The density of Tbet+ tumor-infiltrating T lymphocytes reflects an effective and druggable preexisting adaptive antitumor immune response in colorectal cancer, irrespective of the microsatellite status

Abstract

Purpose: The recent success of anti-PD1 antibody in metastatic colorectal cancer (CRC) patients with microsatellite instability (MSI), known to be associated with an upregulated Th1/Tc1 gene signature, provides new promising therapeutic strategies. However, the partial objective response highlights a crucial need for relevant, easily evaluable, predictive biomarkers. Here we explore whether in situ assessment of Tbet+ tumor infiltrating lymphocytes (TILs) reflects a pre-existing functional antitumor Th1/Tc1/IFNγ response, in relation with clinicopathological features, microsatellite status and expression of immunoregulatory molecules (PD1, PDL1, IDO-1). Methodology: In two independent cohorts of CRC (retrospective n = 80; prospective n = 27) we assessed TILs density (CD3, Tbet, PD1) and expression profile of PDL1 and IDO-1 by immunohistochemistry/image analysis. Furthermore, the prospective cohort allowed to perform ex vivo CRC explant cultures and measure by Elisa the IFNγ response, at baseline and upon anti-PD1 treatment. Results: The density of Tbet+ TILs was significantly higher in MSI CRC, especially in the medullary subtype but also in a subgroup of MSS (microsatellite stable), and positively correlated with PD1 and PDL1 expression, but not with IDO-1. Finally, a high number of Tbet+ TILs was associated with a favorable overall survival. These Tbet+ TILs were functional as their density positively correlated with basal IFNγ levels. In addition, the combined score of Tbet+ PD1+ TILs coupled with IDO-1 expression predicted the magnitude of the IFNγ response upon anti-PD1. Conclusion: Altogether, immunohistochemical quantification of Tbet+ TILs is a reliable and accurate tool to recapitulate a preexisting Th1/Tc1/IFNγ antitumor response that can be reinvigorated by anti-PD1 treatment.

The Caspase-1/IL-18 Axis of the Inflammasome in Tumor Cells: A Modulator of the Th1/Tc1 Response of Tumor-Infiltrating T Lymphocytes in Colorectal Cancer

“…These findings suggest that IL-18 produced by tumor cells can influence the density of the IEL-TIL infiltrate that we and others previously reported to be higher in MSI and a subgroup of MSS CRCs. In addition, these IEL-TILs positively impact overall survival and progression-free survival in our cohort (not shown), consistently with previous studies in CRC [ 2 , 7 ]. In our study, IL-18 was lost in tumor cells in 5% of CRCs and heterogeneously expressed in 10–40% of tumor cells in about 20% of CRC that did not show any distinct clinicopathological or molecular features.…”

“…The microsatellite status of CRC was assessed by IHC using anti-MLH1 (clone E505), MSH2 (clone FE11), MSH6 (clone EP49), and PMS2 (clone EP51) antibodies, as previously described [ 7 ].…”

“…In microsatellite instable (MSI) CRC, which accounts for 15% of all CRCs (as compared to microsatellite stable (MSS) CRC), recent evidence has shown the major role of a T-helper/cytotoxic (Th1/Tc1) gene signature in CRC immune surveillance, caused by frameshift and missense mutations leading to the formation of immunogenic neoepitopes and to an anti-tumor immune response [ 5 , 6 ]. In addition, we recently demonstrated that a preexisting in situ Th1/Tc1 immune response—identified by the expression of the transcription factor T-box expressed in T cells (Tbet)—positively impacted prognosis in MSI as well as in MSS CRC [ 7 ]. This anti-tumor immune response can be counterbalanced by inhibitory signaling pathways including immune checkpoint receptors (ICPs), and their ligands such as the programmed death receptor-1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA4) pathways [ 7 , 8 ], that can be blocked by specific antibodies defined as immune checkpoint inhibitors (ICIs).…”

“…In addition, we recently demonstrated that a preexisting in situ Th1/Tc1 immune response—identified by the expression of the transcription factor T-box expressed in T cells (Tbet)—positively impacted prognosis in MSI as well as in MSS CRC [ 7 ]. This anti-tumor immune response can be counterbalanced by inhibitory signaling pathways including immune checkpoint receptors (ICPs), and their ligands such as the programmed death receptor-1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA4) pathways [ 7 , 8 ], that can be blocked by specific antibodies defined as immune checkpoint inhibitors (ICIs). However, treatment with ICIs only leads to objective and durable responses in 30% to 50% of metastatic MSI CRC cases (mono-or combotherapy) and to a very low response rate in MSS CRC [ 9 , 10 , 11 ].…”

“…In this study, we used two independent cohorts of CRC patients to assess IL-18 expression and caspase-1 activation profiles in tumor cells and studied their relationship with TILs density and microsatellite status. In addition, functional and multiparametric approaches, at the protein and mRNA levels, were performed both on an ex vivo 3D model of CRC explant cultures that maintained cellular interactions able to build innate/adaptive immune responses (previously validated as a good preclinical model [ 7 ]), and on TILs isolated from CRC primary tumors.…”