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Correlation between CD3+ TIL densities calculated from whole slides and from TMA samples.

Correlation between frameshift mutations and CD8+ TIL density in the second series.

Frequency of frameshift mutations within target genes and proportion of tumors harboring these mutations in all malignant cells.

Association between clinical characteristics and number of frameshift mutations.

Supplementary Figure Legend

Supplementary Figure 3. In vitro cytotoxic activity of peripheral TLs from donors harboring or not mutations in TGFBR2, TAF1B and ASTE1 genes.

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+ T cells. However, there has yet to be a clear link established between CD8+ 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+, CD8+, and FOXP3+ TIL densities were quantitated. We found that CD8+ 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+ 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. ©2015 AACR.

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.

HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in human tumors such as neuroblastomas and natural killer (NK) lymphomas. HACE1 has been shown to ubiquitylate Rac1, a protein involved in cell proliferation and G2/M cell cycle progression. The function of HACE1 and the factors involved in its transcriptional regulation are largely unknown in the context of B-cell lymphomas. We show here, by RT-qPCR, that HACE1 gene is constitutively expressed in Normal lymph nodes and in normal B-cells isolated from peripheral blood, contrasting with a strong downregulation of its expression in more than 70% (77/111) of diffuse large B-cell lymphoma (DLBCL) cases and in four tested B-Lymphoma cell lines. HACE1 gene copy number was assessed by quantitative multiplex PCR of short fluorescent fragments (QMPSF) and array for comparative genomic hybridization (aCGH) in 91 DLBCL cases. A HACE1 heterozygous deletion was observed in 38.1% and an homozygous deletion in 2.4% of cases. These deletions were associated with a significant gene expression decrease. The molecular epigenetic mechanisms underlying HACE1 downregulation were also investigated. Using pyrosequencing assays, as compared to normal B-cells, we observed an hypermethylation of HACE1 promoter CpG177 island in 60% (68/111) of DLBCL cases and in all tested B-Lymphoma cell lines. However, no significant correlation between promoter methylation status and gene expression level was demonstrated. Furthermore, RT-qPCR assays revealed that the demethylating agent 5′azacytidine (5′AZA) did not induce a HACE1 gene expression increase in the different cell lines. By contrast, the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and LBH589 strongly reactivated the expression of HACE1 in Ramos, Raji and RL cells in which the CpG 177 island was fully methylated. We next performed ChIP experiments to determine whether HACE1 locus chromatin was in an active or inactive conformation in Ramos cell line, the most sensitive cell line to TSA effect. We found that the chromatin of HACE1 gene promoter region was predominantly in the inactive conformation (methylated H3 histones). TSA treatment was able to reverse this pattern, switching the conformation of HACE1 promoter chromatin to an active one predominantly associated with acetylated H3 histones. The putative role of HACE1 in B-cell lymphomagenesis was further investigated using lentiviral transduction (shHACE1). We demonstrated in Ramos and Raji cells that a down-regulation of HACE1 expression was associated with a significant decrease of apoptosis level and cell cycle arrest in G2/M phase. To conclude, our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and be downregulated by deacetylation and methylation of its promoter region chromatin constituting a potential target for HDAC inhibitors. Citation Format: Abdelilah Bouzelfen, Marion Alcantara, Hafid Kora, Philippe Bertrand, Sylvain Mareschal, Elodie Bohers, Catherine Maingonnat, Philippe Ruminy, Sahil Adriouch, Gaetan Riou, Martin Figeac, Thierry Fest, Christian Bastard, Hervé Tilly, Jean-Baptiste Latouche, Fabrice Jardin. HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis down-regulated by both deletion and epigenetic mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4947. doi:10.1158/1538-7445.AM2015-4947