Archive | 2019
Stratification of Protein Expression across the Pancreatic Ductal Adenocarcinoma Disease Axis to Inform an Early Detection Platform
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an extremely deadly disease with a dismal long-term prognosis. Currently there is an 8% 5-year survival rate for PDAC patients, which is due in part to a lack of early detection methods (Siegal et al, 2017). Notably, the majority of patients present with late stage disease, when therapeutic treatment is not effective. Novel biomarkers that can facilitate early detection of PDAC are needed to catch cancer earlier when there are more treatment options. Our laboratory has identified an overlooked population of tumor cells in the peripheral blood of cancer patients across all stages of disease. These cells are fusion hybrids between macrophages and pancreatic tumor cells, and harbor properties of both parental cell types—these cells are called circulating hybrid cells (CHCs). CHCs express the epithelial protein cytokeratin (CK) and the pan-leukocyte epitope, CD45. We have determined their existence in greater numbers compared to conventionally-defined circulating tumor cells (CTCs) that express CK, but not CD45 in cancer patients. In addition, we identified CHCs in patients with precursor pathologies or precursor lesions across all different stages of PDAC, leading us to expect that specific protein expression across the disease axis could increase specificity of CHCs in an early detection assay. To support this, I will test the hypothesis that distinct protein expression defines different stages of disease in the pancreatic cancer continuum. In utilizing a disease-specific tissue microarray, and antibodies recognizing discrete proteins identified with immunofluorescence, I differentiated states of pancreatic epithelium from normal, pancreatitis, pre-cancer (PanIN1, PanIN2, PanIN3), and cancer. Introduction PDAC is a type of exocrine pancreatic cancer deriving from cells that line ducts in the pancreas (Stark, A., & Eibl, G, 2015). Advanced stages of pancreatic cancer are present in around 50% of patients due to the disease’s heterogeneity of genetic mutations and lack of distinctive symptoms (Adamska et al, 2017). The only reliably curative treatment for patients with pancreatic cancer is surgical resection; however, only precancerous or early stages of PDAC are considered resectable. In order to detect earlier stages of disease efforts must focus on identification of patients with precursors of PDAC such as pancreatic intraepithelial neoplasms (PanINs), mucinous cystic neoplasms (MCNs), and intraductal mucinous cystic neoplasms (IPMNs). Novel biomarkers are needed to detect these precursors, because the most commonly used FDA approved blood biomarker test for PDAC, the carbohydrate antigen 19-9 (CA19-9) (Becker et al, 2014), is not routinely effective. CA19-9 is a poor screening tool for PDAC as it is expressed in low prevalence within the general population and is unreliable as a sensitive readout. Another potential biomarker for PDAC are conventionally defined circulating tumor cells (CTCs) (Poruk et al, 2016). CTCs are identified in the blood of patients with malignant neoplasm, but in very low numbers and rarely in healthy control samples (Poruk et al, 2016. Therefore, CTC-based assays have failed. A novel biomarker, identified in our laboratory, is a circulating tumor cell that is the product of tumor cell-macrophage cell fusion, referred to as circulating hybrid cell (CHC). These CHCs are of interest due to the discovery of their existence in patients with precancerous lesions as well as metastatic pancreatic cancer at levels greater than conventionally defined CTCs (Gast et al, 2018). Although CTCs are also found across PDAC stages, CHCs exist in numbers greater in magnitude than CTCs in the metastatic setting (Gast et al, 2018). Therefore, we hypothesize that distinct protein expression can define the various stages of pancreatic ductal adenocarcinoma. Recognizing there are distinct morphologic differences between the different stages of pancreatic disease, identification of discrete protein profiles that define each stage of cancer can be applied to assays for early detection. Similarly, Schelitter et al identified prognostic relationships between histopathology and molecular profile based on the morphologic stratification of PDACs through mutational status of the four driver genes, KRAS, CDKN2A/p16, SMAD4 and TP53 (Jones et al, 2008). To identify discrete disease-specific protein expression, we generated pancreatic tissue microarrays (TMA) that represented pathology across the disease continuum (normal, inflammation, PanIN, and PDAC). We then validated an extensive number of antibodies with potential differential protein expression, and applied these antibodies to the TMA. Utilizing an immunofluorescence approach, protein expression patterns were visualized from the TMA. Overall, protein expression displayed differential patterns in the epithelium across the disease axis. We identified a subset of antibodies that recognized epithelial cells, and/or cancer cells and therefore have potential to distinguish different stages of pathology across the PDAC disease axis to increase specificity of CHC’s in early detection platforms. Figure 1: A, B) Human macrophage/tumor fusion: Identification of fusion hybrids in PDAC (left) and precancerous neoplasia (PanIN, right). Co-expression of Y-chromosome (red) and tumor epithelial marker cytokeratin (gray) demonstrates macrophage/tumor fusion C, D) Diagram of macrophage fusion event. Schematic of macrophage fusion with cancer cells to create CHCs (C). GFP labeled macrophage fusing with RFP labeled tumor cell resulting in merged phenotype. Fusion event in YFP-Macrophages and RFP-Tumor cells (D). E) Cell fusion hybrids (arrowheads) that co-stain for EPCAM (yellow) and CD45 (green) and have a Y chromosome (white dot) in their nuclei (blue). Arrows denote leukocytes. Reprinted/adapted from Gast, Charles E., et al. “Cell Fusion Potentiates Tumor Heterogeneity and Reveals Circulating Hybrid Cells That Correlate with Stage and Survival.” Science Advances, vol. 4, no. 9, 12 Sept. 2018, doi:10.1126/sciadv.aat7828. © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC) http://creativecommons.org/licenses/by-nc/4.0/ Figure 3: Pancreatic disease morphology (H&E): Healthy to inflamed pancreatic tissue (A) characterized by circular duct structures. Progression through pancreatic intraepithelial neoplasia (PanIN) and PDAC results in increasing ductal irregularity and dysplasia (B,C,D). Adapted from Clinical Cancer Research, 2000, Volume 6/Issue 8, 2969-2972, Ralph H. Hruban, Michael Goggins, Jennifer Parsons, Scott E. Kern, Progression Model for Pancreatic Cancer, with permission from AACR. Modifications by Melissa Wong. A B Figure 2: A) Quantification of CHCs and CTCs across the stages of PDAC development through flow cytometry. CHCs found in the precancerous setting as well as the node -/+ and metastatic setting. B) Immunofluorescent identification of CHCs via the co-expression of tumor epithelial marker cytokeratin and panleukocyte marker CD45 (CK+/CD45+, left) and CTCs (CK+/CD 45-, right). (Gast, et al, 2018). Materials and Methods Human Subjects/Ethics: Tissue samples were collected with approved protocols according to the ethical requirements and regulations of the Oregon Health and Science University (OHSU) review board. All patients gave informed consent. Generation of Tissue Microarray: For tissue preparation, the selected FFPE tissues were marked for the specified tissue section utilized for the cores. A template for the TMA production was prepared with the tissues organized according to the outline: Figure 4: A) Tissue microarray (TMA) layout. Labels a-f and 0-6 were how we were able to designate what specific core/disease stage was being viewed under the microscope (6 cores per disease stage and 7 rows in total of the different stages). B) Hematoxylin and Eosin staining of TMA. A