Abstract A088: Immune profiling of inflamed microsatellite stable colorectal cancer
Authors: Llosa Nicolas Jose , Housseau Franck , Siegel Nicholas , Smith Kellie N. , Fan Hongni , Anders Robert M. , Le Dung , Diaz Luis , Sears Cynthia , Pardoll Drew M. 
Published: November 2016

Abstract
Immunotherapy is an important therapeutic modality that is rapidly turning into standard of care for many cancers. However, early immunotherapy efforts in colorectal cancer (CRC) were ineffective, and only recently did a successful breakthrough lead to improved survival in a small subset of metastatic colorectal cancer patients which were noted to harbor MMR deficiency (microsatellite instability: MSI). Therefore, there is an urgent need for additional biomarkers to identify CRC patients who are predicted to respond to immunotherapy. For the past year we have been interrogating the immune microenvironment of microsatellite stable (MSS) CRC patients to aid our understanding of why a fair proportion of these patients have an inflamed tumor microenvironment (TME). We have banked 140 tumor/normal CRC tissue matched pairs using our recruitment network at Hopkins. Genetic MSI testing was done on 114 of these patients, with 20 (17%) and 94 (83%) being categorized as MSI and MSS, respectively. Our results are therefore representative of the general CRC population which includes 15-20% of MSI+ tumors. We are also currently working with material obtained from CRC patients treated under the Hopkins phase 2 study testing anti PD-1 therapy in patients with MSI tumors. Immunohistochemistry (IHC) and digital image analysis of primary and trial CRC patients: We previously demonstrated the critical geographic association between infiltrating CD8+ T cells and immuno-regulatory molecule expression in the TME of MSI CRC. CRC FFPE tissue sections were stained with anti-CD3 and anti-CD8 antibodies and digital image analysis utilizing the HALO platform was performed.
Inflamed MSS CRC are infiltrated by PD1high T cells which are functionally suppressed: Multiparameter flow cytometry (MFC) performed on lymphocytes freshly isolated from our primary CRC tumors demonstrated that a large proportion of both CD4+ and CD8+ T cells infiltrating what we denominate inflamed MSS tumors express higher levels of PD-1 compared to conventional MSS patients. Very importantly, these PD-1high T cells are capable of producing a large amount of IFN-γ after short term PMA/ionomycin stimulation.
CD8+ T cells co-localize with immune checkpoint expression in inflamed MSS CRC: We further explored the nature of the T-cell infiltrates by performing laser capture microdissection (LCM) on inflamed MSS tumors and MSS patients who stabilized disease during anti PD-1 therapy, separately dissecting the TIL and invasive front compartments and then performed qRT-PCR for selected genes encoding signature T-cell cytokines as well as core transcription factors for each of the three major Th subsets, Th1/Tc1 (type I CTL; TBX21 and IFN-γ are common to Th1 and Tc1), Th2, and Th17. We additionally analyzed genes associated with CTL and Treg and also general inflammatory cytokines. Finally, we analyzed expression of genes encoding both co-inhibitory membrane ligands and receptors (checkpoints) and metabolic enzymes that have been shown to regulate lymphocyte activity. Inflamed MSS tumors exhibited a similar CD8+ T cell infiltration with a Th1/Tc1 immune signature associated with the counter expression of immune checkpoints as observed in MSI patients.
Prediction of MHC class I-restricted Mutation Associated Neoantigens (MANAs): Nonsynonymous mutations in tumor tissue are expected to generate MANAs, which are 8-11 amino-acid peptides generated from proteosomal degradation and recognized by tumor-specific CD8+ T cells in the context of HLA-I restriction. By comparing tumor to normal colon exome we are currently identifying mutations and MANAs in MSS CRC patients who showed high levels of lymphocyte infiltration and PD-1 expression. MANAs in inflamed MSS CRC will be tested for their recognition by autologous TIL.
Analysis of the TCR Vβ repertoire in primary CRC and anti-PD-1-treated CRC: In addition to the IHC characterization of CRC T cell infiltration, we are using TCRVβ clonality analysis of the intra-tumor immune response as a metric of the intensity of the antitumor immune response. We postulate that the number and the frequency of unique TCRVβ sequences in the tumor tissue should reflect the density and the immunogenicity of neoepitopes, respectively. In conclusion, we have identified a subset of MSS CRCs that exhibit an “MSI-like” immunologic microenvironment. The final aim of our studies is to validate that MSS CRC patients with a preexisting anti-tumor immune response can benefit from immunotherapeutic interventions.
Citation Format: Nicolas Jose Llosa, Franck Housseau, Nicholas Siegel, Kellie N. Smith, Hongni Fan, Robert M. Anders, Dung Le, Luis Diaz, Jr., Cynthia Sears, Drew M. Pardoll. Immune profiling of inflamed microsatellite stable colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A088.