Microdissected Epithelium Research Articles

Exploring the Regulators of Keratinization: Role of BMP-2 in Oral Mucosa.

The oral mucosa functions as a physico-chemical and immune barrier to external stimuli, and an adequate width of the keratinized mucosa around the teeth or implants is crucial to maintaining them in a healthy and stable condition. In this study, for the first time, bulk RNA-seq analysis was performed to explore the gene expression of laser microdissected epithelium and lamina propria from mice, aiming to investigate the differences between keratinized and non-keratinized oral mucosa. Based on the differentially expressed genes (DEGs) and Gene Ontology (GO) Enrichment Analysis, bone morphogenetic protein 2 (BMP-2) was identified to be a potential regulator of oral mucosal keratinization. Monoculture and epithelial-mesenchymal cell co-culture models in the air-liquid interface (ALI) indicated that BMP-2 has direct and positive effects on epithelial keratinization and proliferation. We further performed bulk RNA-seq of the ALI monoculture stimulated with BMP-2 in an attempt to identify the downstream factors promoting epithelial keratinization and proliferation. Analysis of the DEGs identified, among others, IGF2, ID1, LTBP1, LOX, SERPINE1, IL24, and MMP1 as key factors. In summary, these results revealed the involvement of a well-known growth factor responsible for bone development, BMP-2, in the mechanism of oral mucosal keratinization and proliferation, and pointed out the possible downstream genes involved in this mechanism.

Mutation and methylation profiles of ectopic and eutopic endometrial tissues.

Adenomyosis and peritoneal endometriosis are common gynecologic lesions; they are characterized by aberrant locations of normal-appearing endometrium in myometrium and peritoneal surface, respectively. Both ectopic lesions are speculated to originate from uterine eutopic endometrium, which is composed of epithelium and stroma, but how these two different tissue types co-evolve in ectopic locations remains unclear. Here, we analyzed exome-wide mutations and global methylation in microdissected epithelium and stroma separately in paired adenomyosis, peritoneal endometriosis, and endometrium to investigate their relationship. Analyses of somatic mutations and their allele frequencies indicate monoclonal development not only in epithelium but also in the stroma of adenomyosis and peritoneal endometriosis. Our preliminary phylogenetic study suggests a plausible clonal derivation in epithelium and stroma of both ectopic and eutopic endometrium from the same founder epithelium-stroma progenitor cells. While a patient-specific methylation landscape is evident, adenomyosis epithelium and stroma can be distinguished from normal-appearing eutopic endometrium epigenetically. In summary, endometrial stroma, like its epithelial counterpart, could be clonal and both ectopic and eutopic endometrium following divergent evolutionary trajectories. Our data also warrant future investigations into the role of endometrial stroma in the pathobiology of endometrium-related disorders. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Upregulation of lipid metabolism genes in the breast prior to cancer diagnosis

Histologically normal tissue adjacent to the tumor can provide insight of the microenvironmental alterations surrounding the cancerous lesion and affecting the progression of the disease. However, little is known about the molecular changes governing cancer initiation in cancer-free breast tissue. Here, we employed laser microdissection and whole-transcriptome profiling of the breast epithelium prior to and post tumor diagnosis to identify the earliest alterations in breast carcinogenesis. Furthermore, a comprehensive analysis of the three tissue compartments (microdissected epithelium, stroma, and adipose tissue) was performed on the breast donated by either healthy subjects or women prior to the clinical manifestation of cancer (labeled “susceptible normal tissue”). Although both susceptible and healthy breast tissues appeared histologically normal, the susceptible breast epithelium displayed a significant upregulation of genes involved in fatty acid uptake/transport (CD36 and AQP7), lipolysis (LIPE), and lipid peroxidation (AKR1C1). Upregulation of lipid metabolism- and fatty acid transport-related genes was observed also in the microdissected susceptible stromal and adipose tissue compartments, respectively, when compared with the matched healthy controls. Moreover, inter-compartmental co-expression analysis showed increased epithelium-adipose tissue crosstalk in the susceptible breasts as compared with healthy controls. Interestingly, reductions in natural killer (NK)-related gene signature and CD45+/CD20+ cell staining were also observed in the stromal compartment of susceptible breasts. Our study yields new insights into the cancer initiation process in the breast. The data suggest that in the early phase of cancer development, metabolic activation of the breast, together with increased epithelium-adipose tissue crosstalk may create a favorable environment for final cell transformation, proliferation, and survival.

Evaluation of NID2 promoter methylation for screening of Oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is an aggressive human malignancy. Because of late diagnosis and recurrence of OSCC, the treatment of patients with OSCC is often ineffective. Thus, finding novel biomarkers of OSCC are essential. Here we derived a methylation marker by utilizing methylation microarray data and testing its capacity in cross-sectional study designed for OSCC detection and screening.MethodsAccording to bioinformatics analysis of total of 27,578 cg sites, cg22881914 of Nidogen 2 (NID2) methylation was selected for evaluation. Next, we confirmed the methylation status by bisulfite sequencing from the microdissected OSCC cells in comparison with the microdissected oral epithelia. Subsequently, we developed a simple technique using real-time PCR with the specific probe to examine the ability for the detection of OSCC in the oral epithelial samples, which included 103 oral rinse and 82 oral swab samples.ResultsBased on the comparison of microdissected tissue, cg22881914 of NID2 was proved to be methylated in most OSCC cells but unmethylated in the normal oral epithelia. Furthermore, the methylated NID2-relied quantitative PCR approach has demonstrated that this marker assists in distinguishing among patients with OSCC from normal oral epithelia, smokers, and patients with oral lichen planus using the non-invasive oral rinse and swab samples.ConclusionsSpecific methylation at cg22881914 of NID2 of OSCC could be used as an important potential marker for detecting OSCC. Thus, to certify the utility of this marker, further studies with a larger sample size are needed.

Discovery and Validation of Novel Biomarkers for Detection of Epithelial Ovarian Cancer.

Detection of epithelial ovarian cancer (EOC) poses a critical medical challenge. However, novel biomarkers for diagnosis remain to be discovered. Therefore, innovative approaches are of the utmost importance for patient outcome. Here, we present a concept for blood-based biomarker discovery, investigating both epithelial and specifically stromal compartments, which have been neglected in search for novel candidates. We queried gene expression profiles of EOC including microdissected epithelium and adjacent stroma from benign and malignant tumours. Genes significantly differentially expressed within either the epithelial or the stromal compartments were retrieved. The expression of genes whose products are secreted yet absent in the blood of healthy donors were validated in tissue and blood from patients with pelvic mass by NanoString analysis. Results were confirmed by the comprehensive gene expression database, CSIOVDB (Ovarian cancer database of Cancer Science Institute Singapore). The top 25% of candidate genes were explored for their biomarker potential, and twelve were able to discriminate between benign and malignant tumours on transcript levels (p < 0.05). Among them T-cell differentiation protein myelin and lymphocyte (MAL), aurora kinase A (AURKA), stroma-derived candidates versican (VCAN), and syndecan-3 (SDC), which performed significantly better than the recently reported biomarker fibroblast growth factor 18 (FGF18) to discern malignant from benign conditions. Furthermore, elevated MAL and AURKA expression levels correlated significantly with a poor prognosis. We identified promising novel candidates and found the stroma of EOC to be a suitable compartment for biomarker discovery.

Abstract P4-04-06: Study of the mutational landscape of normal and pregnant breast to predict pregnancy-associated breast cancer risk

Abstract Background: Amongst the various risk factors for breast cancer (BC), the molecular basis which may explain the correlation between age at first full-term and breast cancer risks is still understudied. Epidemiology studies indicate that an early first full-term pregnancy (before 25 years of age) confers a significant level of protection towards the development of post-menopausal BC compared to the risk in nulliparous or late-parous women. On the other hand, any pregnancy relates to a higher risk in developing cancer during or within one year of pregnancy (pregnancy-associated breast cancer, PABC). Thus, the relation between age of pregnancy and breast cancer risk may be too difficult to explain using only epidemiology data. Aims of the study: With our research study, we aim to study a cohort of 60 normal breast samples of nulliparous and age-matched early- and late-parous women collected from Komen Tissue Bank, University of Indiana, and to create for the first time a mathematical model of cell clone expansion in the normal breast growth. This will allow us to determine how the rates of both cancer drivers, passenger mutations and genetic variations are affected by pregnancy. We then aim to translate this in cancer tissues, and to determine how the rate of the same mutations in both pregnancy and non pregnancy-associated cancers (post-menopausal). At the same time, we intend to create a mouse model which will be used to further validate our model, where driver mutations will be induced in the mammary epithelium of pregnant mice of different ages. This will allow us to test our model of growth of a mutated clone in a pregnancy environment, and to determine what are the molecular changes in the pregnant mammary gland which can trigger a different BC risk in the early-parous cohort. Results: To examine the mutational landscape in the normal parous and nulliparous women, we extracted DNA from laser-capture microdissected epithelium and the stroma, the latter of which will be used to eliminate germ line mutations. We are currently analysing the results from Whole Genome Sequencing at 30x 100pe on a MGISEQ2000 platform on a first set of samples (two nulliparous samples and two age-matched parous samples from both early and late pregnancy). Our procedure for processing and analysis of this data follows the Broad Institute's “GATK Best Practice Guidelines” for use of next generation sequencing (NGS) data. Based on the collected data, we plan to continue with targeted sequencing or whole genome sequencing on the remaining samples. Conclusions: Our study will provide novel information on which areas of the genome are mostly mutated or altered in the normal breast, and will indicate how mutated cells, including mutations in driver genes for breast cancer, and genetic alterations change in the contest of pregnancy. With the mathematical model of clone growth/extinction, we intend to explain how different ages of pregnancy can significantly alter the clone composition in the normal breast and result in a different probability of developing breast cancer. Citation Format: Cereser B, Tabassum N, Carter P, Del Bel Belluz L, Stebbing J. Study of the mutational landscape of normal and pregnant breast to predict pregnancy-associated breast cancer risk [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-04-06.

Abstract 2456: The PIWI-interacting RNA pathway in prostate is regulated by vitamin D and altered in cancer

Abstract Low serum vitamin D is a risk factor for aggressive prostate cancer. piRNAs and their interacting partners, PIWI-like (PIWIL) proteins, are aberrantly expressed in many cancers, but few studies have focused on piRNAs in the prostate. We used laser-capture microdissected (LCM) human prostate epithelium from frozen samples collected for a clinical trial of high dose vitamin D to examine the small non-coding RNA population by next-generation sequencing. The 8 patients for this study were selected for having either high (avg=48.7pg/ml) or low (avg=21.4pg/ml) prostatic vitamin D (1,25(OH)2D) levels. VDR ChIP-seq was performed as well, using human primary epithelial cells treated for 3hr with EtOH or 50nM vitamin D. In LCM-collected prostate epithelium unexpectedly piRNAs represented ~20% of the mapped reads. Of all the small RNA species investigated, piRNAs were the most affected by vitamin D status, increasing in abundance in tissues wish high vitamin D (p=0.036, t-test). The LCM data were compared to small-RNA sequencing data from benign prostate available from The Cancer Genome Atlas (TCGA), which represent whole prostate tissue and may contain stromal bias. The LCM-collected samples had a distinct, more consistent profile of small RNAs than TCGA samples. Two unambiguous piRNAs that were highly expressed in LCM data, but not in TCGA data, were verified as epithelial-specific in prostate by in situ hybridization. Several piRNA-biogenesis genes lie within 100kb of VDR ChIP peaks, including TDRKH, which was found to be vitamin D-regulated experimentally. The expression of piRNA-interacting PIWIL proteins was assessed by RT-qPCR, immunoblot and immunofluorescence on a tissue microarray containing paired benign and cancer tissues (N=23). Of the 4 human PIWIL proteins, PIWIL1, 2 and 4 were present in normal prostate epithelial cells by PCR. PIWIL1 protein was also detected in PrE and RWPE-1 benign cells, but PIWIL1 was not detected in the PC3 prostate cancer cell line. By immunofluorescence, PIWIL1 protein in prostate epithelium significantly decreased in prostate cancer (p=0.016, paired t-test). In conclusion, vitamin D status associated with the expression of piRNA in prostate epithelium in clinical trial samples. PIWIL1 protein levels were lower in prostate cancer, suggesting a pro-differentiation role for the PIWI pathway that may be affected in vitamin D-deficient patients. Citation Format: Bethany Baumann, Shang Gao, Giovanni Lugli, Larisa Nonn. The PIWI-interacting RNA pathway in prostate is regulated by vitamin D and altered in cancer [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 2456.

Abstract 4250: Molecular alterations in the breast associated with early menarche

Abstract Background: Menarche, the onset of the female menstruation, is a marker of pubertal timing. Age at menarche varies widely between girls and is highly dependent on nutritional status and body fat accumulation. The occurrence of menarche at an early age is linked to an increased risk of several adverse health conditions later in life, such as obesity, type-2 diabetes, breast and endometrial cancer, and cardiovascular disease. Indeed, for every one year decrease in age at menarche (from an average age at menarche of 12.5 years), breast cancer risk is increased by 5%. Several genome wide association studies (GWAS) have identified genetic variants (i.e. in the LIN28B gene) that are associated with early age at menarche, however little is known about the changes occurring in the breast tissue of women with early menarche. We hypothesize that early age at menarche results in permanent molecular alterations in the breast tissue and that those abnormalities may contribute to the tissue’s susceptibility to carcinogens and breast cancer development. Methods: To test our hypothesis we used the resources available at the Susan G. Komen Tissue Bank at the Indiana University Simon Cancer Center (KTB). We selected histologically normal breast tissue from healthy, young women with either early (age ≤ 10 years) or late menarche (age ≥ 15 years), and matched for age, race, BMI, and menstrual phase. Breast tissue biopsies from these women were microdissected to isolate the breast epithelium and next generation RNA-sequencing was used to generate a transcriptome profile for each sample. Differential expression was performed using DESeq2 in R. The tissue samples were also evaluated using immunostaining. Results/Conclusions: Preliminary data show significant differences when comparing the transcriptome profiles of the microdissected breast epithelium from the early and late menarche sample cohorts. The tissue from women with early menarche had upregulation of genes associated with defense against oxidative stress and/or infectious bacteria (lactotransferrin [LTF], ceruloplasmin [CP]), cell adhesion, (ITGα11, ITGαX, ITGαM, ITGαL, ITGβ2), immune response (CARD9, LAIR1), and had downregulation of ubiquitination pathways (USP40, AMFR) and lipoprotein metabolism (OSBPL1A, LIPH, PIGN). Immunohistochemical evaluation of markers of oxidative stress (LTF, CP), cell proliferation (Ki67), and immune infiltrates (CD45, CD20, CD8, CD68) is underway. Together, this information will give us the opportunity to better understand early age at menarche as a breast cancer risk factor and advance research for women’s health. Citation Format: Mariah L. Johnson, Natascia Marino, Anna Maria V. Storniolo, Bradley A. Hancock, Milan Radovich, George E. Sandusky. Molecular alterations in the breast associated with early menarche [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 4250. doi:10.1158/1538-7445.AM2017-4250

Abstract 4826: Maternal high butter fat intake heightens mammary cancer risk in offsprings gestationally exposed to bisphenol A at environmentally relevant dose

Abstract In utero exposure to bisphenol A (BPA) at 250ug/kg BW was shown to augment mammary cancer risk in rodent models. High fat diet is widely believed to be a major risk of breast cancer (BCa). It is therefore important to investigate whether maternal high fat intake could potentially heighten mammary cancer risk associated to gestational BPA exposure. In this study, we exposed Sprague Dawley rats with different doses of BPA (2.5-2500ug/kg BW) mixed with high butter-based diet (HBF) in the period from preconception to birth. DMBA-PND50 protocol was followed. In the presence of HBF, we observed non-monotonic effects of BPA in cancer incidence as well as in the number of terminal end buds of PND21 mammary glands. Both results were peaked at a dosage of 25ug/kg BW BPA and the effects were significant when compared with HBF alone group. We further analyzed transcriptomic data of microdissected epithelia of PND21 mammary glands from BPA (25ug/kg BW) group as well as HBF group and revealed that two specific cancer networks involving ERK and androgen receptor signaling were dysregulated. DNA methylation is one of the key mechanisms to dysregulate and impair the transcription of two selected top genes, Car7 and Kcnv2, as supported by our bisulfite-sequencing data. These data suggest that in utero BPA exposure could epigenetically modify gene expression and predispose cancer risk during early development. More importantly, some BPA genes are of high human relevance because seven selected BPA genes could dichotomize patients into two groups with significant difference in overall survival in a TCGA cancer cohort. The prognostic power of the genes was further enhanced in the survival analysis of Caucasian with ER positive patients. In conclusion, our data strongly suggest that BPA predisposes higher cancer risk by dysregulating gene expression during early mammary gland development in the presence of HBF, which could facilitate cancer development later in life. Citation Format: Yuet-Kin Leung, Vinothini Govindarajah, Ana Cheong, Dan Song, Xuegong Zhu, Jun Ying, Ady Kendler, Mario Medvedovic, Scott Belcher, Shuk-Mei Ho. Maternal high butter fat intake heightens mammary cancer risk in offsprings gestationally exposed to bisphenol A at environmentally relevant dose [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 4826. doi:10.1158/1538-7445.AM2017-4826

Cancer-Associated Mutations in Endometriosis without Cancer

BackgroundEndometriosis, defined as the presence of ectopic endometrial stroma and epithelium, affects approximately 10% of reproductive-age women and can cause pelvic pain and infertility. Endometriotic lesions are considered to be benign inflammatory lesions but have cancerlike features such as local invasion and resistance to apoptosis.MethodsWe analyzed deeply infiltrating endometriotic lesions from 27 patients by means of exomewide sequencing (24 patients) or cancer-driver targeted sequencing (3 patients). Mutations were validated with the use of digital genomic methods in microdissected epithelium and stroma. Epithelial and stromal components of lesions from an additional 12 patients were analyzed by means of a droplet digital polymerase-chain-reaction (PCR) assay for recurrent activating KRAS mutations.ResultsExome sequencing revealed somatic mutations in 19 of 24 patients (79%). Five patients harbored known cancer driver mutations in ARID1A, PIK3CA, KRAS, or PPP2R1A, which were validated by Safe-Sequencing System or immunohistochemical analysis. The likelihood of driver genes being affected at this rate in the absence of selection was estimated at P=0.001 (binomial test). Targeted sequencing and a droplet digital PCR assay identified KRAS mutations in 2 of 3 patients and 3 of 12 patients, respectively, with mutations in the epithelium but not the stroma. One patient harbored two different KRAS mutations, c.35G→T and c.35G→C, and another carried identical KRAS c.35G→A mutations in three distinct lesions.ConclusionsWe found that lesions in deep infiltrating endometriosis, which are associated with virtually no risk of malignant transformation, harbor somatic cancer driver mutations. Ten of 39 deep infiltrating lesions (26%) carried driver mutations; all the tested somatic mutations appeared to be confined to the epithelial compartment of endometriotic lesions.

Overexpression of lipid metabolism genes and PBX1 in the contralateral breasts of women with estrogen receptor-negative breast cancer

Risk biomarkers for estrogen receptor (ER)-negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis.

Proteomic characterization of microdissected breast tissue environment provides a protein-level overview of malignant transformation.

Both healthy and cancerous breast tissue is heterogeneous, which is a bottleneck for proteomics‐based biomarker analysis, as it obscures the cellular origin of a measured protein. We therefore aimed at obtaining a protein‐level interpretation of malignant transformation through global proteome analysis of a variety of laser capture microdissected cells originating from benign and malignant breast tissues. We compared proteomic differences between these tissues, both from cells of epithelial origin and the stromal environment, and performed string analysis. Differences in protein abundances corresponded with several hallmarks of cancer, including loss of cell adhesion, transformation to a migratory phenotype, and enhanced energy metabolism. Furthermore, despite enriching for (tumor) epithelial cells, many changes to the extracellular matrix were detected in microdissected cells of epithelial origin. The stromal compartment was heterogeneous and richer in the number of fibroblast and immune cells in malignant sections, compared to benign tissue sections. Furthermore, stroma could be clearly divided into reactive and nonreactive based on extracellular matrix disassembly proteins. We conclude that proteomics analysis of both microdissected epithelium and stroma gives an additional layer of information and more detailed insight into malignant transformation.

Abstract B44: Immune pathway dysregulations in inflammatory breast cancer health disparity

Abstract Background: Inflammatory breast cancer (IBC) is a distinct, aggressive and the most lethal form of breast cancer. Furthermore, data from population-based registries, when stratified by race and compared to other locally advanced breast cancers, suggest that IBC has a disproportionately high incidence, prevalence &amp; shorter median survival time in African Americans (AA) compared to Caucasian American (CA) patients. IBC health disparities that are independent of external factors like differences in income, screening rates, and access to health care suggest a biological component to poor outcomes. IBC pathobiology is also unique wherein instead of a solid tumor, tightly packed tumor cell clusters/emboli are formed with a propensity for lymphatic dissemination. The underlying mechanisms for this extremely aggressive breast cancer phenotype are largely unknown and, therefore, present a major impediment to identifying the molecular underpinnings for health disparities in IBC patients. Methods and Results: In order to understand how IBC cells evade cell death signals in the host microenvironment, we conducted comparative analysis of the largest collection of untreated primary tumor samples of IBC (N=137), along with stage- and subtype-matched non-IBC (N=252) and normal breast tissue. Results reveal that pretreatment tumor tissue samples from IBC patients have a heightened expression of adaptive stress response genes comprised of high levels of the nuclear transcription factor, NFkappaB, and its target genes in the anti-apoptotic and immune/inflammatory pathways that correlate with cell survival. In addition, guided by analysis based on presence of tumor infiltrating leukocytes in IBC (N=69) and non-IBC (N=62) patient samples, we have identified a set of pathways and genes that have the ability to distinguish the immune responses in IBC and non-IBC patients in a subtype independent manner. In particular, interferon alpha/beta and NFκB signaling pathways were differentially regulated in IBC vs. non-IBC. Gene expression driven analysis of enrichment of leukocyte subsets reveal specific differences in plasma cells and effector memory CD8 cells in IBC bulk tumor samples. Preliminary data analysis of gene expression data sets from laser microdissected epithelium and stromal compartments of AA and CA breast cancer patients also show enrichment of plasma cells, effector memory CD4+, and M1 macrophages. Studies are ongoing to characterize these immune profiles that are unique and distinguishable in IBC patients and further identify race-related differences. Most importantly, we discovered that the constitutive NFκB hyperactivation observed in IBC tumor cells is driven by stress-mediated translational upregulation of the most potent anti-apoptotic protein, XIAP. Interestingly, XIAP:NFκB overexpressing cells are highly resistant to immunotherapy mediated cell death. These data strongly suggest that in IBC, host immune phenotype promotes outgrowth of cell death resistant population and specific tumor cell pro-survival signaling mechanisms that enhances invasion and lymphatic dissemination. We have developed novel in vitro IBC tumor emboli culture model and a transgenic mouse bearing red fluorescent lymphatic vasculature that allow recapitulating host factors to define tumor cell signaling mechanisms on immune effector function and the resultant invasive phenotype. Conclusions: As the incidence is rising and targets for therapy are scant, IBC has the potential to become a major public health disparity concern. This study reveals that the immune factors in the host microenvironment may interact with underlying IBC genetics to promote the aggressive nature of the tumor, and more specifically, these factors may be one of the sources of biologic variation between patients of different ethnicities leading to IBC disparity outcomes. Supported in part by Duke IBC Consortium, DCI NIH CAO14236 development funds (GRD), DoD W81XWH-13-1-0047 (GRD). Citation Format: Gayathri R. Devi, Eun-Sil Shelley Hwang, John Stewart, Michael A. Morse, Steven Van Laere. Immune pathway dysregulations in inflammatory breast cancer health disparity. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B44.

Abstract 2619: Novel biomarkers and molecular alterations for breast cancer initiation and susceptibility

Abstract Background: Despite significant advances in diagnosis and treatment, breast cancer remains the leading cause of cancer-related death in women worldwide. Therefore, there is a critical need to identify molecular mechanisms responsible for cancer initiation and progression. To date, there have been attempts to identify biomarkers that can predict progression of pre-malignant lesions (i.e. DCIS) to invasive carcinoma. Our study aims to identify earliest markers of breast cancer initiation. Methods: We evaluated local (breast tissue) and systemic (serum/plasma) molecular alterations associated with breast cancer susceptibility using the unique resources available at the Komen Normal Tissue Bank at IUSCC. Human specimens (serum/plasma and breast tissue) donated by women before their cancer was clinically detectable were used to identify circulating biomarkers that are associated with breast cancer risk. Specimens from age-matched controls were used for comparison. Sera/plasma were analyzed for circulating miRNAs and telomeric level in the cell-free circulating DNA (cfDNA). We employed next generation sequencing to obtain transcriptome in the “normal” breast of women who eventually developed breast cancer and age-matched control normal breast. Results/Discussion: Serum/plasma of women who developed breast cancer showed variation in circulating miRNAs as well as telomeric cfDNA levels as compared to the healthy control group. Among 385 microRNAs detected in circulation, 10 miRNAs were present at different levels in the susceptible group. In addition, susceptible group displayed reduced levels of plasma telomeric cfDNA and telomere shortening in breast epithelium. Transcriptome analysis of microdissected breast epithelium and stroma revealed molecular alterations in the “normal” breast tissue associated with cancer development. Pathway analyses of these differentially expressed genes are underway to determine the mechanisms associated with breast cancer initiation. Conclusion/Impact: Functional analysis of biomarkers identified in this study may help in cancer risk assessment and improvement of preventive therapy. Citation Format: Natascia Marino, Mariah L. Johnson, Hiromi Tanaka, Xi Wu, Theresa Mathieson, Jill E. Henry, Mircea Ivan, Yunlong Liu, Connie Rufenbarger, Anna Maria V. Storniolo. Novel biomarkers and molecular alterations for breast cancer initiation and susceptibility. [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 2619.

Abstract P4-13-01: Trancriptome sequencing of the hisologicaly normal breast epithelium of BRCA mutation carriers

Abstract Background: Meaningful progress in the prevention of breast cancer is unlikely until the risk of the development of breast cancer is translated into specific, quantifiable molecular alterations. Among those women at the greatest risk are BRCA mutations carriers: 55 to 65% of women who inherit a deleterious BRCA1 mutation and approximately 45 % who inherit a deleterious BRCA2 mutation will develop breast cancer by age 70 years. The purpose of this study was to identify the earliest transcriptional alterations present by examining the histologically normal breast epithelia of BRCA mutation carriers. Methods: Epithelia were microdissected and the RNA isolated from the histologically normal breast of 16 frozen tissue cores from known BRCA mutation carriers, who were donors to the Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center. RNA-sequencing was carried out using the Life Technologies SOLiD XL 5500 Platform. RPKM gene expression values from the BRCA specimens and from Komen normal breast epithelium (controls; Pardo et al, Breast Cancer Research, 2014) were merged, quantile normalized, and batch effect corrected. Normalization and differential gene expression was performed using EdgeR v2.11 Results: 6583 RNAs were differentially expressed with the false discovery rate set at 0.1. 1. DNA damage is signaled by a tripartite system that includes kinases, phosphatases and proteins with modular domains, e.g., the BRCA 1 C-terminal (BRCT), that recognize phosphorylated linear motifs in other proteins. Several BRCT domains recognize motifs phosphorylated by kinases that are activated by DNA damage. To identify the proteins active in DNA damage signaling, Monteiro and colleagues generated a protein-protein interaction map for seven proteins with tandem BRCT (Cell Signaling, 2012). A significant proportion of the genes with decreased expression in our data set encode proteins that have been identified in this network. 2. Two of the top biological processes affected by the BRCA mutations in the histologically normal breast tissue are translation, and cellular protein metabolism. 3. There was significantly less expression of genes associated with other hereditary cancer syndromes inducing the genes responsible for Lynch Syndrome: MHL1, MHS2, MSH6 and PMS2; and the multiple endocrine neoplasia genes RET and MEN1. 4. High grade serous carcinoma of the ovary, the histologic subtype associated with BRCA mutation, may originate in the distal Fallopian tube. The list of differentially expressed genes from microdissected breast epithelia was compared with that derived from gene array profiling of microdissected Fallopian tube epithelium from histologically normal BRCA1 mutation carriers and controls. 795 genes were common to both gene sets, of which 354 were regulated similarly, i.e., increased or decreased. MetaCore data analysis revealed CREB1 and c-Myc as the most important regulatory factors in the similar dataset. Conclusions: There are significant transcription alterations in the histologically normal breast tissue of BRCA mutation carriers. These data will have to be corroborated at the protein level and functional level. Once substantiated, they have potential to stratify risk and to serve as targets for prevention. Citation Format: Dadrie Baptiste, MiRan Choi, Zhiping Wang, Yunlong Liu, Milan Radovich, Susan E Clare. Trancriptome sequencing of the hisologicaly normal breast epithelium of BRCA mutation carriers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-13-01.

Abstract PD05-06: Next-generation RNA-sequencing of triple negative breast cancer compared to donated microdissected normal epithelium and adjacent normal tissues

Abstract Background: There currently exists an inadequate understanding of the transcriptional dysregulation that differentiates Triple-Negative Breast Cancer (TNBC) from normal breast epithelium. Currently, a popular comparator in gene expression studies is to use normal tissue adjacent to the primary tumor. Using RNA-seq data of TNBCs and donated microdissected normal breast epithelium from our institution combined with TNBCs and adjacent normal tissues from the The Cancer Genome Atlas (TCGA), we sought to determine the differential regulation of genes between TNBC compared to normal breast epithelium and adjacent normal tissues. Methods: Normal breast tissues from healthy pre-menopausal volunteers with no history of disease were procured from the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. To eliminate bias from stromal tissue, normal tissues were laser capture microdissected for ductal epithelium. RNA from 20 microdissected normal breast tissues and 10 TNBCs from our institution were sequenced on a Life Technologies SOLiD sequencer. This data was combined with RNA-seq data from an additional 77 TNBCs and 10 adjacent normal tissues from the TCGA Data Portal. The merged gene expression values (RPKM) were quantile normalized, batch effect corrected, and submitted for differential gene expression analysis using Partek Genomics Suite. Differentially expressed genes were statistically determined with cutoffs of FDR&amp;lt;5% and Fold Change&amp;gt;±2. Pathway and network analyses utilized Ingenuity Pathway Analysis. Results: Principal components analysis (PCA) of all expressed protein coding genes revealed a close association of adjacent normal tissues to TNBCs whereas microdissected normal breast tissues were starkly separated. In comparing these 3 tissue types, 3841 genes were differentially expressed between TNBC vs. donated microdissected normal of which 1627 genes were also differentially expressed between adjacent normal vs. donated microdissected normal. The PCA along with the differential expression support the notion that adjacent normal gene expression is strongly influenced by the primary tumor, and in a significant set of genes, adjacent normal gene expression mimics the expression of primary tumors. The differential gene expression analysis also revealed 1240 genes that were restricted solely to the TNBC vs. donated microdissected normal that were not detected when TNBCs were compared to adjacent normal tissues. Examination of these genes reveals several potential kinase drug targets, genes involved in notch signaling, metabolism, cell cycle among other key cancer pathways. Preliminary analysis of the non-coding RNA component also reveals the same pattern of dysregulation in cancer associated microRNAs and lincRNAs. Conclusions: Gene expression within adjacent normal tissues is significantly impacted by the primary tumor and donated microdissected normal breast epithelium from healthy volunteers may be an optimal comparator for use in next-generation RNA sequencing breast cancer studies. Use of this comparator may identify therapeutic targets that would not otherwise be detected when using adjacent normal tissue as controls. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-06.

Autoantibodies and decreased expression of the transcription factor ELF‐3 together with increased chemokine pathways support an autoimmune phenotype and altered differentiation in lichen planus located in oral mucosa

The pathogenesis of oral lichen planus (OLP), a chronic inflammatory disease, is not fully understood. It is known that OLP has autoimmune features, and it is suggested to be an autoimmune disease. ELF-3 is involved in differentiation of keratinocytes and deregulated in different tumours and inflammatory diseases. CXCR-3 and its ligands CXCL-10 and CXCL-11 are increased in autoimmune diseases and linked to Th-1 immune response. To analyse and compare expression of ELF-3, CXCR-3, CXCL-10 and CXCL-11 in OLP lesions and controls in whole and microdissected epithelium. Tissue biopsies from 20 patients clinically and histologically diagnosed with OLP and 20 healthy controls were studied using whole tissues or microdissected epithelium. By the use of qRT-PCR, mRNA levels of ELF-3, CXCR-3, CXCL-10 and CXCL-11 were studied. Western blot was used for analysis of ELF-3 protein expression. Sera from 19 OLP patients and 20 controls were analysed with ELISA in search for autoantibodies. Results The upregulation of CXCR-3, CXCL-10 and CXCL-11 found in OLP is similar to previous findings showing an autoimmune phenotype in lichen planus (LP) and lichen sclerosus. Decreased expression of the differentiation-related transcription factor ELF-3 was also seen in OLP lesions, and we further demonstrate presence of circulating autoantibodies against the ELF-3 protein in sera from 3 of 19 (16%) LP patients tested. On the basis of these findings, we confirm that OLP shows features of an autoimmune disease and suggest deregulated differentiation of keratinocytes to be one of the causes of the disease phenotype.

Abstract 114: Study of the high-grade ovarian cancer precursors in the oviduct

Abstract The discovery that many high-grade ovarian cancers originate in the oviduct has generated a paradigm shift in the field of ovarian cancer. The description of new entities in the oviduct that signify early phases of ovarian carcinogenesis, including both serous tubal intraepithelial carcinomas and their precursors the p53 signatures, has provided a tangible sequence of events leading to malignancy that can be linked to DNA damage and p53 mutations. However, it has become clear that other pathway disturbances also play a critical role in the early development of ovarian cancer. We hypothesize that molecular alterations present in morphologically normal oviductal epithelium from ovarian cancer patients reflect the earliest events in ovarian carcinogenesis and may be markers of increased cancer risk as well as targets for risk reduction. Whole-genome transcriptome analysis of laser microdissected oviductal epithelium from histologically normal ovarian cancer patients and controls was performed. We identified a set of aberrantly expressed genes in the oviductal epithelium from women who developed high-grade ovarian cancer and demonstrated some of these genes are also abnormally expressed in the ovarian cancers due to the genomic alterations. We further examined the function of these genes using three-dimensional cell culture system and mouse models. Our work demonstrates the synergistic effects of defective p53 pathway and other newly found pathways in driving early steps of ovarian cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 114. doi:1538-7445.AM2012-114

Ciliary Activity in the Oviduct of Cycling, Pregnant, and Muscarinic Receptor Knockout Mice1

The transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. Because nonneuronally produced acetylcholine influences ciliary beating in the trachea via the muscarinic receptors M2 and M3, we supposed that components of the cholinergic system may also modulate ciliary activity in the oviduct. To address this issue, we analyzed the expression profile of muscarinic receptors (CHRMs) in the murine oviduct by RT-PCR and assessed ciliary beat frequency (CBF) and cilia-driven particle transport speed (PTS) on the mucosal surface of opened oviductal segments in correlation with histomorphological investigations. RT-PCR of laser-assisted microdissected epithelium revealed expression of Chrm subtypes Chrm1 and Chrm3. In opened isthmic segments, particle transport was barely seen, correlating with a significantly lower number of ciliated cells compared to the ampulla. In the ampulla, basal PTS and CBF were high (71 μm/sec and 21 Hz, respectively) both in cycling and pregnant wild-type mice and in mice with targeted deletion of the Chrm genes Chrm1, Chrm3, Chrm4, and Chrm5. In contrast to the trachea, where basal ciliary activity was low and largely enhanced by muscarinic stimulation, muscarinic agonists and antagonists did not affect the high ampullar PTS. Our results imply that this high oviductal autonomous ciliary activity is independent from the intrinsic cholinergic system and serves to maintain optimal clearance of the tube throughout all stages of the estrous cycle and early pregnancy.

Abstract 333: The role of the retinoblastoma pathway (Rb) in high grade serous ovarian varcinoma (HGSC)

Abstract The Rb pathway functions as a cell cycle checkpoint and deregulation of its components, commonly found in malignancies, causes progression from G1 to S phase, promoting cellular proliferation. Because of Rb's central role in checkpoint regulation, abrogation of the pathway can occur through multiple non-redundant mechanisms including Rb loss, hypermethylation or mutation, and CCND1 or CCNE amplification. Emerging evidence shows that tumour types can often be distinguished by particular alterations in one member of the pathway suggesting that different mechanisms of Rb abrogation may regulate tumour behaviour. We hypothesize that Rb pathway deregulation is frequent in HGSC, the most common and most aggressive histotype of ovarian cancer, and that the mechanism of Rb pathway deregulation identifies clinically distinct subgroups of HGSC. Micro-dissected epithelium from HGSC and normal FTE samples were analyzed for differential gene expression using the Affymetrix U133 Plus 2.0 gene-chips, and expression values for p53, p21, p27, p16, CCND1, CCNE and Rb genes determined. Protein expression was assessed by immunohistochemistry (IHC) on tissue microarrays composed of ovarian/tubal carcinomas inclusive of the major histotypes. Digitized stained slides were quantified using automated image analysis and correlated with clinico-pathologic variables including outcome. Rb loss of heterozygosity (LOH) was tested by an Rb diagnostics protocol involving D13S153 and RB1.2 polymorphic marker analyses using PCR amplification, followed by comparisons of the tumour and its corresponding normal sample by MicroGene Clipper sequencers. Gene expression analysis showed statistically significant over-expression of p53, CCNE E2F1/3 and p16 and down-regulation of p21 and CCND1 in HGSC compared to normal fallopian tube epithelium (p&amp;lt;0.001). Protein expression determined by IHC analysis of HGSC revealed a similar pattern of expression when compared to normal fallopian tube, the site of origin of this carcinoma. There were important differences in the expression of these proteins between HGSC subgroups, where up-regulation was observed for p16, CCNE, CCND1 and BIRC5 in 58.3%, 57%, 33.3%, and 58.3% respectively. Rb however, showed no statistically significant differences at the RNA level, but 40% of all HGSC profiled had a significant decrease in protein expression. Interestingly, LOH analysis revealed 76% of HGSC had Rb inactivation at the gene level. Furthermore, we observed statistically significant correlations (p=0.029) between p16 over-expression and Rb protein loss, using Fisher's Exact test. HGSC is characterized by both genetic and protein abrogation in the Rb pathway. Additionally, we observed differences in the mechanism of this G1/S checkpoint inactivation amongst HGSC patient samples which may represent important biological/clinical differences amongst sub-groups of serous cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 333. doi:10.1158/1538-7445.AM2011-333