Early Stages Of Carcinogenesis Research Articles

WNT/Β-CATENIN SIGNALING PATHWAY REGULATES TUMOR-INITIATING CELLS IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

Objectives Head and neck squamous cell carcinoma (HNSCC) is one of the leading cancers, with a 40% 5-year survival rate in advanced cases. HNSCC is notorious for its high recurrence rate and frequent occurrence of synchronous/metachronous primary tumors. Tumor initiating cells (TICs) model was proposed to explain its tumor heterogeneity and frequent recurrences. Lineage tracing is a genetic approach that allows identification of TICs in their native habitats and characterization of their in vivo behavior. Findings Our re-analysis of TCGA data revealed that high expression of AXIN2, a downstream target of Wnt signaling, was significantly correlated with low survival rate of HNSCC. To characterize the Wnt-responsive cell population, we established a carcinogen-induced model using Axin2-CreER reporter mice. After tamoxifen induction, clonal expansion of fluorescent reporter-positive cells (Wnt-responsive tumor cells) was visualized in the basal cell layer of epithelial dysplasia and HNSCC, suggesting the critical role of Wnt in regulating TICs in early stages of carcinogenesis. β-catenin and LEF1 immunofluorescent staining were performed to confirm Wnt activation. Lineage tracing was also accomplished in 3D organoid culture. The fluorescent reporter-positive cells were capable of forming organoids. Furthermore, application of cisplatin enriched AIXN2 cell population. Conclusions Activation of Wnt/β-catenin signaling pathway is an early event in HNSCC carcinogenesis. Lineage tracing using the Axin2-CreER reporter may link TICs properties with a fundamental signaling pathway in normal development. Further research is required to clarify the role of Wnt-responsive TICs in recurrence and therapy resistance of HNSCC.

Nicotine-induced oxidative stress contributes to EMT and stemness during neoplastic transformation through epigenetic modifications in human kidney epithelial cells

Nicotine is a component of cigarette smoke and mounting evidence suggests toxicity and carcinogenicity of tobacco smoke in kidney. Carcinogenicity of nicotine itself in kidney and the underlying molecular mechanisms are not well-understood. Hence, the objective of this study was to determine the carcinogenic effects of chronic nicotine exposure in Hk-2 human kidney epithelial cells. The effects of nicotine exposure on the expression of genes for cellular reprogramming, redox status, and growth signaling pathways were also evaluated to understand the molecular mechanisms. Results revealed that chronic exposure to nicotine induced growth and neoplastic transformation in HK-2 cells. Increased levels of intracellular reactive oxygen species (ROS), acquired stem cell-like sphere formation, and epithelial-mesenchymal-transition (EMT) changes were observed in nicotine exposed cells. Treatment with antioxidant N-acetyl cysteine (NAC) resulted in abrogation of EMT and stemness in HK-2 cells, indicating the role of nicotine-induced ROS in these morphological changes. The result also suggests that ROS controls the stemness through regulation of AKT pathway during early stages of carcinogenesis. Additionally, the expression of epigenetic regulatory genes was altered in nicotine-exposed cells and the changes were reversed by NAC. The epigenetic therapeutics 5-aza-2′-deoxycytidine and Trichostatin A also abrogated the stemness. This suggests the nicotine-induced oxidative stress caused epigenetic alterations contributing to stemness during neoplastic transformation. To our knowledge, this is the first report showing the ROS-mediated epigenetic modifications as the underlying mechanism for carcinogenicity of nicotine in human kidney epithelial cells. This study further suggests the potential of epigenetic therapeutics for pharmacological intervention in nicotine-induced kidney cancer.

Short chain fatty acid butyrate uptake reduces expressions of prostanoid EP4 receptors and their mediation of cyclooxygenase-2 induction in HCA-7 human colon cancer cells

Microbiota produce short chain fatty acids (SCFAs), which are known to maintain gut homeostasis, by the fermentation of dietary fiber in the human colon. Among SCFAs, butyrate has been considered as the most physiologically effective SCFA in colorectal epithelial cells for growth and differentiation. Here we show that the E-type prostanoid 4 (EP4) receptor expression level is regulated by different concentrations of butyrate, but not by other SCFAs, in human colon cancer HCA-7 cells, through sodium-coupled monocarboxylate transporter-1 (SMCT-1)-mediated uptake followed by the activation of histone acetyltransferase: cAMP response element binding protein-binding protein/p300. Of particular interest, the prostanoid EP4 receptors are known to be expressed in normal colorectal crypt epithelial cells and maintain intestinal homeostasis by preserving mucosal integrity, while they are also known to be involved in the early stage of carcinogenesis. Thus, the links between butyrate and the expression of prostanoid EP4 receptors are both important factors for maintaining homeostasis. Based on in silico analysis, almost half of colorectal cancer tissues have lost the expression of SMCT-1 mRNA when compared with healthy corresponding tissues. Therefore, with the collapse of homeostasis systems such as a decrease in the concentration of butyrate in colorectal tissues, or reduced butyrate uptake, there is a possibility of early stage colorectal cancer development; the transformation of normal cells to the cancerous phenotype may be due to the overexpression of prostanoid EP4 receptors followed by excessive cyclooxygenase-2 induction, which are caused by a reduced amount of butyrate and/or its uptake, in/around colorectal epithelial cells.

HPV and skin carcinogenesis

Epidemiological and biological studies provide several lines of evidence for the involvement of cutaneous beta human papillomaviruses (HPVs), together with ultraviolet (UV) radiation, in the development of cutaneous squamous cell carcinoma. These viruses appear to act with a hit-and-run mechanism, being necessary at an early stage of carcinogenesis and being dispensable for the maintenance of the malignant phenotype. Studies in experimental models show that beta HPVs, mainly via the E6 and E7 oncoproteins, are able to promote proliferation and to circumvent cellular stresses induced by UV radiation. These findings support a model of skin carcinogenesis in which beta HPV-infected keratinocytes remain alive despite the accumulation of UV-induced DNA mutations. In this manner, these cells become highly susceptible to progression towards malignancy. Thus, UV radiation is the main driver of skin cancer development, while beta HPVs act as facilitators of the accumulation of UV-induced DNA mutations.

CpG methyl-seq and RNA-seq epigenomic and transcriptomic studies on the preventive effects of Moringa isothiocyanate in mouse epidermal JB6 cells induced by the tumor promoter TPA

Epigenetic mechanisms play an important role in the early stages of carcinogenesis. Moringa isothiocyanate (MIC-1) is a major bioactive component derived from Moringa oleifera that has considerable antioxidant and anti-inflammatory effects. However, how MIC-1 influences epigenomic alterations in TPA-mediated JB6 cell carcinogenic transformation has not been evaluated. In this study, DNA and RNA isolated from TPA-induced JB6 cells in the presence or absence of MIC-1 were subjected to DNA Methyl-seq and RNA-seq to identify differentially methylated regions (DMRs) and differentially expressed genes (DEGs), respectively. When JB6 cells were challenged with TPA alone, there was a significant alteration of DEGs and DMRs; importantly, MIC-1 treatment reversed the patterns of some of the DEGs and DMRs. Transcriptome and CpG methylome profiling was performed in Ingenuity® Pathway Analysis (IPA) software to analyze the altered signaling pathways. Several anti-inflammatory responses, antioxidative stress-related pathways, and anticancer-related pathways were identified to be affected by MIC-1. These pathways included NF-kB, IL-1, LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, p53, and PTEN signaling pathways. Examination of correlations between transcriptomic and CpG methylome profiles yielded a small subset of genes, including the cancer-related genes Tmpt, Tubb3, and Muc2; the GTPases Gchfr and Igtp; and the cell cycle-related gene Cdc7. Taken together, our results show the potential contributions of epigenomic changes in DNA CpG methylation to gene expression to molecular pathways active in TPA-induced JB6 cells and demonstrate that MIC-1 can reverse these changes, supporting the potential preventive/treatment effects of MIC-1 against skin carcinogenesis.

Src in endosomal membranes promotes exosome secretion and tumor progression

c-Src is a membrane-associated tyrosine kinase that has key roles in the signaling transduction that controls cell growth, adhesion, and migration. In the early stage of carcinogenesis, c-Src is activated under the plasma membrane and transduces oncogenic signals. Here we show that c-Src localized to the endosomal membrane has unique functions in c-Src–transformed cells. Our results indicate that activated c-Src in the endosomal membrane promoted the secretion of exosomes, in which c-Src was encapsulated. In addition, the ESCRT-interacting molecule, Alix was identified as a c-Src–interacting protein in exosomes. We revealed that the interaction between the SH3 domain of c-Src and the proline-rich region of Alix activates ESCRT–mediated intra-luminal vesicle (ILV) formation, resulting in the upregulation of exosome secretion in c-Src–transformed cells. We observed also a correlation between malignant phenotypes and Alix–dependent aberrant exosome secretion in Src–upregulated cancer cells. Collectively, our findings provide a unique mechanism for the upregulation of exosomes in cancer cells, as well as new insights into the significance of exosome secretion in cancer progression.

Lactate modulates CD4+ T-cell polarization and induces an immunosuppressive environment, which sustains prostate carcinoma progression via TLR8/miR21 axis.

Leukocyte infiltration plays an active role in controlling tumor development. In the early stages of carcinogenesis, T cells counteract tumor growth. However, in advanced stages, cancer cells and infiltrating stromal components interfere with the immune control and instruct immune cells to support, rather than counteract, tumor malignancy, via cell-cell contact or soluble mediators. In particular, metabolites are emerging as active players in driving immunosuppression. Here we demonstrate that in a prostate cancer model lactate released by glycolytic cancer-associated fibroblasts (CAFs) acts on CD4+ T cells, shaping T-cell polarization. In particular, CAFs exposure (i) reduces the percentage of the antitumoral Th1 subset, inducing a lactate-dependent, SIRT1-mediated deacetylation/degradation of T-bet transcription factor; (ii) increases Treg cells, driving naive T cells polarization, through a lactate-based NF-kB activation and FoxP3 expression. In turn, this metabolic-based CAF-immunomodulated environment exerts a pro-invasive effect on prostate cancer cells, by activating a previously unexplored miR21/TLR8 axis that sustains cancer malignancy.

Estimation of expression of beta-human chorionic gonadotropin levels through progression of disease from normal to epithelial dysplasia to malignancy.

Background:Gonadotropins have been extensively studied in trophoblastic and nontrophoblastic tumors of breast, gastric, bladder, parathyroid, renal cell and cervical carcinomas, with a significant increase in tissue expressions. Serum levels of beta-human chorionic gonadotropin (β-hCG) and its tissue expression were found more in oral squamous cell carcinoma (OSCC) patients with a significant diagnostic and prognostic value. No such study has been done on oral epithelial dysplasia (OED).Aims and Objective:To evaluate the expression of β-hCG in OED and the feasibility of using this marker for early diagnosis and to see its progression from normal to dysplasia to malignancy.Materials and Methods:The study population consisted of thirty histologically confirmed cases of OED and thirty cases of OSCC. Fifteen normal tissues were also included in the study. All the tissue samples were subjected to immunohistochemical (IHC) staining using antimouse β-hCG antibody.Results:The IHC expression of β-hCG was completely negative in normal cases (Group 1 [n = 15]), whereas 13 (43.3%) cases of OED (Group 2 [n = 30]) and 13 cases (43.3%) of OSCC (Group 3 [n = 30]) showed diffuse cytoplasmic staining in dysplastic surface epithelium and epithelial islands of OSCC. This difference was statistically significant with P = 0.007.Conclusion:We conclude that the expression of β-hCG increased from normal mucosa to dysplasia to OSCC, suggesting that it is involved in the early stage of carcinogenesis and progression of the disease.

MECHANISTIC MODELING PREDICTS ANTI-CARCINOGENIC RADIATION EFFECTS ON INTERCELLULAR SIGNALING IN VITRO TURN PRO-CARCINOGENIC IN VIVO.

Oncogenic transformed cells represent an in vitro system mimicking early-stage carcinogenesis. These precancerous cells are subject to a selective removal via apoptosis induced by neighbor cells. By modulating the underpinning intercellular signaling mediated by cytokines and reactive oxygen/nitrogen species, ionizing radiation enhances this removal of precancerous cells in vitro, at doses from a few mGy to a few Gy. However, epidemiological data demonstrate that radiation exposure induces cancer, at least above 100 mGy. Mechanistic modeling of the given anti-carcinogenic process explains this discrepancy: The model reproduces in vitro data on apoptosis and its enhancement by radiation. For in vivo-like conditions with signal lifetimes shorter and cell densities higher than in vitro, radiation is predicted to reduce this anti-carcinogenic mechanism. Early-stage lesions that would be turned dormant or completely removed may grow large and escape this control mechanism upon irradiation.

Comparison of oxidative stress on DNA, protein and lipids in patients with actinic keratosis, Bowen's disease and squamous cell carcinoma.

Detailed mechanisms on the effect of oxidative stress (OS), an etiological factor involved in photocarcinogenesis, remain to be fully elucidated. We used immunohistochemical methods to study OS in the DNA, protein and lipids of patients with actinic keratosis (AK), Bowen's disease (BD) and squamous cell carcinoma (SCC). Between January 2009 and December 2014, we treated 230 patients; 79 had AK, 61 had (BD) and 90 had cutaneous SCC; 28 healthy subjects served as the normal controls. OS on DNA, protein and lipids was assessed by the expression of 8-hydroxydeoxyguanosine (8-OHdG), dityrosine (DT) and malondialdehyde (MDA), respectively. 8-OHdG was significantly overexpressed in AK and BD lesions compared with surrounding non-lesional tissue, SCC lesions and the healthy controls. DT was more highly expressed in AK, BD and SCC than in the controls. There was no significant difference among AK, BD and SCC. The expression of MDA was higher in AK, BD and SCC lesions than the controls; SCC showed the highest expression. Our observations suggest that DNA oxidation plays an important role in the early stage of carcinogenesis, that protein oxidation is involved in all stages of carcinogenesis and that lipid oxidation is strongly implicated in the late stages of carcinogenesis.

Immunological dysfunction in chronic arsenic exposure: From subclinical condition to skin cancer.

Exposure to arsenic is a global health issue. Long-term arsenic exposure may associate with various cancers and many other pathological effects. Over 100 million people worldwide are exposed to arsenic particularly in countries such as Bangladesh, Chile, China, India, Mexico, Taiwan and the USA. Drinking of water contaminated with arsenic is the major route of human exposure. Skin lesions are considered to be the most common adverse effects associated with chronic arsenic exposure. Skin lesions usually develop with the latency period spanning more than 20 years from first exposure. Arsenic-induced Bowen's disease, the most frequently encountered carcinoma in situ resulting from chronic arsenic exposure, is characterized by multiple and recrudescent lesions. Long-term arsenic exposure results in impaired immunity in susceptible individuals. In the prenatal stage, enhanced placental inflammatory responses and reduced placental T cells by arsenic may result in decreased thymic size and functions in newborns. In childhood, arsenic exposure may reduce peripheral CD4+ cells and interleukin-2 secretion which leads to susceptibility to opportunistic infections. There was an impairment of macrophage function and oxidative DNA damage of peripheral polymorphonuclear leukocytes in adults with skin lesions. In arsenic-induced Bowen's disease lesions, a decrease in the number and functions of Langerhans cells and, in parallel, a selective CD4+ cell apoptosis was noticed. These findings provide scientific evidence for understanding the phenomenon of arsenic-induced immune escape in the early stage of carcinogenesis and a reasonable explanation for multiple and recrudescent arsenic cancers in the skin.

Expression of stem cell markers Nanog and Nestin in lip squamous cell carcinoma and actinic cheilitis.

To investigate the presence of NANOG, NESTIN, and β-tubulin in lip squamous cell carcinoma (LSCC), actinic cheilitis (AC), and normal epithelium (NE). Thirty cases of LSCC, thirty cases of AC (both analyzed according to the WHO classification and AC according to the binary classification), and twenty cases of NE were submitted to an immunohistochemical study. NANOG was more expressed in the nuclei of AC compared to NE (p=0.007), as well as in high-risk AC cases (p=0.017) and well-differentiated LSCCs (no significance). There was an accumulation of nuclear NANOG from mild to moderate and severe ACs. NESTIN was significantly less present in NE compared to AC (p=0.001) and LSCC (p=0.003). There was a higher expression in severe dysplasia or high-risk AC and well-differentiated LSCC. These results indicate an upregulation of NANOG and NESTIN in the early stages of carcinogenesis. β-tubulin was intensely present in all lesions. The results suggest an upregulation of NANOG and NESTIN in the biological behavior these diseases, mainly in the transformation from AC to LSCC.

Associations of dietary fat with risk of early neoplasia in the proximal colon in a population-based case-control study.

Excess dietary fat consumption is strongly associated with the risk of colorectal cancer, but less is known about its role in the earliest stages of carcinogenesis, particularly within the proximal colon. In the following case-control study, we evaluated the relationship between the intake of dietary fats and the frequency of early proximal neoplasia [aberrant crypt foci (ACF) or polyps], detectable by high-definition colonoscopy with contrast dye-spray. Average-risk screening individuals underwent a high-definition colonoscopy procedure as part of larger ongoing clinical study of precancerous lesions in the proximal colon. Dietary fat intake was assessed using the Block Brief Food Frequency Questionnaire, which estimates average dietary intake based on 70 food items. The diets of individuals with no endoscopically identifiable lesions (n = 36) were compared to those with either ACF or polyps detected in the proximal colon. In multivariate analysis, high dietary intake of total polyunsaturated fatty acids (PUFAs) and intake of omega-6 and omega-3 fatty acids were positively associated with neoplastic lesions in the proximal colon. When comparing ACF and polyp groups separately, a positive association was observed for both proximal polyps (OR 2.28; CI 1.16-7.09) and ACF (OR 2.86; CI 1.16-7.09) for total PUFA intake. Furthermore, the prevalence of proximal ACF was increased with higher intake of omega-6 (OR 3.54; CI 1.32-9.47) and omega-3 fatty acids (OR 2.29; CI 1.02-5.13), although there was no discernible difference in the omega-6/omega-3 ratio. These results suggest that dietary PUFAs may be positively associated with risk of early neoplasia in the proximal colon. This study provides further evidence that dietary PUFA composition may play an important role in altering the microenvironment within the human colon.

P-109 - Functional fluorescence probe for lipid derived radicals detection

Lipids are easily oxidized by reactive oxygen species, resulting in lipid peroxidation and metabolic products such as lipid-derived electrophiles. These products have been reported to induce inflammation, angiogenesis and ferroptosis. Here, the initial products of lipid oxidation are lipid-derived radicals, the most upstream products that can cause extensive chain reactions affecting lipid peroxidation. Hence, the ability to detect lipid radicals would provide information about one of most important class of molecules causing cellular and tissue damage in a wide range of diseases. In this study, I will present the first fluorescence probe for lipid radicals, NBD-Pen, with high selectivity and sensitivity. To demonstrate bioavailability of NBD-Pen, we tested it in an animal model in which hepatocellular carcinoma was induced by the hepatic procarcinogen diethylnitrosamine (DEN). Furthermore, we found that lipid radical inhibitor significantly relieved inflammation at the early stage of carcinogenesis, as well as following induction of tumor formation by DEN. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo.

Tocopherols inhibit esophageal carcinogenesis through attenuating NF-κB activation and CXCR3-mediated inflammation.

Esophageal cancer is one of the common causes of cancer mortality in the world. The predominant histological subtype, esophageal squamous cell carcinoma (ESCC), often results in poor prognosis due to the lack of effective approaches for the early diagnosis and treatment, highlighting the need for preventive intervention against this disease. Here we report that dietary tocopherols significantly prevents esophageal carcinogenesis by inhibiting the activation of NF-κB and the subsequent interaction of chemokine CXCL9/10/11 with their receptor CXCR3 in ESCC induced by N-nitrosomethylbenzylamine (NMBA) in murine models. Dietary supplementation with 0.15% α-tocopherol (α-T), δ-tocopherol (δ-T), or γ-tocopherol rich mixture (γ-TmT) markedly suppressed the production of pro-inflammatory cytokines, as well as the induction of CXCR3+ effector T cells (CD4+ Th1 and CD8+ CTLs) infiltration, especially at the early stage of carcinogenesis. In experiments in vivo and in vitro, these events were tightly correlated with the blockade of NF-κB activation. Our results show that tocopherols decrease carcinogenesis through inhibiting NF-κB and CXCR3 signaling, as well as related inflammation in early premalignant lesions. This pathway may offer a novel target for chemoprevention of esophageal cancer.

G-101 Human papillomaviruses and carcinogenesis: well-established and novel models

Human papillomaviruses (HPVs) infect the cutaneous or mucosal epithelia. More than 200 HPV types have been isolated so far, and they are classified phylogenetically as genera and species. Persistent infections by the mucosal high-risk (HR) HPV types from genus alpha have been clearly associated with cancer development of the genital and upper respiratory tracts. The products of two early genes, E6 and E7, are the major HR HPV oncoproteins, being essential in all steps of the carcinogenic process. They exert their functions by interacting with a large number of cellular proteins, including the products of tumour suppressor genes, and altering their properties. Biological and epidemiological data indicate that beta HPV types, together with ultraviolet (UV) radiation, promote non-melanoma skin cancer development. However, in contrast to the HR mucosal HPV types, cutaneous beta HPV types appear to be required only at an early stage of carcinogenesis, facilitating the accumulation of UV-induced DNA mutations. Several findings also suggest that these HPV types and other carcinogens may synergize in the induction of malignancies at different anatomical sites.

Feature-based analysis of cell nuclei structure for classification of histopathological images

Pathological examination of histopathological images remains the main method of cancer diagnosis. We proposed a novel automatic feature-based analysis scheme for classification of the histopathological images. An animal model of intestinal carcinogenesis-multiple intestinal neoplasia mouse model was used to evaluate the feasibility of the nuclear structure feature for detecting early-stage carcinogenesis and assessing cancer risk. Firstly, the cell nuclei are segmented based on collaborate cell localization and the improved morphology method. Then several types of features, including shape features, statistical features and textual features (Gabor and Markov random field features) are extracted. Feature selection methods including wrapper, filter and the maximum relevance-minimum multicollinearity are applied to obtain the optimal feature set. Experimental results show that the proposed segmentation method can automatically segment histopathological images and has effective segmentation results. The maximum relevance-minimum multicollinearity method outperformed all other methods in term of classification accuracy. The textual features can effectively improve the characterization of cell nuclei structure and feature selection methods can get better classification results.

CXCR4 Is a Potential Target for Diagnostic PET/CT Imaging in Barrett's Dysplasia and Esophageal Adenocarcinoma

Purpose: Barrett's esophagus represents an early stage in carcinogenesis leading to esophageal adenocarcinoma. Considerable evidence supports a major role for chronic inflammation and diverse chemokine pathways in the development of Barrett's esophagus and esophageal adenocarcinoma.Experimental Design: Here we utilized an IL1β transgenic mouse model of Barrett's esophagus and esophageal adenocarcinoma and human patient imaging to analyze the importance of CXCR4-expressing cells during esophageal carcinogenesis.Results: IL1β overexpression induces chronic esophageal inflammation and recapitulates the progression to Barrett's esophagus and esophageal adenocarcinoma. CXCR4 expression is increased in both epithelial and immune cells during disease progression in pL2-IL1β mice and also elevated in esophageal adenocarcinoma patient biopsy samples. Specific recruitment of CXCR4-positive (CXCR4+) immune cells correlated with dysplasia progression, suggesting that this immune population may be a key contributor to esophageal carcinogenesis. Similarly, with progression to dysplasia, there were increased numbers of CXCR4+ columnar epithelial cells at the squamocolumnar junction (SCJ). These findings were supported by stronger CXCR4-related signal intensity in ex vivo fluorescence imaging and autoradiography with advanced dysplasia. Pilot CXCR4-directed PET/CT imaging studies in patients with esophageal cancer demonstrate the potential utility of CXCR4 imaging for the diagnosis and staging of esophageal cancer.Conclusion: In conclusion, the recruitment of CXCR4+ immune cells and expansion of CXCR4+ epithelial cells in esophageal dysplasia and cancer highlight the potential of CXCR4 as a biomarker and molecular target for diagnostic imaging of the tumor microenvironment in esophageal adenocarcinoma. Clin Cancer Res; 24(5); 1048-61. ©2017 AACR.

Abstract P2-02-08: Validation of an autoantibody blood test for the detection of early breast cancer (BC), particularly hormone receptor positive BC

Abstract Background: Breast cancer (BC) remains the most common type of cancer in women, with an incidence of 1.6 million cases per year worldwide. Early detection through mammographic screening reduces BC mortality by ˜20%, which while highly significant still leaves 80% of BC deaths unchanged. Furthermore, mammographic screening is only applicable to women between 50-70 years, in which only about one third of BCs occurs. There is an urgent need to identify a test, preferably a blood test showing high sensitivity and specificity for early BC across all ages of patients and all tumour types. Advances in understanding of the human autoantibody (AAB) response to early cancers has allowed us to harness this biological phenomenon for early cancer detection. . Aim: To identify a panel of tumor associated antigens (TAAs) which would detect AABs in the blood with high sensitivity and specificity for early BC: enabling cancer/normal discrimination. Methods: Serum samples from 120 BC patients and matched controls were tested against a panel of 60 multiple TAAs using an optimised new multiplex microarray platform. A sub-group of 60 samples were also tested for AABs to estrogen receptor (ER). The selected TAAs were spotted onto a glass slide surface in an automated, highly reproducible system platform. If serum autoantibodies are present they bind to one or more of the TAA spots. Bound antibodies are detected with a fluorescent reporter and signal intensity measured using GenePix pro-6. Data analysis: A Monte Carlo Simulation method was employed to define the best panel of the antigens with optimised cutoff points in each assay that would yield the highest sensitivity and specificity to discriminate BC patients from controls. AAB positivity in the BC group was analysed by tumor size (≤20mm versus >20mm), histological grade, lymph node status (positive or negative) and ER status (positive or negative) Results: Using a panel of 12 TAAs, AABs were detected in pre-op blood of 34/60 (57%) primary BC patients compared to 9/59 controls (15%) (p=0.000003); one control sample data was unavailable. This gave a sensitivity of 57% and specificity of 85%. Median age of BC patients was 59yrs (20-81) versus 59yrs (28-81) in controls. There was no significant difference for AAB detection when compared to tumour size, grade, lymph node status or ER status. In the sub-group of 60 patients where ER antigen was measured using a panel of 8 TAAs AABs were detected in 20/29 BC patients compared to 2/30 controls (p=3.5e-7). This represents a sensitivity of 69% with a specificity of 93%. Conclusions: These results confirmed our hypothesis that AABs can be detected in women of all ages with early BC. AABs were not related to tumour size, grade, lymph node status or ER status. If a panel of AAB assays can be validated it opens the possibility of a blood test for detection of early BC. Future direction of this research will be i) validation studies of a panel of AABs for detection of early BC, ii) detection of AABs at the earliest stages of carcinogenesis and iii) a panel of AABs which detect ER positive BC thereby enabling stratified chemoprevention in a high risk group combined with mammographic screening. Citation Format: Negm OH, Ahmed O, Figueredo GP, Hamed MR, Pollard L, Garibaldi J, Sewell H, Robertson JF. Validation of an autoantibody blood test for the detection of early breast cancer (BC), particularly hormone receptor positive BC [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-08.

β-HPV Infection Correlates with Early Stages of Carcinogenesis in Skin Tumors and Patient-Derived Xenografts from a Kidney Transplant Recipient Cohort.

Many malignancies that occur in high excess in kidney transplant recipients (KTRs) are due to viruses that thrive in the setting of immunosuppression. Keratinocyte carcinoma (KC), the most frequently occurring cancer type in KTR, has been associated with skin infection by human papillomavirus (HPV) from the beta genus. In this report, we extend our previous investigation aimed at identifying the presence of active β-HPV infection in skin tumors from KTRs through detection of viral protein expression. Using a combination of antibodies raised against the E4 and L1 proteins of the β-genotypes, we were able to visualize infection in five tumors [one keratoacanthoma (KA), three actinic keratoses (AKs), and one seborrheic keratoses (SKs)] that were all removed from two patients who had been both transplanted twice, had developed multiple KCs, and presented with a long history of immunosuppression (>30 years). These infected tissues displayed intraepidermal hyperplasia and increased expression of the ΔNp63 protein, which extended into the upper epithelial layers. In addition, using a xenograft model system in nude mice displaying a humanized stromal bed in the site of grafting, we successfully engrafted three AKs, two of which were derived from the aforementioned KTRs and displayed β-HPV infection in the original tumor. Of note, one AK-derived xenograft, along with its ensuing lymph node metastasis, was diagnosed as squamous cell carcinoma (SCC). In the latter, both β-HPV infection and ΔNp63 expression were no longer detectable. Although the overall success rate of engrafting was very low, the results of this study show for the first time that β-HPV+ and ΔNp63+ intraepidermal hyperplasia can indeed progress to an aggressive SCC able to metastasize. Consistent with a series of reports attributing a causative role of β-HPV at early stages of skin carcinogenesis through ΔNp63 induction and increased keratinocytes stemness, here we provide in vivo evidence that these events are also occurring in the affected skin of KTRs. Due to these β-HPV-driven molecular pathways, the nascent tumor cell is able to acquire a high enough number of carcinogenic insults that its proliferation and survival will eventually become independent of viral gene expression.