Skin cancer is the most common cancer in the United States, with 5.5 million annual cases. It is strongly associated with ultraviolet (UV) radiation that generates many mutations. There is thus a need to investigate what mutations drive skin cancer. The CDKN2A gene encodes the p16 tumor suppressor protein, and silencing of the CDKN2A gene plays a key role in cancer progression. Our previous mouse study showed that chronic UV irradiation to mouse skin induced mutations at the mouse Cdkn2a/p16 promoter. However, the impact of the mutations at this promoter on cancer progression remained unclear. We hypothesized that these UV-induced mutations at the Cdkn2a/p16 promoter may inhibit the binding of transcription factors, thereby suppressing the expression of the p16 tumor suppressor and leading to cancer development. The mutations at the Cdkn2a/p16 promoter were found in the DNA sequence that was similar to the ETS transcription factor binding motif. Also, it has been shown that ETS2 is expressed in the skin. Thus, we tested whether mouse ETS2 binds to the mouse Cdkn2a/p16 promoter by measuring light signals from luciferase that is expressed under the control of the promoter. Overexpression of mouse ETS2 resulted in higher light signals than no overexpression control, indicating that ETS2 binds to the mouse Cdkn2a/p16 promoter. We also tested mutated promoters that carry the UV-induced mutations. With overexpression of mouse ETS2, the mutated Cdkn2a/p16 promoter showed lower light signals than wild-type promoter, implying that the mutation at the promoter inhibits the binding of ETS2 transcription factor to the promoter. Although further investigations are needed, these results suggest that UV-induced mutations affect the binding of transcription factors. This study highlights the importance of investigating promoter mutations that may contribute to cancer progression.

Merkel cell carcinoma (MCC) is a rare and aggressive cancer of the skin with a mortality rate of ~30%. In the US, most MCC tumors arise from integration of the Merkel cell polyomavirus (MCPyV) DNA into a host chromosome, leading to expression of viral T-Antigen (T-Ag) oncoproteins that drive tumorigenesis. Though current treatment options have significantly improved MCC prognosis, new therapies are needed to address recurrent/resistant disease. While T-Ag-specific antibodies are usually detected in the blood of patients with virus-driven MCC, the role of these antibodies in tumor immunity remains unclear. Here, we analyzed blood samples from 100 MCC patients prior to definitive treatment, 51 of whom had high titers of antibodies recognizing the T-Ags. These 51 high titer samples were assessed for binding across two domains of the T-Ag. Suprisingly, we found that patients who had high titers of antibodies binding both regions of the T-Ag had worse MCC control than patients whose antibodies predominantly bound one region (median PFS 5.5 vs. 14.2 months, p=0.003). These data suggest that careful mapping of circulating antibody reactivity to different regions of T-Ag can serve as a biomarker to identify high-risk patients for which a more aggressive treatment regimen is needed. Future work is also focused on understanding the immune response resulting in differential response to T-Ag domains.

Merkel cell carcinoma (MCC) is rare and aggressive skin cancer with high risk of metastasis. Developments of PD-1/PD-L1 immunotherapy has significantly improved treatment outcomes of metastatic MCC. Unfortunately, only 50% of patients demonstrate long-term responses. Previous studies by our lab have demonstrated that infiltration of CD8+ T cells in MCC tumors correlates to long-term responses. However, the impact of other innate immune cells and lymphocytes remains unclear. I hypothesize that the spatial interaction of pro-inflammatory cells (primarily T and B cells) associate with better outcomes in the absence of suppressive macrophages that disrupt this immunological crosstalk. To investigate this, I have been using QuPath, a spatial analysis software, to examine the microenvironments of 75 MCC tumors. These tumors have been stained with 18-marker antibody panel to identify subsets of B, T, and innate immune cells. Using QuPath, I have been quantifying different immune cell populations, as well as investigating their spatial relationship to each other. Using this data, I have been working with a PhD student and Dr. Paul Nghiem, to correlate this immunological data to clinical outcomes. Our preliminary data demonstrates that high B-cell infiltration may correlate with better clinical outcomes. While this is promising, I plan to expand my studies, investigating the impact of other innate immune cells on immunotherapy outcomes. I will then work to validate my studies by investigating a different cohort of 35 MCC tumors taken from a recent immunotherapy clinical trial. By investigating the tumor microenvironment, my goal is to help identify which population of cells associate with better responses. This in turn may lead to a diagnostic assay that may help assess cancer aggressiveness, which physicians can use to tailor treatment course. In addition, this information can help guide future drug development, showcasing which cells are the most important to target and/or enhance.

Merkel cell carcinoma (MCC), a rare and aggressive skin cancer, shows approximately 60% response to immune-checkpoint inhibitors (ICIs) in immunocompetent patients. The efficacy of ICIs in immunosuppressed patients, who generally have poorer MCC prognoses, is less clear. This retrospective study assesses ICI outcomes in both immunocompetent and immunosuppressed MCC patients, and across various immunosuppression types. In this project, I determined the cohort of 183 patients with advanced MCC who were treated with first-line ICIs from a Seattle-based data registry. I collected the following data from analyzing patients’ medical records: treatment response, immunosuppressive status, disease-specific and overall survival. I coordinated with the statisticians for Kaplan-Meier analyses of the data, and am now working on the manuscript. The results show that initial response rates to ICIs were comparable between immunosuppressed (50%) and immunocompetent (61.5%) patients (p=0.17). After starting ICI treatment, immunosuppressed patients experienced a 65% increased rate of disease progression compared to immunocompetent patients (Hazard Ratio [HR]=1.65, p=0.04), with their median time to disease progression being 11.2 months, versus 32.9 months in the immunocompetent group. Outcomes also varied by immunosuppression subtype, with chronic lymphocytic leukemia patients having the lowest chance of response (20%, 2/10) and highest progression risk. In our cohort, immunotherapy often elicited an initial response in immunosuppressed MCC patients; however, the duration of this response was significantly shorter. Despite limited duration in immunosuppressed patients, ICIs still offer a high response rate for patients with advanced MCC, regardless of immunosuppression type. It is crucial to have a comprehensive prognosis discussion with patients before initiating treatment.