Inter-patient and Intra-tumor Heterogeneity in the Sensitivity to Tumor-targeted Immunity in Colorectal Cancer

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1 54 Jpn. J. Clin. Immunol., 40 (1)54~59 (2017)C 2017 The Japan Society for Clinical Immunology The Memorial Thesis of the Best Poster Award (Recommended article) Recommender: Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Prof. Yutaka KAWAKAMI Inter-patient and Intra-tumor Heterogeneity in the Sensitivity to Tumor-targeted Immunity in Colorectal Cancer Tsubasa Miyauchi, Tomonori Yaguchi and Yutaka Kawakami Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine (Accepted January 17, 2017) summary Efficacy of immune checkpoint inhibitors such as PD-1 antibody for colorectal cancer remains to be proved except in microsatellite-instability-high (MSI-H) cases. While the objective response rate of MSI-H cases was 40%, that of microsatellite-stable (MSS) cases was 0%, showing that response rate to immune checkpoint inhibitors varies even among the microsatellite status. Some possible mechanisms that confer each patient variation in the response to immunotherapy should be considered. We focused on the combination of inter-patient heterogeneity and intra-tumor heterogeneity as a determining factor of immune reaction. An example of intra-tumor heterogeneity is the low expression of tumor antigen by CD271 + cells in melanoma. It is not surprising that similar mechanism exists in CRC. Other related intra-tumor heterogeneity includes EMT and autophagy, both molecular mechanisms that are thought to promote immune-evading phenotype. Besides the microsatellite status, inter-patient heterogeneity in response to tumor immunity includes hypermutator phenotype, which is driven by POLE mutation, intrinsic cytokine production, and microbiota in the gut. Key words colorectal cancer; tumor immunology; immune checkpoint inhibitors; tumor heterogeneity Background In colorectal cancer, prognostic value of immune cell infiltrate in the tumor tissue have been reported 1 3). Intensity of the levels of immune cell invasion influenced an outcome of not only immunotherapy but also chemotherapy and even surgery. These findings had made us believe that blockade of negative feedback pathways utilized by cancer cells to escape from host immunity, by means of anti-pd1 therapy for example, facilitate the immune cell infiltration leading to clinical benefits in colorectal cancer and many other types of cancer including melanoma, non-small cell lung cancer 4), Hodgkin s lymphoma 5, 6), head and neck squamous cell carcinoma 7) and bladder cancer 8). However, in colorectal cancer, unlike other types of cancer, only the patients deficient of mismatch repair (MMR) gene showed response to PD-1 antibody administration 9). MMR deficiency, which in turn bring microsatellite instability-high phenotype (MSI-H), was reported to be caused by Lynch syndrome, in which germline mutation of MMR on one allele followed by somatic inactivation of second allele, or sporadic cases which result from epigenomic silencing mainly following to BRAF mutation in serrated polyp 10, 11). It has been reported that pre-existing anti-tumor immune reactions greatly affects the sensitivity to anti-pd1 therapy 12). Therefore, since MSI-H colon cancer harbor larger numbers of immunogenic mutations and neo-antigens 2, 13), high responsiveness to immune checkpoint inhibitors could be plausible 9). However, statistical analysis of MSI-H status predicted clinical benefit of immune checkpoint blockade, not all the patients with MSI-H colorectal cancer responded to anti-pd1 therapy, and the disease progression of some MSS cases were slightly inhibited with administration of PD1 antibody 9) and little is known about the mechanisms underlying the heterogeneity of sensitivity to immune checkpoint inhibitors, within same microsatellite status. Indeed, even in MSI cases the response rate remains at 40%. In this review, we introduce possible mechanisms which may affect the sensitivity to tumor-targeted immunity and immune checkpoint inhibitors.

2 MIYAUCHI Heterogeneity in immune response to Colorectal Cancer 55 Inter-patient heterogeneity and intra-tumor heterogeneity involved in immune reaction against colorectal cancer. In this review, we focus on the tumor heterogeneity involved in tumor-targeted immune reaction. Genetic and phenotypic variations are observed among individuals with the same tumor type (inter-patients heterogeneity). Within a tumor, there is subclonal diversity of tumor cells (intra-tumor heterogeneity) 14). Inter-patient heterogeneity in tumor immunity result from genetic background involved in carcinogenesis, such as mismatch repair gene deficiency and POLE mutation, and personal disease-modifying factors, such as composition of intestinal bacterial flora 15, 16), smoking status and pre-existing disease. These factors of inter-patient heterogeneity were previously evaluated by analyzing therapeutic response data, clustering analysis preceded by DNA microarray analysis, and whole exome sequencing which revealed DNA mutation load found in each patient (Table 1). On the other hand, intra-tumor heterogeneity is caused by genetic instability and exogenous selection from treatment and focal immune reaction, forcing the tumor tissue to form subclones during cancer evolution. The resulting intra-tumor heterogeneity may be involved in varying levels of immune reaction against colorectal cancer. 1. Inter-patient heterogeneity which make a difference in intensity and profile of tumor-targeted immune reaction Hypermutator phenotype in CRC other than MMR gene deficiency found in a subset of CRC patients Many mechanisms of somatic hypermutation other than MMR deficiency such as mutations in the DNA polymerase-ε encoded by POLE and exposure to exogenous or endogenous mutagens, have been reported 17). However, the significance of these mechanisms in the context of the reactivity of immune checkpoint inhibitors remains to be clarified. Giannakis et al. studied colorectal cancer cases with POLE exonuclease domain mutations and reported a significantly higher neoantigen load in comparison to MSS cancers 18). Tumor microenvironment of endometrial cancer with POLE mutation or MSI were immunologically active and characterized by the abundance of tumor specific neo-antigens and high levels of TILs infiltration 19 21), leading to over-expression of PD-1 and PD-L1 19) in endometrial cancer. In colorectal cancer, Ahn reported that there were no significant difference in the number of TILs with cytotoxic T lymphocyte phenotype between POLE-mutant MSS and POLE-wild type MSS colorectal cancer 22). However the TILs were mostly positive for CD45RO and/or CD3 and some were PD1 positive. PD1 positive immune cells tended to accumulate greater in POLE-mutant colorectal cancer than POLE-wild type. Besides, recent studies demonstrated that a family of Table 1. Inter-patient heterogeneity which make a difference in intensity and profile of tumor-targeted immune reaction in CRC Mechanism of heterogeneity Roles in tumor immunity Clinical significance Mismatch repair deficiency Increased mutation including neo-antigens Favorable prognoses of MSI-H patients Higher immune cell infiltrates in MSI-H cases POLE mutation Increased mutation including neo-antigens Prognostic value and high sensitivity to immune checkpoint inhibitors never been proved yet High numbers of immune cell infiltrates observed in POLE-mt cases Chromosomal instability Intestinal bacterial flora Chromosomal deletion resulting in a particular cytokine depletion LOH may be possible mechanism of loosing the neoantigen-presenting machinery Epigenetic effects (CpG island methylator phenotype) on MLH1 silencing Treg and Th17 induction Decreased production of CXCL13 and IL15 resulting in weakened CXCR5 + Tfh and CD8 + T cells proliferation, associated with poor prognoses Loss of neoantigen-presenting mechanism may provide a chance of relapse Fusobacterium is preferentially found in MSI-H CRC

3 56 日本臨床免疫学会会誌 (Vol. 40 No. 1) APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing, could bring clusters of mutation in human tumors. In mice, Blanc et al. showed tumorigenic activities of APOBEC in rodent intestinal tumor 23). From TCGA database, Roberts et al reported the APOBEC mutation in various types of cancer, including bladder, cervical breast, head and neck, and lung cancer, but not in colorectal cancer 24), indicating that APOBEC in CRC remains controversial. Inter-patient difference in chromosomal instability and gene mutations which directly affect cytokine and chemokine production, and presentation of neo-antigens Bindia et al. reported that genomic instability of the CXCL13 was one mechanism, which determines the intensity of Tfh and B cell infiltration 25). CXCL13 and IL21 were pivotal factors for the Tfh/B cell axis that correlated with survival. Mlecnik et al. performed an analysis of chromosomal instability and gene expression of 59 cytokines and receptors in colorectal cancers. They revealed that metastatic patients showed high frequency of deletions of cytokines encoded on chromosome 4. One of the genes deleted was IL15 and its deletion corresponded with decreased IL15 expression, a higher risk of relapse, and worse prognosis 26). These findings demonstrate chromosomal instability as a mechanism in tumor that changes its cytokine production. E Tran et al. demonstrated the neo-antigen KRAS G12D was recognized by HLA-C*08:02-restricted tumor-infiltrating lymphocytes that were composed of multiple clones, each of which specifically targeted KRAS G12D 27). They also reported a case of HLA-C* 08:02 patients with 7 lung metastatic lesions of CRC treated by adopted immunotherapy using their KRAS G12D-directed HLA-C*08:02-restricted tumor-infiltrating lymphocytes. Although objective regression of all metastatic lesions was observed after the treatment, one of these lesions progressed 9 months after the therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I MHC. These findings showed inter-tumor heterogeneity formed by the loss of molecules needed for antigen presentation 28). 2. Intra-tumor heterogeneity which confer difference in sensitivity to tumor-targeted immunity Surface CD antigens for identifying certain subpopulation in cancer Intra-tumor heterogeneity has been demonstrated to depend on patterns of gene expression and methylation, rather than genetic alterations, leading to a novel classification of colorectal cancer that may affect future clinical practice 29). Phenotypic heterogeneity defined by surface antigens were reported in many types of cancer such as melanoma 30), but it has been controversial that whether a particular subpopulation harboring such immunological phenotypes affect the sensitivity to tumor-targeted immunity. Some CD antigens were reported for defining a particular characteristic subpopulation (Table 2). However, their properties were mainly elucidated not from the tumor-mesenchymal interaction but their genetic back- Table 2. CD antigens which form intra-tumor heterogeneity in CRC CD antigens Role in intra-tumor heterogeneity references CD24 CD26 CD44s CD44v6 CD44v9 CD133 CD151 CD166 CD110 CD271 CD318 CD326 (EpCAM) CSC: cancer stem cell, especially metastasis initiating cell marker, act as antioxidant mechanism, involved in Akt activation CD151 cells harboring metastatic potential used with CD44 and EpCAM Tendency to metastasize to liver, poor prognostic factor CD271 cells show high viability Tendency to metastasize to lung, poor prognostic factor Sahlberg et al., 2014 Pang et al., 2010 Dalerba et al., 2007 Todaro et al., 2014 Saya et al., 2011 Ricci-Vitiani et al., 2007 Kamijo et al., 2014 Semenza et al., 2008 Dalerba et al., 2007 Gao et al., 2013 Zuli yang et al., 2014 Gao et al., 2013 Dalerba et al., 2007

4 MIYAUCHI Heterogeneity in immune response to Colorectal Cancer 57 ground or epigenetic properties. Markers that have been described to identify colorectal cancer stem cells (CSCs) include EpCAM + /CD44 + /CD ), LGR5 + 32), and CD44v6 + 33). Some markers are associated with migration of cancer cells to distant organs and development of CRC metastasis, as confirmed by the inverse correlation between overall survival and the marker expression 33). For example, Gao et al. demonstrated that CD110 + and CDCP1 + cells were included in CD133 + subpopulation, which was reported as cancer stem cell marker in colorectal cancer, pancreatic adenocarcinoma and glioma, and seems to be preferential to metastasize to the liver and lung, respectively 34). Furthermore, some markers are associated with immunological phenotype of cancer cells, for example, in melanoma CD271 + subpopulation which are known as melanoma stem cell population, low expression of tumor antigens, MART-1, MAGE- C1 and thyrosinase, contribute to evasion from host s immunosurveillance 35). In 2015, Guinney, Vermeulen and Tejpar et al. analyzed across six independent classification systems and aggregated their data to re-classify into four consensus molecular subtypes (CMSs) with distinct features. Gene set mrna enrichment analysis revealed CMS1 subgroup characterized by hypermutation, microsatellite unstable, enrichment for BRAF mutation, and strong immune activation showed relatively low expression of caner stem cell-related genes 36). Although it remains unclear whether low expression of the CSCrelated genes itself contributes to immunogenic features, subpopulations in cancer tissue may have immunosuppressive effects on TILs. The surface markers which define the immunosuppressive subpopulation can be useful for both diagnosis and indication for immunotherapy. EMT and immune-resistance Epithelial-mesenchymal transition (EMT) is essential process for distant metastasis of cancer. During EMT, epithelial cells express mesenchymal genes, such as SNAIL and TWIST, that causes their phenotype to shift from epithelial to mesenchyme-like characteristics, allowing the cells to invade tumor stroma and vessels. Kudo-Saito et al. demonstrated that overexpression of SNAIL in cancer cells accelerates metastasis not only by enhanced invasion but also induction of immunosuppression 37) via production of CCL2 and subsequent induction of immunoregulatory dendritic cells (regdc) 38). However, in lung adenocarcinoma with EGFR mutation, immunohistochemical analysis revealed significant association between PD-L1 expression and EMT phenotype characterized by ZEB1, SNAIL, SLUG, or Vimentin expression. Additionally, the cases with EMT phenotype and PD-L1 expression showed the higher levels of PD-1 + cells and CD8 + T cells infiltration. This study suggested that patients with EGFR-mutant lung adenocarcinoma with EMT phenotype may benefit from PD-1/PD-L1 blockade therapy 39). Conclusion Variety in response to immune checkpoint inhibitors among patients with colorectal cancer may due to inter-patient heterogeneity and intra-tumor heterogeneity. Although the components of both heterogeneities influence each other, it is necessary to clarify the immunologic meaning of each component to reveal underlying mechanisms targeted for further immunotherapy. Recent comprehensive analysis by using large database such as TCGA is expected to highlight more axis to evaluate patients heterogeneity. Acknowledgement This work was supported by Grants-in-aid for Scientific Research ( and 15K09783) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, the Project for Cancer Research And Therapeutic Evolution (P-CREATE) (16cm h 0001) from Japan Agency for Medical Research and Development (AMED), and the research grants for life sciences and medicine from Keio University Medical Science Fund. We thank Mr. JeongHoon Park for preparation of the manuscript. References 1) Atreya, I., Neurath, M.F.: Immune cells in colorectal cancer: prognostic relevance and therapeutic strategies. Expert Rev Anticancer Ther. 8: , ) Galon, J., et al.: Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 313: , 2006.

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