Dottorato in INNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Titolo della tesi: Immune profile of cancer patients to improve selection and efficacy of immunotherapeutic strategies

Over the years, the clinical outcome of cancer patients has remarkably improved with the introduction of immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs), that by targeting immune system restore an efficient anti-tumor immune response. Despite the potential of immunotherapy as cancer leading treatment, initial response rates with ICIs are however limited and depend on the host pre-existing anti-cancer immunity and the degree of immunosuppression present in the patient. Most of patients fail to respond and to increase the number of responder patients is necessary a more in-depth understanding of the underlying immunity and the identification of biomarkers. In this research project we investigate and characterize the immune system of cancer patients (mRCC, NSCLC, HNSCC, UM) before and during treatment with TKIs or ICIs, in order to investigate the relation between the circulating immune profile, the tumor microenvironment (TME), the gut microbiome and clinical outcome. The aim was the identification of possible biomarkers/immune profile able to select patients and improve clinical outcome. We assessed immunological analysis to evaluate exhausted/activated circulating T cells by cytofluorimetric assay, 14 immune checkpoint-related proteins and 20 inflammation cytokines/chemokines using Luminex assay. The immunological profile was correlated with survival (PFS and OS), clinical parameters and response to treatment. Gut microbiota composition was evaluated through metagenomic analysis and immunohistochemistry was used to characterize tumor microenvironment. Our results demonstrated that TKIs and ICIs modulate immune system. We observed a decrease of soluble immune checkpoint molecules in serum of mRCC (sPDL2, sHVEM, sPD1, sGITR) and NSCLC patients (sPD1, sPDL2) during treatment (p<0.05). In particular, the decrease of sPD1 and sPDL2 resulted associated with response to treatments (p=0.03 and p=0.01, respectively). Moreover, the immune profile of responder (R) patients was characterized by low levels of soluble protein (sCTLA4, sIFNY, sPD-L1 in mRCC vs sCD137, sTIM3, sPDL1, sPDL2 in NSCLC) (p<0.05) and by a high proportion of eubiosis-associated gut metabolites. These data resulted also associated with better clinical status, PS=0 (performance status). Profiling circulating immune cells in patients undergoing ICI treatment (anti-PD1) we identified CD3+CD137+ and CD3+CD8+CD137+ T cells that correlate with improved response to therapy. The percentage of both CD3+CD137+ and CD3+CD8+CD137+ T population was higher in R patients (p=0.03 and p=0.02) and correlated to a better survival in terms of PFS and OS (p<0.05). Moreover, R patients had higher levels of CD3+CD137+PD1+ and of CD8+CD137+PD1+ lymphocytes (p=0.02 and p=0.01), but only CD3+CD137+PD1+ resulted associated with a low number of metastasis (p=0.01) and longer survival (OS)(p=0.015). Instead, the high concentration of the immunosuppressive sCD137 in the serum was associated with a lower PFS (p=0.038) and OS (p=0.012). In tumor microenvironment, patients with a complete clinical response showed a high percentage of CD137+ and CD8+ T cells. Results were validated in an independent cohort of metastatic cancer patients. These results identified immunological parameters that, independently from tumor setting and administered therapy, predict clinical outcome of cancer patients, monitor immune response and help clinicians in the decision-making.