T cells expressing antigen-specific T-cell receptors (TCRs) can mediate effective tumor regression, but they often also are accompanied by autoimmune responses. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo , we used a unique self-antigen system comprising seven human melanoma gp100(209–217)-specific TCRs spanning physiological affinities (1–100 μM). We found that in vitro and in vivo T-cell responses are determined by TCR affinity, except in one case that was compensated by substantial CD8 involvement. Strikingly, we found that T-cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 µM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Together, these results suggest that a relatively low-affinity threshold is necessary for the immune system to avoid self-damage, given the close relationship between antitumor activity and autoimmunity. The low threshold, in turn, indicates that adoptive T-cell therapy treatment strategies using in vitro-generated high-affinity TCRs do not necessarily improve efficacy.

Background Identification of primary melanoma patients at the highest risk of recurrence remains a critical challenge, and monitoring for recurrent disease is limited to costly imaging studies. We recently reported our array-based discovery of prognostic serum miRNAs in melanoma. In the current study, we examine the clinical utility of these serum-based miRNAs for prognosis as well as detection of melanoma recurrence. Methods Serum levels of 12 miRNAs were tested using qRT-PCR at diagnosis in 283 melanoma patients (training cohort n=201, independent validation n=82; median FU 68.8 months). A refined miRNA signature was chosen and evaluated. We also tested the potential clinical utility of the miRNAs in early detection and monitoring of recurrence using multiple longitudinal samples (pre- and post-recurrence) in a subset of 82 patients (n=225). In addition, we integrated our miRNA signature with publicly available TCGA data to examine the relevance of these miRNAs to melanoma biology. Results Four miRNAs (miR-150, miR-30d, miR-15b, and miR-425) in combination with stage separated patients by recurrence-free and overall survival and improved prediction of recurrence over stage alone in both training and validation cohorts (training RFS and OS p<0.001, validation RFS p<0.001, OS p=0.005). Serum miR-15b levels significantly increased over time in recurrent patients (p<0.001), adjusting for endogenous controls as well as age, gender, and initial stage. In non-recurrent patients, miR-15b levels were not significantly changed with time (p=0.17). Conclusion Data demonstrate that serum miRNAs can improve melanoma patient stratification over stage and support further testing of miR-15b to guide patient surveillance.

Previous reports have demonstrated a role for hedgehog signaling in melanoma progression, prompting us to explore the therapeutic benefit of targeting this pathway in melanoma. We profiled a panel of human melanoma cell lines and control melanocytes for altered expression of hedgehog pathway members and determined the consequences of both genetic and pharmacological inhibition of the hedgehog pathway activator Smoothened (SMO) in melanoma, both in vitro and in vivo. We also examined the relationship between altered expression of hedgehog pathway mediators and survival in a well-characterized cohort of metastatic melanoma patients with prospectively collected follow up information. Studies revealed that over 40% of the melanoma cell lines examined harbored significantly elevated levels of the hedgehog pathway mediators SMO, GLI2, and PTCH1 compared to melanocytes (p < 0.05). SMO inhibition using siRNA and the small molecule inhibitor, NVP-LDE-225, suppressed melanoma growth in vitro, particularly in those cell lines with moderate SMO and GLI2 expression. NVP-LDE-225 also induced apoptosis in vitro and inhibited melanoma growth in a xenograft model. Gene expression data also revealed evidence of compensatory up-regulation of two other developmental pathways, Notch and WNT, in response to hedgehog pathway inhibition. Pharmacological and genetic SMO inhibition also downregulated genes involved in human embryonic stem cell pluripotency. Finally, increased SMO expression and decreased expression of the hedgehog pathway repressor GLI3 correlated with shorter post recurrence survival in metastatic melanoma patients. Our data demonstrate that hedgehog pathway inhibition might be a promising targeted therapy in appropriately selected metastatic melanoma patients.

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