Abstract B21: Tertiary lymphoid organs within pancreatic ductal carcinoma tissue are a favorable prognosticator, being strongly associated with blood vessels not infiltrated by cancer cells
Authors: Hiraoka Nobuyoshi 
Published: December 2015

Abstract
Purpose: It has been speculated that immune system relates closely to vascular system in cancer stroma, although there has been few studies in human cancers. Host immunity plays critical roles in tumor surveillance. Tertiary lymphoid organs (TLOs) are induced in various inflamed tissues including cancers, although it remains poorly characterized how TLOs are affected by cancer invasion and related to vascular system. The aim of this study was to investigate the clinicopathological and pathobiological characteristics of tumor microenvironment in pancreatic ductal carcinoma (PDC) with TLOs.
Methods: We examined PDCs to investigate the clinicopathological impact of TLOs and their association with tumor-infiltrating immune cells, the cytokine milieu, and tissue characteristics including vascular system. Two cohorts were used, which included consecutive patients (n=308 and 226) who underwent initial surgical resection for PDC in our center between 1990 and 2004, and between 2005 and 2009, respectively. TLOs are organized lymphoid structures similar to lymph nodes and Peyer's patches, although TLOs are not encapsulated and supplied by afferent lymphatics. We defined tumor-associated TLOs as either intratumoral, i.e. a TLO located within the PDC tissue, or peritumoral, i.e. a TLO located outside the PDC tissue and attached to it. Tumor area was detected by microscopic observation and was marked with ink on tissue slides, and then we evaluated the position of TLOs. If a TLO existed on the line of tumor border, it was counted as a peritumoral TLO. There was no patient having PDC without any TLOs and almost all the patients had peritumoral TLOs. Patients were divided into two groups by the presence or absence of intratumoral TLOs for survival and correlation analyses. Kaplan-Meier analysis and Cox regression multivariate survival analysis were performed. After immunohistochemistry to detect each immune cells and vessels, quantitative evaluation of tumor-infiltrating immune cells or vessels was carried out. Double or triple immunofluorescence was also performed to detect different types of vessels. Expression of genes related to immune-microenvironment was measured by real-time RT-PCR.
Results: There were two different localizations of PDC-associated TLOs, intratumoral and peritumoral. 15.9% (49/308) of PDCs had intratumoral TLOs. A better outcome was observed in patients with intratumoral TLOs, and this was independent of other survival factors. The results were confirmed by our investigation using another cohort.
The PDC tissues with intratumoral TLOs showed significantly higher infiltration of CD8+ T, CD4+ T, and CD20+ B cells and lower infiltration of immunosuppressive cells such as FOXP3+ regulatory T cells and CD163+ M2 macrophages, as well as significantly higher expression of Th1- and Th17-related genes.
TLOs had a strong association with arterioles, venules, and nerves. These vessels were significantly reduced their numbers and were frequently involved by cancer invasion in PDC tissues without intratumoral TLOs compared to PDC tissues with intratumoral TLOs. The numbers of CD31+ endothelial cells in PDC tissue with intratumoral TLOs were higher than those in PDC tissue without intratumoral TLOs. The PDC tissues with intratumoral TLOs also had significantly frequent capillaries consisting of ERG+VE-cadherin+ endothelial cells covered by alpha-SMA+ pericytes. Conversely, the density of endothelial cells composed of abnormal blood vessels lacking a covering of pericytes was higher in PDC tissue without intratumoral TLOs.
Conclusions: Our results suggest that the presence of intratumoral TLOs represents a microenvironment that is less susceptible to cancer invasion, has an active immune reaction, and shows a relatively intact vascular network retained.
Citation Format: Nobuyoshi Hiraoka. Tertiary lymphoid organs within pancreatic ductal carcinoma tissue are a favorable prognosticator, being strongly associated with blood vessels not infiltrated by cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B21.