Colorectal cancers with microsatellite instability (MSI) represent 15% of all colorectal cancers, including Lynch syndrome as the most frequent hereditary form of this disease. Notably, MSI colorectal cancers have a higher density of tumor-infiltrating lymphocytes (TIL) than other colorectal cancers. This feature is thought to reflect the accumulation of frameshift mutations in sequences that are repeated within gene coding regions, thereby leading to the synthesis of neoantigens recognized by CD8 þ T cells. However, there has yet to be a clear link established between CD8 þ TIL density and frameshift mutations in colorectal cancer. In this study, we examined this link in 103 MSI colorectal cancers from two independent cohorts where frameshift mutations in 19 genes were analyzed and CD3 þ , CD8 þ , and FOXP3 þ TIL densities were quantitated. We found that CD8 þ TIL density correlated positively with the total number of frameshift mutations. TIL densities increased when frameshift mutations were present within the ASTE1, HNF1A, or TCF7L2 genes, increasing even further when at least one of these frameshift mutations was present in all tumor cells. Through in vitro assays using engineered antigen-presenting cells, we were able to stimulate peripheral cytotoxic T cells obtained from colorectal cancer patients with peptides derived from frameshift mutations found in their tumors. Taken together, our results highlight the importance of a CD8 þ T cell immune response against MSI colorectal cancer-specific neoantigens, establishing a preclinical rationale to target them as a personalized cellular immunotherapy strategy, an especially appealing goal for patients with Lynch syndrome. Cancer Res; 75(17); 3446-55. Ó2015 AACR.

Colorectal cancers with microsatellite instability are characterized by an important density of tumor-infiltrating lymphocytes and a good prognosis. Microsatellite instability results from the inactivation of the DNA mismatch repair system and induces secondary somatic frameshift mutations within target genes harboring repeat sequences in their coding frame. By disrupting the open reading frame, frameshift mutations can result in the appearance of potentially immunogenic neopeptides. To determine the frameshift mutations inducing a T-cell response during the development of a tumor with microsatellite instability, we studied in 61 colorectal cancer patients with microsatellite instability, using a fluorescent multiplex PCR comparative analysis, the relative frequency of frameshift mutations within 19 target genes and analyzed the correlation of these frameshift mutations with the density of CD3 þ tumor-infiltrating lymphocytes. The four most frequently mutated genes were ACVR2 (92%), TAF1B (84%), ASTE1/HT001 (80%) and TGFBR2 (77%). The vast majority (95%) of the tumors exhibited at least three frameshift mutations, and the number of frameshift mutations was associated with tumor progression (TNM stage, wall invasion and tumor diameter). Tumor-infiltrating lymphocyte density was associated with the overall number of frameshift mutations and with the presence of frameshift mutations within two target genes, namely ASTE1/HT001 and PTEN. These results strongly argue for the clinical relevance of immunotherapy of colorectal cancers with microsatellite instability.

Carcinoembryonic antigen (CEA) is a potential target for antigen-specific immunotherapy, as it is frequently overexpressed in human carcinomas. Moreover, an epitope derived from CEA, designated CAP1 (YLSGANLNL), has been proposed as naturally processed and presented by tumors in the human leukocyte antigen (HLA)-A*0201 context. Our aim was to fully characterize and assess the clinical relevance of the HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) response against CEA. Stable and potent artificial antigen presenting cells (AAPCs) were used to evaluate T-cell response against CEA. These cells efficiently activate CTLs against tumor-derived epitopes after transduction with the antigenic peptides or full-length proteins. We found that AAPCs genetically modified to express CAP1, the agonist peptide CAP1-6D, or the whole CEA protein were not able to activate CAP1-specific CTLs from HLA-A*0201+ healthy donors or patients with colorectal carcinoma, even after multiple stimulations. In addition, we showed that a CAP1-specific T-cell clone, obtained after multiple stimulations of T cells of a HLA-A*0201+ healthy donor in vitro with autologous antigen presenting cells, recognized CEA(-) HLA-A*0201+ tumors transduced with a minigene encoding CAP1 but failed to react against HLA-A*0201+ tumor cells expressing CEA. Finally, AAPCs expressing the whole CEA protein did not induce any specific CTL response against CEA+ HLA-A*0201+ tumor cells highlighting the potential difficulty of mounting an efficacious T-cell response against this autoantigen. Altogether, our data indicate that CAP1 is not efficiently processed and presented by CEA+ tumor cells, and therefore, is not an appropriate target for T-cell-based immunotherapy.

BackgroundColorectal cancers (CRCs) with microsatellite instability (MSI) are associated with a good prognosis and a high density of tumor-infiltrating lymphocytes (TILs). We have undertaken to determine the link between TIL densities and MSI CRC histologic features.Patients and MethodsUsing tissue microarrays, T-cell sub-population infiltration, including T cells (CD3), cytotoxic T cells (CD8) and regulatory T cells (FoxP3) were studied in 86 MSI CRCs. We separately analyzed TILs of the stromal and epithelial compartments in the tumor center, the tumoral invasion margin and associated normal tissue.ResultsFor FoxP3+ TIL density in the tumor center stromal compartment, we found a strong negative correlation with T4 stage (p = 0.01), node invasion (p<0.001) and VELIPI (vascular emboli, lymphatic invasion and perinervous invasion) criteria (p = 0.002).ConclusionThe strong correlation between regulatory T cell density and the absence of VELIPI criteria suggests that this sub-group of T cells is preferentially associated with less invasive tumors.