and activity followed by BRCA1 mutation carriers and non-carriers. Western blot analysis of mitochondrial proteins regulating glycolysis and oxidative phosphorylation showed differential expression based on mutation status. Transmission electron microscopy of BRCA2 mutation carriers showed increased disrupted mitochondrial networks when compared to wild type, suggesting that mutation status may contribute to increased mitochondrial fission. These observations reveal an entirely new aspect of BRCA1 and BRCA2 biology and further studies will disaggregate the effects of BRCA1 and BRCA2 on mitochondrial function, versus DNA damage repair. Ultimately, this work will provide new insight into the adaptive changes within FT cells which promote neoplastic transformation and inform how high-grade serous carcinoma (HGSC) cells distinctively respond to microenvironmental stimuli. Citation Format: Jovanka Ravix, Iru Paudel, Gauthami Pulivendala, Ramlogan Sowamber, Elizabeth Smith, Matthew Schlumbrecht, David Lombard, Sophia George. BRCA 1/2 haploinsufficiency-associated metabolic dysfunction in benign fallopian tube epithelia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7064.

Abstract 4777: A small-molecule SMUG1 activator enhances repair of 5-hydroxymethyl-2’-deoxyuridine-mediated pyrimidine lesions in DNA

Abstract Background: Pharmacological upregulation of DNA repair pathways presents a potentially promising approach for cancer prevention in populations that are genetically high-risk. SMUG1, an enzyme involved in base excision repair, removes uracil and certain oxidized bases from DNA to mitigate adverse genotoxic and mutagenic effects and represents a promising target for cancer therapy. A growing body of evidence suggests that SMUG1 is involved in tumorigenesis, and deficiency of SMUG1 correlates with poor prognosis in several cancers. We hypothesize that upregulation of SMUG1 by a small molecule activator may be a viable strategy for reducing tumorigenesis in at-risk populations. We report on the properties of SU0547 - a SMUG1 activator that was derived from the EGFR inhibitor drug gefitinib - which shows reduced kinase inhibition and potent activation of SMUG1 both in vitro and in human cell lines. Methods: MTT proliferation assays were used to screen for toxicity and to determine the effect of SU0547 on 5-hydroxymethyl-2’-deoxyuridine (5-hmdU)-treated cell lines. The effect of the compound on SMUG1 expression was determined by Western blot and RT-qPCR while its effect on DSBs via γH2AX expression in 5-hmdU-treated cells was determined by immunofluorescence and Western blot. Results: SU0547 activates SMUG1 by 347 ± 50% in vitro at 100 nM, with an AC50 of 2.55 ± 0.62 µM. We examined whether it can modulate SMUG1 activity in a panel of human cell lines by artificially inducing DNA damage with the native SMUG1 substrate 5-hmdU and treating cells with the activator. In all five human cancer cell lines tested, SU0547 caused the cells to become significantly more resistant to 5-hmdU by factors of 2.1 to 5.3-fold. A series of CRISPR-Cas9 generated SMUG1 KO cell lines showed resistance to 5-hmdU and SU0547 had no effect on these treated cells, confirming that this activator acts on endogenous SMUG1. To determine the effect of the activator on double-strand breaks (DSBs), cells were treated with 5-hmdU with or without the compound and DSBs were visualized and quantitated by examining γH2AX foci. 5-hmdU treatment induced γH2AX foci while SU0547 treatment significantly reduced these foci to near or below baseline levels. These results were confirmed using Western blot quantitation of γH2AX. Conclusions: We envision that this activator will serve as a useful tool for further studying the role of SMUG1 in mediating 5-hmdU toxicity and will enable the testing of our hypothesis that enhancing DNA repair processes may serve to inhibit mutagenesis and slow progression of pre-neoplastic lesions to invasive cancers associated with genomic instability. Citation Format: Lisa A. McPherson, Yixuan Gao, Shanthi Adimoolam, Samyuktha Suresh, David L. Wilson, Ishani Das, Elizabeth R. Park, Christine S. Ng, Yong Woong Jun, James M. Ford, Eric T. Kool. A small-molecule SMUG1 activator enhances repair of 5-hydroxymethyl-2’-deoxyuridine-mediated pyrimidine lesions in DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4777.

Abstract 1434: Modeling the 3q26 OncCassette using genetically engineered mice

Abstract Lung squamous cell carcinoma (LUSC) is a devastating disease, accounting for ~30% of all lung cancer diagnoses and ~40,000 deaths annually in the United States alone. LUSC patients suffer from high prevalence of relapse and a dismal 5-year survival of only ~24%. The lack of animal models that reflect the salient features of human disease to assess the safety and efficacy of drugs, and to explore the underlying molecular mechanisms of LUSC tumor biology, is a major impediment for LUSC patients. We have identified concomitant 3q26 copy number gain (CNG) and TP53 loss as a defining genetic alteration present in ~90% of LUSC tumors. Furthermore, we have identified and characterized three 3q26 oncogenes (PRKCI, ECT2 and SOX2) that genetically and biochemically collaborate to drive LUSC tumor formation. Using syngeneic mouse modeling, we have demonstrated that coordinate overexpression of Prkci, Ect2 and Sox2, and loss of Trp53 in ex vivo cultures of lung basal stem cells (LBSCs; a cell of origin for LUSC) drives transformation of these cells into tumors of exclusively LUSC histopathology. These findings lead to the hypothesis that PRKCI, ECT2 and SOX2 represent a multigenic driver of 3q26 CNG-driven LUSC, which we term the 3q26 OncCassette. Based in these findings, we have developed and begun characterizing the first autochthonous genetically engineered mouse model (GEMM) to study 3q26 OncCassette-driven tumorigenesis. Our novel immune competent GEMM will provide critical insight into tumor initiation and progression of preneoplastic lesions to malignant LUSC and allow characterization of the immune landscape in these tumors. Citation Format: Kayleah M. Meneses, Yi Liu, Capella R. Weems, Lee Jamieson, Duy T. Nguyen, Verline Justilien, Nicole R. Murray, Alan P. Fields. Modeling the 3q26 OncCassette using genetically engineered mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1434.

Abstract A029: Development of a new iPSC-derived organoid platform to model FTE-derived HGSOC onset and progression

Abstract A029: Development of a new iPSC-derived organoid platform to model FTE-derived HGSOC onset and progression

Abstract B041: Modeling the malignant transformation of fallopian tube epithelium driven by extracellular vesicles cargos in an organ-on-chip microphysiological system

Abstract Epithelial ovarian cancer (EOC) remains the leading cause of death from gynecological malignancy and the fifth leading cause of all cancer-related deaths among American women. The most common and lethal subtype, high-grade serous ovarian cancer (HGSOC) tends to originate from the fallopian tube. Precursor lesions of HGSOC such as secretory cell expansion (SCE), the loss of PAX2, PTEN, p53 mutation and serous tubal intraepithelial carcinoma (STIC) are often found at the fallopian tube epithelium (FTE). However, the mechanism of how neoplastic lesions disseminate in FTE and eventually colonize the ovary is unclear. We are interested in understanding if ovarian cancer initiation in the FTE is mediated by extracellular vesicles (EVs), which are nanosized biovesicles secreted by all living cells. EVs serve as vehicles for nucleic acids, proteins, and lipids. Cancer cells commonly secrete more EVs than healthy cells and shift their EV cargos to promote tumorigenesis. The alteration of molecular content of tumor-derived EV can potentially indicate disease states and serve as a new class of cancer biomarkers. Hence, our hypothesis is that EV cargos secreted by tumor cells contain factors promoting preneoplastic lesions in the FTE. Our goal is to interrogate the role of EVs in the initiation of ovarian cancer in the FTE at the early stage, for which there is no adequate in vitro model. We created a microfluidic model using a 3D dynamic culture system termed PREDICT-MOS to enable the direct interaction of hFTE with EVs and the long-term culture of the fallopian tube explant. We are interested to 1) test the role of EVs to drive preneoplastic transformation of hFTE progenitor cells and 2) to test whether tumor derived-EVs shift EV proteins cargos secreted by hFTE. The hFTE cultured on PREDICT-MOS is directly exposed to OVCAR3-derived EVs and embedded for transcriptomic profiling utilizing GeoMx Digital Spatial Profiler to identify differentially expressed genes in both ciliated and secretory cells within the epithelium. In parallel, we are characterizing the hFTE EV protein content with or without OVCAR3 EV treatment to detect the alteration in hFTE cargos using mass spectrometry proteomics. Our preliminary transcriptomic data from 8 patient fallopian tube samples demonstrates the deregulation of pathways associated with inflammation (TNF and NF-kappa B signaling), immune suppression (PD-1 checkpoint signaling) and epithelium morphogenesis. In addition, we have identified 295 proteins as the results of the first proteomic characterization of hFTE-derived EV proteins, which can serve as a “baseline” to evaluate whether the fallopian tube shifts its sEV cargo during ovarian cancer carcinogenesis. Further validation of transcriptomic findings on translational level using immunohistochemistry and identification of OVCAR3 EV protein cargos that induce the transcriptomic changes are needed to establish the role of EVs in the initiation of HGSOC in the fallopian tube. This project is supported by the Ovarian Cancer Research Fund Alliance Program Grant. Citation Format: Didi Zha, Jared Sipes, Sagar Rayamajhi, Leonidas E. Bantis, Angela Russo, Harsh Pathak, Andrew K. Godwin, Joanna E. Burdette. Modeling the malignant transformation of fallopian tube epithelium driven by extracellular vesicles cargos in an organ-on-chip microphysiological system [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B041.

Smad4 restricts injury-provoked biliary proliferation and carcinogenesis.

Cholangiocarcinoma (CCA) is a deadly and heterogeneous type of cancer characterized by a spectrum of epidemiologic associations as well as genetic and epigenetic alterations. We seek to understand how these features inter-relate in the earliest phase of cancer development and through the course of disease progression. For this, we studied murine models of liver injury integrating the most commonly occurring gene mutations of CCA - including Kras, Tp53, Arid1a and Smad4 - as well as murine hepatobiliary cancer models and derived primary cell lines based on these mutations. Among commonly mutated genes in CCA, we found that Smad4 functions uniquely to restrict reactive cholangiocyte expansion to liver injury through restraint of the proliferative response. Inactivation of Smad4 accelerates carcinogenesis, provoking pre-neoplastic biliary lesions and CCA development in an injury setting. Expression analyses of Smad4-perturbed reactive cholangiocytes and CCA lines demonstrated shared enriched pathways, including cell-cycle regulation, MYC signaling and oxidative phosphorylation, suggesting that Smad4 may act via these mechanisms to regulate cholangiocyte proliferation and progression to CCA. Overall, we showed that TGFβ/SMAD4 signaling serves as a critical barrier restraining cholangiocyte expansion and malignant transformation in states of biliary injury.

Computer-aided diagnosis in real-time endoscopy for all stages of gastric carcinogenesis: Development and validation study.

Using endoscopic images, we have previously developed computer-aided diagnosis models to predict the histopathology of gastric neoplasms. However, no model that categorizes every stage of gastric carcinogenesis has been published. In this study, a deep-learning-based diagnosis model was developed and validated to automatically classify all stages of gastric carcinogenesis, including atrophy and intestinal metaplasia, in endoscopy images. A total of 18,701 endoscopic images were collected retrospectively and randomly divided into train, validation, and internal-test datasets in an 8:1:1 ratio. The primary outcome was lesion-classification accuracy in six categories: normal/atrophy/intestinal metaplasia/dysplasia/early /advanced gastric cancer. External-validation of performance in the established model used 1427 novel images from other institutions that were not used in training, validation, or internal-tests. The internal-test lesion-classification accuracy was 91.2% (95% confidence interval: 89.9%-92.5%). For performance validation, the established model achieved an accuracy of 82.3% (80.3%-84.3%). The external-test per-class receiver operating characteristic in the diagnosis of atrophy and intestinal metaplasia was 93.4±0% and 91.3±0%, respectively. The established model demonstrated high performance in the diagnosis of preneoplastic lesions (atrophy and intestinal metaplasia) as well as gastric neoplasms.

To study ERG expression in benign, pre-neoplastic, and neoplastic lesions of prostate

Background: Prostate cancer (PCa) is the second most common malignancy in men and the sixth leading cause of death. Clinical course of PCa is highly variable and current clinico pathological parameters are unable to predict accurately the course of the disease because of genetic heterogeneity seen in this tumor. Aims and Objectives: The aim of the study was to study the prevalence of E26 transformation-specific-related gene (ERG) expression in benign and malignant lesions of prostate in Northern Indian patients. Whether ERG expression using immunohistochemical (IHC) methods can also be used for differentiating various lesions of the prostate gland is also evaluated. Materials and Methods: This is an observational study conducted on 50 biopsy specimens encompassing different lesions of prostate. The study was carried out in the Department of Pathology at Integral Institute of Medical Sciences and Research, Lucknow, India, during the period January 2020–September 2022. Results: Amongst 50 cases of prostatic lesions included in the study, there were 20 cases of benign prostatic hyperplasia (BPH), 17 cases of benign mimicker lesions and 13 cases of PCa. ERG expression was absent in all cases of BPH and benign mimicker lesions. ERG expression was seen in 38.5% of cases of PCa. Conclusion: In our study, none of the patients of BPH or benign mimicker lesion expressed ERG on IHC, while 38.5% of patients of PCa showed positive staining. This figure differs from reports from other parts of the world. ERG expressions can be a potential therapeutic target for personalized treatment in a subset of patients of PCa. However, it was not found to be a sensitive marker for the diagnosis of PCa.

Norepinephrine induces anoikis resistance in high-grade serous ovarian cancer precursor cells.

High-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to stess-induced sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines were able to survive in ultra-low-attachment (ULA) culture in a β-adrenergic receptor-dependent (β-AR-dependent) manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE were abrogated using the β-AR blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony-stimulating factor 2. These findings provide mechanistic insight and identify targets that may be regulated by ovary-derived NE in early HGSC.

Interleukin-18 produced by gastric epithelial cells protects against pre-neoplastic lesions in Helicobacter pylori infection in mice.

Interleukin-18 produced by gastric epithelial cells protects against pre-neoplastic lesions in Helicobacter pylori infection in mice.

Spatial profiling reveals tissue-specific neuro-immune interactions in gastroenteropancreatic neuroendocrine tumors.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous malignancies that arise from complex cellular interactions within the tissue microenvironment. Here, we sought to decipher tumor-derived signals from the surrounding microenvironment by applying digital spatial profiling (DSP) to hormone-secreting and non-functional GEP-NETs. By combining this approach with in vitro studies of human-derived organoids, we demonstrated the convergence of cell autonomous immune and pro-inflammatory proteins that suggests their role in neuroendocrine differentiation and tumorigenesis. DSP was used to evaluate the expression of 40 neural- and immune-related proteins in surgically resected duodenal and pancreatic NETs (n = 20) primarily consisting of gastrinomas (18/20). A total of 279 regions of interest were examined between tumors, adjacent normal and abnormal-appearing epithelium, and the surrounding stroma. The results were stratified by tissue type and multiple endocrine neoplasia I (MEN1) status, whereas protein expression was validated by immunohistochemistry (IHC). A tumor immune cell autonomous inflammatory signature was further evaluated by IHC and RNAscope, while functional pro-inflammatory signaling was confirmed using patient-derived duodenal organoids. Gastrin-secreting and non-functional pancreatic NETs showed a higher abundance of immune cell markers and immune infiltrate compared with duodenal gastrinomas. Compared with non-MEN1 tumors, MEN1 gastrinomas and preneoplastic lesions showed strong immune exclusion and upregulated expression of neuropathological proteins. Despite a paucity of immune cells, duodenal gastrinomas expressed the pro-inflammatory and pro-neural factor IL-17B. Treatment of human duodenal organoids with IL-17B activated NF-κB and STAT3 signaling and induced the expression of neuroendocrine markers. In conclusion, multiplexed spatial protein analysis identified tissue-specific neuro-immune signatures in GEP-NETs. Duodenal gastrinomas are characterized by an immunologically cold microenvironment that permits cellular reprogramming and neoplastic transformation of the preneoplastic epithelium. Moreover, duodenal gastrinomas cell autonomously express immune and pro-inflammatory factors, including tumor-derived IL-17B, that stimulate the neuroendocrine phenotype. © 2024 The Pathological Society of Great Britain and Ireland.