Abstract A36: The pattern of exosomal marker (CD63) expression using immunohistochemistry (IHC) in malignant and non-malignant (normal, inflammatory and pre-malignant) ductal cells in resected pancreatic tissues
Authors: Bhardwaj Arun , Patel Girijesh Kumar , Laurini Javier Ariel , Roveda Kelly , Tan Marcus , Singh Seema , Taylor William , Singh Ajay P. 
Published: December 2016

Background: Exosomes are membrane bound nano-vesicles (30-150 nm) of endocytic origin. Exosomes contain functional biomolecules including nucleic acids, lipids and proteins, which can be transferred to other cells upon fusion, macropinocytosis or receptor-mediated endocytosis. Exosomes are considered as an important mediator of intercellular communication, and play pivotal roles in facilitating cancer progression and metastasis. Exosomal membranes are enriched in endosome-specific tetraspanins (CD9, CD63, CD81), membrane transport and fusion proteins (flotillin, GTPase) and multiple vesicular bodies biogenesis-related proteins (Alix, TSG101). Immunohistochemical (IHC) staining for the exosomal markers CD9 and CD63 was used as a method to detect exosomal expression. We explored the pattern of exosomal marker (CD63) expression using IHC in malignant and non-malignant ductal cells in resected pancreatic tissues.

Methods: Between 2013 and 2015, twenty-three patients underwent pancreatic resection at the University of South Alabama Medical Center. Two sections were obtained from every resected specimen. One was obtained from the pathological pancreatic ducts and one was obtained from the adjacent normal ducts. IHC for CD63, a specific exosomal marker, was performed on 5µm sections from the resected pancreatic tissue using specific monoclonal antibody. Two pathologists evaluated the CD63 staining independently. The intensity of cytoplasmic staining in the ductal cells was graded from 1 to 3: 1 (weak), 2 (moderate) and 3 (strong). The percentage of cells stained was estimated on each section in 10% increments. For each tissue section, a Q-score was calculated by multiplying the percentage of positive cells by the intensity of the staining. The average Q-score was calculated for each section. Unpaired t test was used to assess if the difference in expression was considered statistically significant.

Results: The median age was 59 years (range 35-80). Fifty seven percent of the patients were white and 43% were African Americans. Sixty one percent of the patients were females. Eleven patients (48%) had malignant ductal cells in the resected pancreas (7 pancreatic adenocarcinoma, 3 pancreatic neuroendocrine tumors and 1 ampullary adenocarcinoma). Twelve patients had no malignant ductal cells (6 intraductal papillary mucinous neoplasm and pancreatic intraepithelial neoplasm and 6 acute/chronic pancreatitis). The mean Q score of the pathological pancreatic ducts is 219 while the mean Q score of the adjacent normal ducts is 169 (N=23, two-tailed P value is 0.0146). The mean Q score of the malignant pancreatic ducts is 262. Their adjacent normal ducts mean Q score is 148 (N=11, two-tailed P value is less than 0.0001). The mean Q score of the malignant pancreatic ducts is 262. The mean Q score of the premalignant pathologic pancreatic ducts is 218(N=6, two-tailed P value is 0.0017). The mean Q score of the non-malignant pathological pancreatic ducts is 178. Their adjacent normal ducts mean Q score is 188 (N=12, two-tailed P value is 0.707).

Conclusion: Immunohistochemical staining for the exosomal marker CD63 in resected pancreatic tissues is feasible. Using IHC, the expression of CD63 is higher in malignant pancreatic ducts compared to non-malignant pathologic pancreatic ducts (premalignant and inflammatory) and adjacent normal pancreatic ducts. Staining for CD9 is ongoing and will be reported in the meeting.

Citation Format: Moh’d Khushman, Arun Bhardwaj, Girijesh Kumar Patel, Javier Ariel Laurini, Kelly Roveda, Marcus Tan, Seema Singh, William Taylor, Ajay P. Singh.{Authors}. The pattern of exosomal marker (CD63) expression using immunohistochemistry (IHC) in malignant and non-malignant (normal, inflammatory and pre-malignant) ductal cells in resected pancreatic tissues. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A36.