Abstract B03: Molecular profiling of immune activation associated with melanoma regression induced by diphencyprone
Authors: Gulati Nicholas , Coit Daniel G. , Fuentes-Duculan Judilyn , Gilleaudeau Patricia , Sullivan-Whalen Mary , Suárez-Fariñas Mayte , Krueger James G. 
Published: July 2015

Abstract
Topical diphencyprone (DPCP), a hapten that causes delayed-type hypersensitivity reactions, has been used to treat cutaneous metastases in melanoma patients, with an 84% regression rate and minimal side effects observed in a 50-patient case series. However, the immunological mechanisms underlying these cases of regression are not well understood. Here we report the case of a 91-year-old female with a history of primary melanoma treated by excision, followed by several recurrences initially responsive to imiquimod, presenting to us with two unresponsive, unresectable cutaneous melanoma metastases on her left lower leg. After 14 weeks of topical DPCP application twice weekly, the patient's melanoma lesions first became very inflamed and then were no longer clinically visible, and we confirmed the absence of melanoma by analysis of biopsy tissue. Immunohistochemistry for Melan-A as well as qRT-PCR analysis of various melanocyte/melanoma markers both demonstrated high levels in the pre-treatment melanoma biopsy, which returned to peri-lesional (non-melanoma) skin levels following DPCP treatment. The patient remains well five months after finishing her DPCP treatment regimen with no cutaneous metastases visible. To better ascertain the mechanisms involved in immune-mediated tumor regression, we studied biopsy tissues at various time points. Previous work from our group has demonstrated that gene expression markers of all major T cell subsets (Th1, Th2, Th9, Th17, Th22, and regulatory T cells) increase at 3 days after a single application of DPCP in healthy volunteers. However, this previous report did not examine the effects of repeated applications of DPCP, as is traditionally required when DPCP is used as a treatment (for clinical conditions that include alopecia areata and warts in addition to melanoma). In our current study, we noted that DPCP led to extensive immune cell infiltrates (by H&E staining as well as immunohistochemistry for CD3, CD11c, and CD163), both after a single and repeated applications of DPCP. We found that the qRT-PCR measures of various Th1-related genes (IFNG, IL12p35, IL12p40, IL12RB1, CXCL9, CXCL10, and CXCL11) increased over time whereas Th2 T cell (IL4, IL5, and IL13) and regulatory T cell (Foxp3) markers increased initially but then decreased. Other T cell subsets [Th9 (IL9), Th17 (IL17A), and Th22 (IL22)] were also increased following a single application of DPCP but decreased with chronic application. In contrast, IL24 increased further with chronic DPCP application. This overall shift towards Th1 polarization and IL24 induction would be expected to promote anti-neoplastic effects, and reaffirms the utility of repeated DPCP applications in melanoma treatment. Another potential anti-neoplastic effector we found to be increased upon DPCP application was granulysin, which co-localized with NKp46+ natural killer cells more than CD8+ cytotoxic T cells by two-color immunofluorescence. To further examine the differences between a single and repeated applications of DPCP, we compared our microarray data from the peri-lesional site 3 days after the first application of DPCP with the peri-lesional site on the last day of DPCP treatment (day 98, after 14 weeks of twice weekly applications). The genes uniquely upregulated at day 98 included natural killer cell markers such as granzyme H, and we histologically confirmed the presence of increased natural killer cells at this time point compared to day 3. Overall, here we provide comprehensive histological and gene expression profiling of a case of successful immune-mediated melanoma metastasis regression induced by DPCP. Our study of several biopsies over time revealed shifts in T cell polarization that could have important implications for cancer immunotherapy. Future study of additional patients (both responders and non-responders to DPCP) will likely provide valuable insight into the roles played by different immune cell subsets in promoting tumor regression.
Citation Format: Nicholas Gulati, Daniel G. Coit, Judilyn Fuentes-Duculan, Patricia Gilleaudeau, Mary Sullivan-Whalen, Mayte Suárez-Fariñas, James G. Krueger. Molecular profiling of immune activation associated with melanoma regression induced by diphencyprone. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B03.