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.

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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.

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