ITEM: Dissecting phenotypic heterogeneity of human melanoma: building a rationale for active immunotherapies

Tumor cells may differ within a cancer patient. This tumor heterogeneity is commonly seen in metastatic melanoma and likely plays a major role in resistance to therapy, immunotherapy included. In fact, during immunotherapy, many patients experience "mixed response", with some tumor lesions regressing and other ones progressing. Current immunotherapies induce, in most patients, immune response against the more common and immunogenic differentiated tumor cell variants, not being likely able to "see" antigens expressed by "stem cell-like" ones. This may lead to cancer relapse. The aim of this project was to identify new target antigens for immunotherapy and biomarkers for clinical outcome. We have analysed material from approximately 90 melanoma patients that have received immunotherapy. Biopsies have been analysed for gene expression, and at protein level. We have also analysed data from databases. We have assessed if specific proteins may be good therapeutic targets. Regarding biomarkers, we have focused on immune-gene-profiles in tumor. The results reveal that the patients have very different immune profiles. We have categorized them into immune-defined subgroups and also developed a draft immune-score. Based on the results, we have initated further studies aimed at establishing biomarkers for clinical outcome. These biomarkers may be used for improved and personalised therapy. We have identified antigens expressed in melanoma cell populations, that may be good targets for immunotherapy against melanoma, and may allow for therapy that overcome cancer cells that are resistant to current treatment. Further work is required to validate these targets and their potential clinical value. The project is a collaboration between research groups in Italy, France, the Netherlands and Norway.

Prosjektet har gitt informasjon om nye aktuelle angrepspunkter for immunterapi, noe som særlig kan være viktig for pasienter som ikke responderer på dagens behandling. Videre har prosjketet gitt ny informasjon om immun-genprofil i melanomsvulster og deres relasjon til klinisk effekt av immunterapi. Denne informasjonen kan benyttes til utvikling av biomarkører, som kan forutsi sjanse for effekt og bivirkniger av immunterapi, og dermed benyttes til persontilpasset behandling. Vi har også sammenlignet immun-gen-profil i ulike svulster fra samme pasient, noe som er viktig for å bestemme om pasientene må utsettes for ny biopsitaking, eller om gamle biopsier kan brukes til valg av behandling.

Inter- and intralesional tumor heterogeneity is commonly seen in metastatic melanoma, likely playing a major role in resistance to therapy, immunotherapy included. In fact, during immunotherapy, many patients experience "mixed response", with some tumor lesions regressing and other ones progressing. Whereas vaccines targeting a few antigens can "passively" select for antigen-negative tumor cells, it is not fully elucidated why even broader spectrum immunotherapies (whole tumor vaccines or immunomodulating antibodies) fail in clearing the tumor in most cases. Both lack of effectiveness and late tumor relapse after immunotherapy might be due to the presence, in the tumor bulk, of tumor cells with stemness properties and a phenotype different from their differentiated counterpart. These variants are less represented in most melanoma patients and less immunogenic, being hardly targeted by specific immunity. Moreover, it has been recently shown that tumor-specific cytotoxic T lymphocytes (CTL) induce dedifferentiation of melanoma cells, lending further support to this hypothesis. Current immunotherapies induce, in most patients, immune response against the more common and immunogenic differentiated tumor cell variants, not being likely able to "see" antigens expressed by "stem cell-like" ones. Moreover, DC vaccines loaded with tumor lysate elicit immunity against the pretherapy tumor tissue, not taking into account dynamic changes induced by the treatment itself on tumor cells. This research project aims to identify, by RNA sequencing of biopsies and single melanoma cells, genes expressed by distinct melanoma and stromal/immune cell subpopulations and to validate their encoded proteins as antigenic targets. The identification of these targets and biomarkers will ultimately allow designing new and more effective immunotherapeutic approaches that, by concurrently targeting the different melanoma cell variants, will reduce both primary resistance to therapy and late relapse after initial response.