Abstract

BackgroundMicrosatellite unstable colorectal cancers (MSI+ CRCs) expressing PD-L1, respond to anti-PD-1 or anti-PD-L1 checkpoint blockade, whereas microsatellite-stable tumors do not respond the same. Our aim was to examine how the immune landscape relates to different aspects of the CRC’s biology, including neoepitope burden.MethodsWe used TCGA data to stratify patients based on a cytolytic T-cell activity expression index and correlated immune cytolytic activity (CYT) with mutational, structural, and neoepitope features of each tumor sample. The expression of several immune checkpoints was verified in an independent cohort of 72 CRC patients, relative to their MSI status, using immunohistochemistry and RT-qPCR.ResultsCRC exhibits a range of intertumoral cytolytic T-cell activity, with lower cytolytic levels in the tumor, compared to the normal tissue. We separated CRC patients into CYT-high and CYT-low subgroups. High cytolytic activity correlated with increased mutational load in colon tumors, the count of MHC-I/−II classically defined and alternatively defined neoepitopes, high microsatellite instability and deregulated expression of several inhibitory immune checkpoints (VISTA, TIGIT, PD-1, IDO1, CTLA-4, and PD-L1, among others). Many immune checkpoint molecules (IDO1, LAG3, TIGIT, VISTA, PD-1, PD-L1 and CTLA-4) expressed significantly higher in MSI+ CRCs compared to MSS tumors. The expression of Treg markers was also significantly higher in CYT-high tumors. Both individual and simultaneous high levels of CTLA-4 and PD-L1 had a positive effect on the patients’ overall survival. On the reverse, simultaneous low expression of both genes led to a significant shift towards negative effect. Assessed globally, CYT-low CRCs contained more recurrent somatic copy number alterations. PD-L1 protein was absent in most samples in the independent cohort and stained lowly in 33% of MSI CRCs. PD-L1+ CRCs stained moderately for CD8 and weakly for FOXP3. CYT-high colon tumors had higher TIL load, whereas CYT-high rectum tumors had higher TAN load compared to their CYT-low counterparts.ConclusionsOverall, we highlight the link between different genetic events and the immune microenvironment in CRC, taking into consideration the status of microsatellite instability. Our data provide further evidence that MSI+ and CYT-high tumors are better candidates for combinatorial checkpoint inhibition.

Highlights

  • Microsatellite unstable colorectal cancers (MSI+ Colorectal cancer (CRC)) expressing PD-L1, respond to anti-PD-1 or antiPD-L1 checkpoint blockade, whereas microsatellite-stable tumors do not respond the same

  • CRC stratification based on the immune cytolytic T-cell activity To evaluate intertumoral immune cytolytic T-cell activity across the colon (COAD) and rectum (READ)

  • We investigated whether the tumor-infiltrating lymphocytes (TILs) load or that of tumor-associated neutrophils (TAN) differs between the two cytolytic CRC subsets

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Summary

Introduction

Microsatellite unstable colorectal cancers (MSI+ CRCs) expressing PD-L1, respond to anti-PD-1 or antiPD-L1 checkpoint blockade, whereas microsatellite-stable tumors do not respond the same. The patient’s antitumoral immune cytolytic activity (CYT), calculated as the geometric mean of the expression of the genes granzyme A (GZMA) and perforin 1 (PRF1), is associated with improved patient survival [6, 7]. GZMA is a tryptase that leads to caspase-independent apoptosis, while PRF1 is a pore-forming enzyme that facilitates the entry of granzymes into the target cells. Both effector molecules are considerably over-expressed upon CD8+ T cell activation [15] and during productive clinical responses to anti-CTLA-4 or anti-PD-L1 treatment [16, 17]

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