Normal Tissue Epithelium Research Articles

Abstract 1061: Diagnostic application of a novel blood CDH17 immunoassay in the detection of colorectal adenoma

Abstract Background: Colorectal cancer (CRC) is the most prevalent gastrointestinal (GI) cancer and contribute significantly to global cancer deaths. The mechanism of CRC development is a complex multistage process from hyperplastic lesions to the formation of adenomas, followed by the progression into malignancy. Hence, early detection during hyperplastic and adenomatous stages warrants new screening tests for effective interception of malignancy progression. Cadherin-17 (CDH17) is a biomarker characterized by its overexpression in several GI cancers, but restricted expression in intestinal mucosal epithelium of normal tissues. We have developed a blood immunoassay using a panel of RUO grade antibodies that unveiled CDH17 as a potential marker with high detection sensitivity and specificity for detecting adenomas. Our immunoassay is useful in identifying high-risk individuals with adenomas that may develop into CRC, which can significantly improve the diagnostic efficiency of colonoscopy that is currently lacking. Methods: The study recruited 66 patients (54 adenoma and 12 hyperplasia) and 39 normal samples (no abnormality by colonoscopy). CDH17 level in plasma samples were measured using the CDH17 immunoassay. Results: The novel CDH17 diagnostic assay showed an operating dynamic range from 90ng to 123pg. We observed a progressive and significant increase in the circulating CDH17 level from normal samples to the hyperplasia group, followed by the adenoma group (mean: 1.66ng/mL, 3.20ng/mL, 4.93ng/mL, respectively P<0.0001). The assay performance by ROC curve demonstrated an AUC of >0.9, with a sensitivity of 85% and specificity of 86%. NPV and PPV were 84% and 87%, respectively. The assay outperforms two market available FIT-DNA tests at predicting adenomas (Table. 1). Conclusion: The novel CDH17 immunoassay is potentially a powerful tool for the detection of aberrant plasma CDH17 expression, improving the surveillance and the detection of high-risk individuals for CRC. CDH17 immunoassay outperforms market availalble FIT-DNA tests at detecting hyperplasia and adenoma FIT-DNA test 1 FIT-DNA test 2 CDH17 blood immunoassay Age 40-74 Age 50-84 Age 45-93 Sensitivity (%) Advanced adenoma 63.5 42.4 85.0 Non-advanced adenoma (i.e Hyperplasia) Unavailable 17.2 58.3 Specificity (%) Normal control (no abnormality by colonoscopy) 87.1 89.8 86.1 NPV (%) Advanced adenoma Unavailable Unavailable 83.8 Non-advanced adenoma (i.e Hyperplasia) Unavailable Unavailable 86.1 PPV (%) Advanced adenoma Unavailable Unavailable 87.2 Non-advanced adenoma (i.e Hyperplasia) Unavailable Unavailable 58.3 Citation Format: Ng Lui, Felix Hon, Stella Sun, Pauline Leung, David Ho, John Moon Luk, Dominic Chi-Chung Foo. Diagnostic application of a novel blood CDH17 immunoassay in the detection of colorectal adenoma [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 1061.

Establishment of a Monoclonal Antibody That Recognizes Cysteine-Rich Domain 1 of Human CD271.

CD271 is a common receptor for all neurotrophins that is localized to neurons, endothelial cells, and the basal layer of the epithelium in normal tissue. Recently, we and others reported that CD271 plays essential roles in the development of squamous cell carcinoma, especially in tumor-initiating cells. Since little is known about how CD271 regulates cancer cell initiation and proliferation, antibodies that recognize different domains of CD271 are needed to enable investigation. Therefore, this study aimed to develop an antihuman CD271 antibody by immunizing mice with a CD271 antigen produced by a baculovirus. The antibody was named hCD271mAb#13, and it recognized cysteine-rich domain 1 with a higher affinity than the commercially available antibody ME20.4. We determined that hCD271mAb#13 is suitable for flow cytometry, Western blotting, immunocytochemistry, and immunohistochemistry of formalin-fixed paraffin-embedded tissue. Use of hCD271mAb#13 for CD271 labeling could enable detailed analyses of cancer cell regulation and other biological processes.

A Digital Pathology-Based Shotgun-Proteomics Approach to Biomarker Discovery in Colorectal Cancer

Background: Biomarkers in colorectal cancer are scarce, especially for patients with Stage 2 disease. The aim of our study was to identify potential prognostic biomarkers from colorectal cancers using a novel combination of approaches, whereby digital pathology is coupled to shotgun proteomics followed by validation of candidates by immunohistochemistry (IHC) using digital image analysis (DIA). Methods and Results: Tissue cores were punched from formalin-fixed paraffin-embedded colorectal cancers from patients with Stage 2 and 3 disease (n = 26, each). Protein extraction and liquid chromatography-mass spectrometry (MS) followed by analysis using three different methods were performed. Fold changes were evaluated. The candidate biomarker was validated by IHC on a series of 413 colorectal cancers from surgically treated patients using a next-generation tissue microarray. DIA was performed by using a pan-cytokeratin serial alignment and quantifying staining within the tumor and normal tissue epithelium. Analysis was done in QuPath and Brightness_Max scores were used for statistical analysis and clinicopathological associations. MS identified 1947 proteins with at least two unique peptides. To reinforce the validity of the biomarker candidates, only proteins showing a significant (P < 0.05) fold-change using all three analysis methods were considered. Eight were identified, and of these, cathepsin B was selected for further validation. DIA revealed strong associations between higher cathepsin B expression and less aggressive tumor features, including tumor node metastasis stage and lymphatic vessel and venous vessel invasion (P < 0.001, all). Cathepsin B was associated with more favorable survival in univariate analysis only. Conclusions: Our results present a novel approach to biomarker discovery that includes MS and digital pathology. Cathepsin B expression analyzed by DIA within the tumor epithelial compartment was identified as a strong feature of less aggressive tumor behavior and favorable outcome, a finding that should be further investigated on a more functional level.

Expression of plakophilin 3 in diffuse malignant pleural mesothelioma.

Diffuse malignant pleural mesothelioma (DMPM) is the most common primary malignant pleural neoplasm still posing major diagnostic, prognostic and therapeutic challenges. Plakophilins are structural proteins considered to be important for cell stability and adhesion in both tumor and normal tissues. Plakophilin 3 is a protein present in desmosomes of stratified and simple epithelia of normal tissues with presence in malignant cells of various tumors where it participates in the process of tumorigenesis. The aim of this study was to investigate the expression of plakophilin 3 protein in DMPM, but also to study its prognostic significance and relation to histologically accessible parameters of aggressive growth. Archival samples of tissue with established diagnosis of DMPM and samples of normal pleural tissue were used. Tumor samples were classified into three histological types of DMPM (epithelioid, sarcomatoid and biphasic). Additional subclassification of epithelioid mesotheliomas into nine patterns based on the prevalent histological component of the tumor was then performed. After immunohistochemical staining, cytoplasmic and membrane immunopositivity of tumor cells was assesed by scoring the intensity of the staining from 0 (no staining) to 4 (very strong staining). Prognostic value and expression of plakophilin 3 with consideration to histologically estimated aggression in tumor growth were then statistically analyzed using non- parametric tests. The results demonstrated higher level of plakophilin 3 expression in tumor samples with histologically more aggressive tumor growth, but no significant prognostic value. According to our study, plakophilin 3 appears to be involved in tumor invasion in malignant mesothelioma.

Precise determination of toll-like receptor (TLR) 7 expression in multiple human tumor types

TLR7 agonists are being progressed as potential immunotherapeutics for the treatment of cancer. TLR7 agonism is believed to trigger a plasmacytoid dendritic cell driven immune response which drives anti-tumor efficacy. In addition to modulation of the immune system, TLR7 may be involved in tumor progression due to expression of TLR7 by tumor cells themselves [1]. To determine the precise distribution of TLR7 in different tumor types, a comprehensive validation of anti-TLR7 antibodies was conducted by immuohistochemistry (IHC). One antibody (Epitomics, 3923-1), out of five selected from scientific citations, proved specific for TLR7 following analyses by western blot of cell lysates, and by IHC of formalin fixed paraffin embedded (FFPE) cell pellets, prepared from HEK293 cells either stably transfected, mock-transfected or non-transfected with TLR7. 3923-1 was validated further across human spleen, lymph node and tonsil, revealing the expected tissue and cellular localization for these lymphoid organs. Assessment of TLR7 expression in a selection of tumor samples revealed non-specific tumor cell staining for the rejected antibodies, compared with minimal tumor staining for 3923-1. To explore further the distribution of TLR7, 5 tissue microarrays comprising 18 different human tumor types (6-25 patients/tumor type, triplicate cores) and 14 normal tissues (5 donors/tissue type, duplicate cores) were examined by IHC using 3923-1. Staining for TLR7 was scored by a pathologist (4-point scale: 0 negative, 1+ weak, 2+ moderate, 3+ strong) for tumor and immune cell compartments. Out of 18 tumor types examined, 5 were negative for tumor cell expression of TLR7 (ovarian, glioma, thyroid, liver, renal) and 9 exhibited a proportion (4%-36%) of patients with weak staining (breast, lung, colorectal, pancreatic, gastric, head & neck, melanoma, esophageal, endometrial). Moderate staining was observed for 11%-17% of sarcoma, prostate and bladder tumors. Corresponding normal tissue epithelium was largely negative for TLR7. Importantly, an increased density of immune infiltrates was observed in tumor tissues compared with normal tissues, and a greater proportion of the immune infiltrates were TLR7 positive. These data demonstrate that TLR7 may be less frequently expressed by tumor cells than suggested by the literature and that all tumor types exhibit a marked TLR7 positive immune cell infiltrate. Together, these data identify tumour types that might benefit from TLR7 therapy and may guide patient selection.

Abstract 3670: Toll-like receptor (TLR) 7 expression in the human tumor microenvironment

Abstract Following the success of imiquimod, agents targeting TLR7 are being progressed as potential immunotherapeutics. Stimulation of TLR7 initiates a plasmacytoid dendritic cell driven immune response which drives efficacy. In addition to a role in regulating immune cell responses, TLR7 has also been reported to play a role in regulating tumor cell function (1,2). To investigate the expression of TLR7, by both tumor cells and by tumor infiltrating immune cells, we have used immunohistochemistry (IHC). Initially, a comprehensive validation of commercially available anti-TLR7 antibodies was conducted revealing one antibody (Epitomics, 3923-1) with specificity for TLR7 in western blots of cell lysates, and by immunocytochemistry of formalin fixed paraffin embedded (FFPE) cell pellets, of HEK293 cells stably transfected with TLR7, mock-transfected or non-transfected. 3923-1 was validated further for IHC across FFPE sections of human spleen, lymph node and tonsil demonstrating expected tissue and cellular localization. A comparison of staining for TLR7 in whole sections of FFPE tumors revealed non-specific tumor staining using antibodies that failed the validation process compared with the staining pattern observed for 3923-1. Subsequently, five tissue microarrays (TMAs) comprising 18 different human tumor types (6-25 patients/tumor type, triplicate cores) and 14 normal tissues (5 donors/tissue type, duplicate cores) were stained by IHC for TLR7 expression using 3923-1. Pathologist scores for tumor cells revealed 5/18 tumor types negative for TLR7 (ovarian, glioma, thyroid, liver, renal) and 9/18 tumor types with a proportion of patients (4%-36%) exhibiting weak (1+) staining (breast, lung, colorectal, pancreatic, gastric, head &amp; neck, melanoma, esophageal, endometrial). Furthermore, 11-17% of sarcoma, prostate and bladder tumors displayed moderate (2+) staining for TLR7, whereas corresponding normal tissue epithelium was largely negative for TLR7. Importantly, an increased density of immune infiltrates was observed in tumor tissues compared with normal tissues, and a greater proportion of the immune infiltrate in tumors was TLR7 positive. The conclusion drawn is that TLR7 may be less frequently expressed by tumor cells than reported previously and that tumors exhibit a marked TLR7 positive immune cell infiltrate. These data identify tumour types which might benefit from TLR7 therapy and may guide patient selection. 1. Grimm M et al. Eur J Cancer (2010):2849-57. 2. Cherfils-Vicini J et al. J Clin Invest (2010):1285-97. Citation Format: Nicola Haughton, Foster Emily, Christopher Womack, Simon Barry, Setsuko Yamamoto, Masashi Murata, Marie Cumberbatch. Toll-like receptor (TLR) 7 expression in the human tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3670. doi:10.1158/1538-7445.AM2014-3670

The Application of Normal, SV40 T-antigen-immortalised and Tumour-derived Oral Keratinocytes, under Serum-free Conditions, to the Study of the Probability of Cancer Progression as a Result of Environmental Exposure to Chemicals

In vitro models are currently not considered to be suitable replacements for animals in experiments to assess the multiple factors that underlie the development of cancer as a result of environmental exposure to chemicals. An evaluation was conducted on the potential use of normal keratinocytes, the SV40 T-antigen-immortalised keratinocyte cell line, SVpgC2a, and the carcinoma cell line, SqCC/Y1, alone and in combination, and under standardised serum-free culture conditions, to study oral cancer progression. In addition, features considered to be central to cancer development as a result of environmental exposure to chemicals, were analysed. Genomic expression, and enzymatic and functional data from the cell lines reflected many aspects of the transition of normal tissue epithelium, via dysplasia, to full malignancy. The composite cell line model develops aberrances in proliferation, terminal differentiation and apoptosis, in a similar manner to oral cancer progression in vivo. Transcript and protein profiling links aberrations in multiple gene ontologies, molecular networks and tumour biomarker genes (some proposed previously, and some new) in oral carcinoma development. Typical specific changes include the loss of tumour-suppressor p53 function and of sensitivity to retinoids. Environmental agents associated with the aetiology of oral cancer differ in their requirements for metabolic activation, and cause toxic effects to cells in both the normal and the transformed states. The results suggest that the model might be useful for studies on the sensitivity of cells to chemicals at different stages of cancer progression, including many aspects of the integrated roles of cytotoxicity and genotoxicity. Overall, the properties of the SVpgC2a and SqCC/Y1 cell lines, relative to normal epithelial cells in monolayer or organotypic culture, support their potential applicability to mechanistic studies on cancer risk factors, including, in particular, the definition of critical toxicity effects and dose-effect relationships.

A subgroup of HOX Abd-B gene is differentially expressed in cervical cancer

The HOX genes are a family of transcription factors that bind to specific sequences of DNA in target genes regulating their expression. The role of HOX genes in adult cell differentiation is still obscure, but growing evidence suggests that they may play an important role in the development of cancer. In order to study the role of the HOX Abd-B genes in cervical cancer, we analyzed their expression in cervical tissues. Reverse transcription–polymerase chain reaction and RNAin situhybridization were used to detect HOX Abd-B messenger RNA expression in nine normal cervical tissues and ten cervical carcinomas. The normal tissues were human papillomavirus (HPV) negative, whereas all invasive carcinomas included were HPV16 positive. In this study, we show that HOXA9, A10, A11, A13, B9, D11, and D13 genes are expressed in both the epithelium of normal tissues and neoplastic cells from squamous cervical carcinomas. Interestingly, the HOXC10 and D12 genes were not expressed in any cervical tissues; however, HOXB13, C9, C11, C12, C13, D9, and D10 genes were expressed only in the tumoral tissues but not in the normal cervix. Our findings suggest that the expression of HOXB13, D9, D10, and HOXC cluster (HOXC9, C11–C13) genes might be an important step involved in cervical cancer.

A subgroup of HOX Abd-B gene is differentially expressed in cervical cancer

The HOX genes are a family of transcription factors that bind to specific sequences of DNA in target genes regulating their expression. The role of HOX genes in adult cell differentiation is still obscure, but growing evidence suggests that they may play an important role in the development of cancer. In order to study the role of the HOX Abd-B genes in cervical cancer, we analyzed their expression in cervical tissues. Reverse transcription-polymerase chain reaction and RNA in situ hybridization were used to detect HOX Abd-B messenger RNA expression in nine normal cervical tissues and ten cervical carcinomas. The normal tissues were human papillomavirus (HPV) negative, whereas all invasive carcinomas included were HPV16 positive. In this study, we show that HOXA9, A10, A11, A13, B9, D11, and D13 genes are expressed in both the epithelium of normal tissues and neoplastic cells from squamous cervical carcinomas. Interestingly, the HOXC10 and D12 genes were not expressed in any cervical tissues; however, HOXB13, C9, C11, C12, C13, D9, and D10 genes were expressed only in the tumoral tissues but not in the normal cervix. Our findings suggest that the expression of HOXB13, D9, D10, and HOXC cluster (HOXC9, C11-C13) genes might be an important step involved in cervical cancer.

Increased angiotensin II type-2 receptor density in hyperplasia, DCIS and invasive carcinoma of the breast is paralleled with increased iNOS expression.

Binding of angiotensin II on the angiotensin II type-1 receptor induces cell growth, while triggering via the angiotensin II type-2 (AT(2)) receptor causes an opposing effect of growth inhibition and apoptosis. AT(2) receptor stimulation has also been shown to enhance inducible nitric oxide synthase (iNOS) expression, an enzyme associated with cancer. To study the involvement of the angiotensin II receptors and iNOS in the carcinogenesis of human breast, we visualised both factors in tissues from patients with hyperplasia, ductal carcinoma in situ (DCIS) and invasive carcinoma using immunocytochemistry and in situ hybridisation. In normal ducts, levels for AT(2) protein and mRNA are low, but these are markedly increased in all pathological tissues. While in normal tissue both negative and positive ducts are found, the staining patterns in hyperplasia, DCIS and invasive carcinoma have a homogeneous positive appearance. Similarly, iNOS enzyme expression was very low in the ductal epithelium of normal tissues, but highly increased in all pathologies, with the highest expression found in hyperplastic ducts. Three human cell lines were assayed for the presence of AT(2) receptor. Normal HMec 1001-3 cells were weakly positive, but only one of the adenocarcinoma cell lines, designated SK-BR-3, was shown to express both AT(2) protein and its mRNA. We show that AT(2) receptor and iNOS overexpression are associated with breast disease, further confirming the involvement of the components of the renin-angiotensin system in the aetiology of breast cancer.

Expression of MHC products and leucocyte differentiation antigens in gynaecological neoplasms: An immunohistological analysis of the tumour cells and infiltrating leucocytes

Monoclonal antibodies directed against monomorphic determinants of Class I and Class II products of the major histocompatibility complex (MHC) and against leucocyte differentiation antigens were used in an indirect immunoperoxidase technique to compare their expression in normal and malignant disease of the ovary, cervix and endometrium. MHC Class I products, strongly expressed on normal ovarian epithelium, were uniformly absent from 7/8 ovarian carcinomas of varying histology. Lack of Class I expression was also a feature of 6/10 cervical carcinomas and of 4/8 endometrial carcinomas, in comparison with their respective normal tissues. Relative to normal tissue epithelium MHC Class II products, could be either lost or gained, the pattern of expression being either uniform or heterogeneous. Leucocytes were sparse in normal ovary but more numerous in cervix and endometrium. In tumours, with few exceptions, they were abundant, though usually confined to the stroma. T cells, largely of cytotoxic/suppressor (OKT8) phenotype, tended to predominate though in some tumours, particularly cervical carcinoma, large numbers of macrophages and to a lesser extent, B cells were sometimes detected. By contrast, leucocytes of natural killer (NK) phenotype were virtually non-existent in any tumour or normal tissue. The ingress of leucocytes into gynaecological neoplasms does not appear to be a random event and may be evoked by an immune response against tumour-associated antigens. However, the relationship between in situ mononuclear cell infiltration and MHC expression on epithelial tumour cells is complex and remains to be elucidated.

Human tumor cell membrane glycoprotein associated with protein kinase activity.

A mouse MAb3 50H.19 raised against the human melanoma cell line MEL-T binds to carcinoma cell lines, carcinoma biopsy material, and certain epithelia of normal tissues. It immunoprecipitates two components from carcinoma cell lines, a major component of 22 kd which is O-glycosylated and a minor one of 24 kd which is additionally N-glycosylated. The immunocomplexed 50H.19 antigen exhibits protein kinase activity with substrate-specificity for casein and phosvitin, but not for histones. It phosphorylates on serine and threonine, but not tyrosine residues. Enzyme activity is cyclic AMP-independent.

Langerhans' cells in human cervical epithelium: effects of wart virus infection and intraepithelial neoplasia.

The distribution of Langerhans' cells and T and B lymphocytes in cervical epithelium affected by wart virus infection (WVI) and intraepithelial neoplasia (CIN) has been studied by immunohistological techniques with a panel of monoclonal antibodies. In comparison with normal tissue epithelium infected by wart virus consistently showed a partial depletion or absence of both Langerhans' cells and T lymphocytes. The few Langerhans' cells which remained showed loss of their normal dendritic processes and were confined to the vicinity of the basement membrane. In contrast, epithelium from patients with CIN showed an increase in spindle-shaped Langerhans' cells associated with increased numbers of stromal and intraepithelial lymphoid cells. These findings suggest that there is a marked difference in host immune responses in these two conditions and may explain why WVI tends to persist in the epithelium while CIN is often associated with a lymphoid reaction.