Abstract 4576: Study of the immune contexture in advanced pancreatic neuroendocrine tumors reveals tumor-associated macrophages as promoters of poor survival
Authors: Francisco-Cruz Alejandro , Uraoka Naohiro , Liu Suyu , Parra Edwin R. , Solis Luisa M. , Mino Barbara , Dasari Arvind , Rodriguez-Canales Jaime , Overman Michael J. , Loree Jonathan M. , Yao James C. , Wistuba Ignacio I. , Halperin Daniel M. , Estrella Jeannelyn S. 
Published: August 2019

Introduction. PanNETs are characterized by heterogeneous but largely indolent growth, leading to advanced stage at diagnosis, difficulty predicting outcomes, and insufficiently effective treatments. A better understanding of PanNETs immune context is needed for rational immunotherapy strategies. The aim of this study was to characterize immune cell infiltrates within primary tumors, understand the correlation of immune infiltration with genes associated with PanNET development, and clinical-pathological features.

Material and methods. Formalin-fixed paraffin-embedded (FFPE) surgically resected primary tumor specimens from 53 patients with metastatic PanNETs were evaluated for DAXX, ATRX, and immune-cell markers (CD8, CD4, CD45RO, FOXP3, ICOS, OX40, PD-1, LAG3, TIM-3, B7-H3, B7-H4, PD-L1, VISTA, and CD68) by immunohistochemistry (IHC). Intratumoral lymphocyte-enriched areas (LEA), defined by CD8 hot-spots, and macrophage-enriched areas (MEA), defined by CD68 hot spots, were selected for image analysis and cell densities were quantitated. We considered higher densities more than the third quartile value and low density as less or equal than the third quartile value for all the markers. 47 cases from the same FFPE tumor tissue blocks were used for paired-end RNA sequencing (HiSeq 4000 Sequencing System), and exome sequencing (T200 Platform) to MEN1, SETD2, MUTYH, CHEK2, BRCA2, ALT, DAXX, ATRX, PTEN, TSC1, and TSC2 genes. Differences between variables were analyzed by non-parametric t-test and Kaplan-Meier curves for time-to-event using SPSS statistical software version 24.

Results. Overall, higher densities of CD8, CD4, CD68, and B7-H3 were found compared with the other markers. We found a significant correlation between CD8 in LEA with CD4 (r=0.7), FOXP3 (r=0.5), CD45RO (r=0.6), ICOS (r=0.5) and PD-1 (r=0.5) cell densities. In addition, CD68 in MEA had significant and positive correlation with TIM-3 (r=0.6) cell densities. Higher TIM-3 cell densities correlated with higher levels of TIM-3 (P=&lt;0.00001), CD163 (P=0.004), and CSF1R (P=0.02) mRNAs. Patients with high CD68 and TIM-3 densities showed worse disease-specific survival (DSS). The ratio between CD68, TIM-3, B7-H3 and FOXP3 cell densities to CD8 positive cells was significantly higher in patients with loss of ATRX nuclear expression. We observed different gene mutations on those PanNET samples but only PTEN mutant tumors were associated with higher densities of CD68 and worst DSS compared with lowest CD68 and WT PTEN mutant tumors (P=0.002).

Conclusions. TAMs were significantly correlated with inferior DSS in PanNETs. TAM depletion may, therefore, present an appealing and rational target for immunotherapeutic approaches in NETs. This work was funded by a Conquer Cancer Foundation of ASCO Career Development Award, and a grant from the Neuroendocrine Tumor Research Foundation.

Citation Format: Alejandro Francisco-Cruz, Naohiro Uraoka, Suyu Liu, Edwin R. Parra, Luisa M. Solis, Barbara Mino, Arvind Dasari, Jaime Rodriguez-Canales, Michael J. Overman, Jonathan M. Loree, James C. Yao, Ignacio I. Wistuba, Daniel M. Halperin, Jeannelyn S. Estrella. Study of the immune contexture in advanced pancreatic neuroendocrine tumors reveals tumor-associated macrophages as promoters of poor survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4576.