Abstract 2658: Immunological and mutational landscape of gallbladder adenocarcinoma
Authors: Carapeto Fernando Cintra Lopes , Soto Luisa Maren Solis , Foo Wai Chin , Lu Wei , Fawaz Eduardo A. Vinuela , Villaseca Miguel A. , Pizarro Eduardo Alberto Vega , Araya Juan C. , Wistuba Ignacio I. , Milind Javle , Shubham Pant , Kwong Lawrence 
Published: September 2020

Abstract
Introduction Gallbladder adenocarcinoma (GBC) is a relatively rare malignancy with high mortality rate due to late stage discovery. At advanced-stages, there are limited treatment options, and most patients develop resistance to chemotherapy. Adapting the recent major advances in cancer immunotherapy specifically to GBC will require a deep understanding of the immune landscape. Our aim is to characterize Immune Checkpoint markers (IC), Tumor infiltrating Lymphocytes (TILs), and Myeloid cells (MCs), and associate them with cancer genomic alterations, clinic-pathological information, and patient survival.
Methods We selected formalin-fixed and paraffin-embedded tumor tissues from surgically resected primary GBC (n=97 patients) without neoadjuvant therapy to construct a tissue microarray. Tumor samples were collected from two institutions (MD Anderson Cancer Center (MDACC), USA, n=53; Sotero del Rio Hospital (SRH), Chile, n=44). We performed immunohistochemistry with the following biomarkers: IC (PD-1, PD-L1, LAG-3, TIM-3, B7-H3, B7-H4, VISTA, ICOS, OX-40, IDO1), TILs (CD3, CD4, CD8, CD45RO, CD57, GZB, FOXP-3, CD20), and MCs (CD68, CD11b, CD14). The evaluation of PD-L1, B7-H3, B7-H4, and IDO1 was performed in malignant cells and scored as percentage of positive cells. The TILs and IC were scored as cell densities (n/mm2) using digital image analysis. Tumors were classified in 4 types based on PD-L1 expression and CD8 densities (cut-off median): I (PD-L1+/CD8high), II (PD-L1-/CD8low), III (PD-L1+/CD8low), and IV (PD-L1-/CD8high). Next Generation sequencing platform T200 was used to access genomic alterations in a subset of cases (n=58).
Results Expression of PD-L1, B7-H3, B7-H4, and IDO1 (cutoff ≥1%) was 4, 12, 7, and 22 % respectively. From all tumors, 49% were type IV, 47% type II, 3% type I, and 1% type III. Comparing MDACC and SRH cohort, densities of CD3, CD4, TIM3, and PD-1 were higher in tumors from SRH (p&lt;0.001) while CD68 was higher in tumors from MDACC (p&lt;0.001). Higher CD57 was observed in pT1/pT2 tumors compared to pT3/pT4 (pT, pathological T) (p=0.003). Lower densities of VISTA (p =0.005 Long rank test) (cut-off: top quartile) correlated with higher overall survival rates in SRH cohort. The most frequent genomic alterations were (%): mutation in TP53 (38), SMAD4 (10), KRAS (10), ARID2 (9), BRCA2 (5); and amplifications in CCNE1 (14), ERRB2 (12) and MDM2/CDK4 (7). Mutations in ARID1A/ARID2/PBRM1 pathway had lower TIM3 densities (p=0.004), mutations in BRAF/KRAS had lower CD3, PD1 and ICOS densities (p=0.048; 0.004 and 0.0001 respectively), and mutations on PTEN/PI3KCA had lower CD68 densities (p=0.033). Amplifications of CCNE1 and ERBB2 had lower densities of OX40 (p=0.013; 0.029 respectively).
Conclusions We characterize the immunological landscape of gallbladder adenocarcinoma. Most tumors were immune ignorant or tolerant (Type II or IV). Different genomic alterations may have distinct immune patterns.
Citation Format: Fernando Cintra Lopes Carapeto, Luisa Maren Solis Soto, Wai Chin Foo, Wei Lu, Eduardo A. Vinuela Fawaz, Miguel A. Villaseca, Eduardo Alberto Vega Pizarro, Juan C. Araya, Ignacio I. Wistuba, Javle Milind, Pant Shubham, Lawrence Kwong. Immunological and mutational landscape of gallbladder adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2658.