1. Dr Céline Bossard Department of Pathology, University Hospital, Nantes celine.bossard@chu-nantes.fr Subordination of the neoplastic progression to the immune system

2. Tumor immunity : an old concept with new clinical applications Ehrlich in 1909 proposed this idea : « tumors are not entirely « self », and immune- mediated recognition of autologous tumor cells could be able to eliminate transformed cells » A concept formalized subsequently by Thomas and Burnett: « the immune surveillance » to refer the recognition and destruction of newly appearing tumor cells which are seen as foreign by the immune system But the development of cancer implies - an imperfect immune surveillance - mechanisms of tumor immune escape, representing potential new therapeutic targets (ex : anti PD-1, PD-L1 antibodies)

3. Clinical evidences for tumour immunity Spontaneous regression of melanoma associated with an in situ T cell clonal expansion (Ferradini et al, 1993) The increased risk of tumour development in immunosuppressed patients (primary or secondary immunodeficiencies) : - tumours of viral origin : > B-cell lymphoma induce by Epstein Barr Virus, > Kaposi’s sarcoma and HHV8, > carcinoma of the anogenital regions and HPV - tumours with no known viral etiology : > melanoma, sarcoma, lung adenocarcinoma…. The improved prognosis of tumours containing a high number of tumour- infiltrating lymphocytes (TIL: T cells, NK cells) : > melanoma (identification of various melanoma-specific Ag) > colon, breast, ovarian adenocarcinomas, head and neck carcinomas…..

4. The tumor microenvironment contains more or less immune cells Stroma : a complex microenvironnement composed of : - blood and lymphatic vessels - an inflammatory milieu consisting of immune cells (of the innate and adaptive immunity) and their secretory products  type, density and location of immune cells varie between each tumour and influence tumour progression differently Immune cells at the invasive margin of the tumor In the center, in the stroma In close contact with tumor cells

5. Immune cells in close contact with tumour cells (intra-epithelial TIL)

6. The major effector cell in anti-tumor immunity: the Cytotoxic T Lymphocyte (CTL) Cell mediated immunity is the major anti tumor mechanism

7. Prognostic Value of Tumor Infiltrating Lymphocytes in the Vertical Growth Phase of Primary Cutaneous Melanoma Claudio G. Clemente et al, 1996 High number of TIL Low number of TIL  Presence of high number of TIL is a positive prognostic factor

8. Intratumoral T Cells, Recurrence, and Survival in Epithelial Ovarian Cancer Lin Zhang et al, 2003, N Eng J Med CD8 immunostaining : intra- tumoral and peritumoral CD8+ T cells The presence of intratumoral T cells correlated with delayed recurrence or delayed death

9. Type, Density, and Location of Immune Cells Within Human Colorectal Tumors Predict Clinical Outcome. J Galon et al, 2006  Patients with a homogeneous increased expression of genes for Th1 adaptive immunity (CD3, CD8, GrB, IFNg) in the tumour have a better prognosis.

10. Type, Density, and Location of Immune Cells Within Human Colorectal Tumors Predict Clinical Outcome. J Galon et al, 2006 Bossard et al, 2012 Int J Cancer The high density of CD3+ TIL is associated with a better prognosis, whatever their localization Center of the tumour Invasive margin CD8+ IEL-TIL

11. ….T cells infiltrate has a prognostic discriminatory power that is superior to the standard TNM staging system…. The immune score as a new possible approach for the cancer staging in colorectal carcinomas Galon et al, 2012 J Translational Medicine

12. NK cells : an effector cell of the innate immunity Intra-epithelial CD244+ NK cells in colorectal adenocarcinoma C Bossard, 2012, Int J Cancer They may provide the first line of defense agains tumor cells NK cells can lyse a wide range of human tumors, including some tumors that seem to be nonimmunogenic for T cells - tumours that fail to express MHC class I Ag cannot be recognized by CTL, but by NK cells that destroy cells without normal expression of MHC class I Ag Some studies have demonstrated the favorable prognostic impact of NK cells infiltration in various type of tumors : - colorectal cancer (Coca et al, Cancer, 1997) - gastric cancer (Ishigami et al, Cancer Lett, 2000)

13. Tumor antigens Classified depending on their molecular structure and source They can induce or not a specific immune response

14. 1 - Products of mutated oncogenes and tumor suppressor genes Neoplastic transformation results from genetic alterations, and some of them may lead to the expression of cell surface Ag (with known or unknown function), seen as non-self by the immune system. Tumor-specific Ag (p53, RAS, BRAF, b-catenin, and many others not related to the transformed phenotype as in MSI tumors….) not expressed by normal cells Shared by many different tumors Recognized as non-self proteins by the immune system These self-mutant proteins can induce specific CTL, but these CTL don’t elicit a protective response

15. The product of an unmutated oncogene is overexpressed in a subset a breast cancers : HER2/NEU oncogene Can be assessed by immunohistochemistry and/or FISH The target of a monoclonal antibody : Trastuzumab (Herceptin)

16. 2 - Overexpressed or aberrantly expressed normal proteins Structurally normal protein overexpressed in tumor cells = normal self-Ag - ex : tyrosinase in melanoma cell (expressed at a low level only in normal melanocyte) > T cells in patients with melanoma recognize peptides derived from tyrosinase > development of tyrosinase vaccine (clinical trials ongoing)

17. Cancer-testis antigen: Ag only expressed in testis but without antigenic expression at the cell surface (no MHC class I expression by sperm !), but with deregulated expression in tumor cells > A tumor-specific Ag ex : the MAGE family of genes (Melanoma Antigen Gene); MAGE Ag are found in melanoma, lung, liver, stomach….. > several MAGE Ag are used in tumor vaccine trials

18. Tumor antigen produced by oncogenic viruses Somes virus are associated with cancers (HPV and cervix, HBV, HCV and hepatocarcinoma, EBV and lymphomas….) Produced proteins are recognized as foreign and induced a strong CTL response (DNA viruses +++) > vaccines against HPV Ags are effective to prevent cervical cancer +++

19. Oncofetal/embryonic antigens Ag only expressed during the embryogenesis but not in normal adult tissues ex : CEA (carcinoembryonic Ag in colon cancer) and α foetoprotein in liver cancer > used as serum markers of cancer

20. Altered cell surface glycolipids or glycoproteins Most human tumors expressed higher than normal levels and/or altered forms of glycolipids or glycoproteins Can be used as diagnostic markers and/or targets for therapy These molecules include gangliosides, mucins, bloog group Ag ex : CA-125, CA 19-9, MUC-1

21. Cell type-specific differentiation antigens Differentiation Ag are specific molecules expressed in particular lineages or at a differentiation stage of various cell types = normal self Ag Tumor cell can express a differentiation Ag normaly expressed by the normal cell counterpart They do not induce immune response > use for identifying the tissue of origin for a morphologically undifferentiated tumor (IHC) > use as a target for immunotherapy : anti-CD20 antibody (Rituximab) used in B-cell lymphomas expressing CD20 as normal B lymphocytes

22. How do tumor cells escape from the immune system in immunocompetent hosts? The immune evasion by tumor

23. Dendritic cell T cells Tumor cell MHCTCR B7 CD28 CTLA-4 Inhibitory signal Activation signal Peripheral tissueLymph node TCR MHC class I Negative regulation From Ribas A in N Eng J Med, 2012:366;26 PD-1 Several escape mechanisms have been proposed X X PD-L1 PD-L2 ✔ TGFb

24. CD94 HLA-E/β2m – HLA-E/β2m + P=0.02 Overexpression of HLA-E/β2m by tumour cells in CRC is associated with a high number of inhibitory CD94/NKG2 +IEL-TIL, and with a poor prognosis HLA-E NK cell and CTL Tumour cell β2M LYSIS Bossard et al, Int J Cancer, 2012

25. Conclusion Type, density and location of immune cells within a tumour can predict the prognosis in some tumours > proposition of an « immune score » for the classification of cancer Host immune cells have essential roles in regulating tumour growth in the tumour microenvironment. Host immune cells provide a great opportunity for therapeutic and preventive interventions > need to boost the effective and specific anti-tumor immune response, and to inhibit the mechanisms of tumor immune evasion