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.

Adoptive transfer of in vitro activated and expanded antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic strategy for infectious diseases and cancers. Obtaining in vitro a sufficient amount of highly specific cytotoxic cells and capable of retaining cytotoxic activity in vivo remains problematic. We studied the role of Toll-Like Receptor-8 (TLR8) engagement on peripheral CTLs activated with melanoma antigen MART-1-expressing artificial antigen-presenting cells (AAPCs). After a 3-week co-culture, 3–27% of specific CTLs were consistently obtained. CTLs expressed TLR8 in the intracellular compartment and at the cell surface. Specific CTLs activated with a TLR8 agonist (CL075) 24 h before the end of the culture displayed neither any change in their production levels of molecules involved in cytotoxicity (IFN-γ, Granzyme B, and TNF-α) nor major significant change in their cell surface phenotype. However, these TLR8-stimulated lymphocytes displayed increased cytotoxic activity against specific peptide-pulsed target cells related to an increase in specific anti-melanoma CTL functional avidity. TLR8 engagement on CTLs could, therefore, be useful in different immunotherapy strategies.

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Colorectal cancers with microsatellite instability (MSI-CRCs) represent 15% of all CRCs, including Lynch syndrome, the most frequent hereditary form of this disease. Notably, MSI-CRCs have a higher density of tumor-infiltrating lymphocytes (TILs) than other CRCs. This feature is thought to reflect an accumulation of frameshift mutations in coding repeat sequences, leading to the synthesis of neo-antigens, expressed only by tumor cells and presented at their cell surface on HLA class I molecules, as immunogenic neo-peptides recognized by CD8+ T cells. However, a clear link between CD8+ TIL density and frameshift mutations in MSI-CRCs has yet to be established. With this aim, we first looked for this link in 103 MSI-CRCs from two independent tumor cohorts. Frameshift mutations in 19 genes were analyzed, using 2 multiplex PCRs, and CD3+, CD8+ and FOXP3+ TIL densities were quantified by immunohistochemistry, using tissue microarrays. We found that CD3+ and CD8+ TIL densities, but not FOXP3+ TIL density, were positively correlated with the total number of frameshift mutations, and that CD8+ TIL density was especially higher when a frameshift mutation was present in ASTE1, HNF1A or TCF7L2 gene, reinforcing the hypothesis according to which anti-tumor frameshift mutation-derived neo-antigen-specific CD8+ T lymphocytes (TLs) could infiltrate MSI colorectal tumors. Based on these results, we secondly undertook to exploit this natural anti-tumor immune response for the treatment of MSI-CRCs. We pre-clinically developed a personalized cellular adoptive immunotherapy strategy based on the characterization of frameshift mutations in a given patient's tumor and the stimulation of this patient's TLs against neo-peptides derived from these mutations. To detect MSI-colorectal tumor mutations, within repeated coding sequences, we used our 2 multiplex PCRs. To activate specific cytotoxic TLs in vitro, we constructed Artificial Antigen Presenting Cells (AAPCs), expressing the main costimulatory molecules, B7.1, ICAM-1 and LFA-3, and efficiently presenting a transgene-encoded peptide on the most frequently expressed HLA class I molecule, HLA-A2.1. In the tumor of the first HLA-A2+ MSI-CRC Lynch patient included in this functional study, we detected a single nucleotide deletion in coding repeat sequences of TGFBR2, TAF1B and ASTE1 genes, leading to the putative synthesis of 3 neo-peptides predicted to have a high affinity for the HLA-A2.1 molecule. We cultured this patient's TLs with AAPCs expressing each one of these frameshift mutation-derived peptides. After expansion, activated TLs were able to specifically kill cells, including MSI-CRC tumor cells, presenting the relevant peptides. Then, we performed similar experiments on 2 other MSI-CRC HLA-A2+ Lynch patients and on 3 HLA-A2+ control donors. After specific activation with the same AAPCs, only MSI-CRC HLA-A2+ Lynch patients' activated peripheral TLs could recognize neo-peptides derived from frameshift mutations present in their tumor. Taken together, our results establish a preclinical rationale for developing personalized cellular adoptive immunotherapy strategies based on the use of our AAPCs to treat MSI-CRCs, an especially appealing goal for Lynch syndrome patients. Citation Format: Pauline Maby, Mohamad Hamieh, Hafid Kora, David Tougeron, Bernhard Mlecnik, Gabriela Bindea, Helen K. Angell, Tessa Fredriksen, Nicolas Elie, Emilie Fauquembergue, Aurélie Drouet, Jérôme Leprince, Jacques Benichou, Jacques Mauillon, Florence Le Pessot, Richard Sesboüé, Thierry Frébourg, Jérôme Galon, Jean-Baptiste Latouche. Towards personalized cellular adoptive immunotherapy targeting tumor specific neo-antigens in microsatellite unstable colorectal cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A114.