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Abstract Objectives To investigate current practices and attitudes regarding use of adjuvant immunotherapy and prognostic gene expression profile (GEP) testing among melanoma medical and surgical oncologists. Methods An anonymous RedCap‐based survey was emailed to ~300 melanoma experts. Results Respondents generally favored adjuvant immunotherapy over observation (73% for all Stage IIIA, 50% for Stage IIB/IIC) and cited a minimum 10‐year recurrence risk of 11%–20% (48%) or 21%–30% (33%) to justify treatment, but acknowledged that risks of serious adverse events may outweigh potential benefits for some Stage IIB/IIC patients. While GEP test results did not strongly influence decision‐making regarding follow‐up or intervention, most were receptive to randomized trials using GEP testing to identify subsets of Stage IIB/IIC (74%) and Stage IB/IIA (54%) patients who may not or may, respectively, benefit from adjuvant therapy. Conclusion Although most respondents do not routinely use GEP testing, many would participate in clinical trials to determine clinical utility.

Abstract Background Multiple primary melanoma (MPM) is known to be associated with familial melanoma. However, the association between MPM and other personal and familial cancers is not well documented. The objective of this study was to evaluate the association between MPM and personal history of other cancers or cancer history among first‐degree relatives (FDRs). Methods We performed a retrospective case–control study including cases with gender‐matched MPM and single primary melanoma (SPM) at a 1:2 ratio from the University of Pittsburgh Cancer Institute Melanoma Center Biological Sample and Nevus Bank. The associations between MPM and other cancers were evaluated using univariable and multivariable logistic regression models. Results In total, 378 patients (44.2% men; median age 52 years) were enrolled, including 252 with SPM and 126 with MPM. In comparison to patients with SPM, patients with MPM were more likely to have squamous cell carcinoma (odds ratio [OR] 1.95, 95% confidence interval [CI] 1.001–3.79, p = 0.047) and prostate cancer (OR 2.72, 95% CI 1.07–7.01, p = 0.034). FDRs of patients with MPM had higher prevalence of melanoma (OR 2.37, 95% CI 1.31–4.28, p = 0.004) and prostate cancer (OR 2.92, 95% CI 1.47–6.14, p = 0.002) but not other cancers. In multivariable analysis, the association remained significant between MPM and squamous cell carcinoma (OR 2.18, 95% CI 1.08–4.39, p = 0.028), prostate cancer (OR 2.85, 95% CI 1.09–7.54, p = 0.032), FDR history of melanoma (OR 2.37, 95% CI 1.31–4.29, p = 0.004), and FDR history of prostate cancer (OR 3.26, 95% CI 1.59–6.83, p = 0.001). Conclusions Patients with MPM have a higher prevalence of personal and FDR histories of nonmelanoma skin cancers and prostate cancer.

BackgroundPre-clinical studies have shown that metformin reduces intratumoral hypoxia, improves T-cell function, and increases sensitivity to PD-1 blockade, and metformin exposure has been associated with improved clinical outcomes in various types of cancer. However, the impact of this drug in diabetic melanoma patients has not yet been fully elucidated.MethodsWe reviewed 4,790 diabetic patients with stage I-IV cutaneous melanoma treated at the UPMC-Hillman Cancer Center and Memorial Sloan Kettering Cancer Center between 1996-2020. The primary endpoints included recurrence rates, progression free survival (PFS), and overall survival (OS) with and without metformin exposure. Tabulated variables included BRAF mutational status, immunotherapy (IMT) by type, and incidence of brain metastases.ResultsThe five-year incidence of recurrence in stage I/II patients was significantly reduced with metformin exposure (32.3% vs 47.7%, p=0.012). The five-year recurrence rate for stage III patients was also significantly reduced (58.3% vs 77.3%, p=0.013) in the metformin cohort. OS was numerically increased in nearly all stages exposed to metformin, though this did not reach statistical significance. The incidence of brain metastases was significantly lower in the metformin cohort (8.9% vs 14.6%, p=0.039).ConclusionThis is the first study to demonstrate significantly improved clinical outcomes in diabetic melanoma patients exposed to metformin. Overall, these results provide further rationale for ongoing clinical trials studying the potential augmentation of checkpoint blockade with metformin in advanced melanoma.

BackgroundProinflammatory chemokines/cytokines support development and maturation of tertiary lymphoid structures (TLS) within the tumor microenvironment (TME). In the current study, we sought to investigate the prognostic value of TLS-associated chemokines/cytokines (TLS-kines) expression levels in melanoma patients by performing serum protein and tissue transcriptomic analyses, and to then correlate these data with patients clinicopathological and TME characteristics.MethodsLevels of TLS-kines in patients’ sera were quantitated using a custom Luminex Multiplex Assay. The Cancer Genomic Atlas melanoma cohort (TCGA-SKCM) and a Moffitt Melanoma cohort were used for tissue transcriptomic analyses. Associations between target analytes and survival outcomes, clinicopathological variables, and correlations between TLS-kines were statistically analyzed.ResultsSerum of 95 patients with melanoma were evaluated; 48 (50%) female, median age of 63, IQR 51-70 years. Serum levels of APRIL/TNFSF13 were positively correlated with levels of both CXCL10 and CXCL13. In multivariate analyses, high levels of serum APRIL/TNFSF13 were associated with improved event-free survival after adjusting for age and stage (HR = 0.64, 95% CI 0.43-0.95; p = 0.03). High expression of APRIL/TNFSF13 tumor transcripts was significantly associated with improved OS in TCGA-SKCM (HR = 0.69, 95% CI 0.52-0.93; p = 0.01) and in Moffitt Melanoma patients (HR = 0.51, 95% CI: 0.32-0.82; p = 0.006). Further incorporation of CXCL13 and CXCL10 tumor transcript levels in a 3-gene index revealed that high APRIL/CXCL10/CXCL13 expression was associated with improved OS in the TCGA SKCM cohort (HR = 0.42, 95% CI 0.19-0.94; p = 0.035). Melanoma differentially expressed genes positively associated with high APRIL/CXCL10/CXCL13 tumor expression were linked to tumor infiltration by a diverse array of proinflammatory immune cell types.ConclusionSerum protein and tumor transcript levels of APRIL/TNFSF13 are associated with improved survival outcomes. Patients exhibiting high coordinate expression of APRIL/CXCL10/CXCL13 transcripts in their tumors displayed superior OS. Further investigation of TLS-kine expression profiles related to clinical outcomes in larger cohort studies is warranted.

Abstract Background The effectiveness of MAPK pathway inhibitors (MAPKi) used to treat patients with BRAF-mutant melanoma is limited by a range of resistance mechanisms, including soluble TNF (solTNF)-mediated NF-kB signaling. solTNF preferentially signals through type-1 TNF receptor (TNFR1), however, it can also bind to TNFR2, a receptor that is primarily expressed on leukocytes. Here, we investigate the TNFR2 expression pattern on human BRAFV600E+ melanomas and its role in solTNF-driven resistance reprogramming to MAPKi. Methods Flow cytometry was used to test TNFR1, TNFR2 and CD271 expression on, as well as NF-kB phosphorylation in human BRAF-mutant melanoma. The ability of melanoma cell lines to acquire MAPKi resistance in response to recombinant or macrophage-derived TNF was evaluated using the MTT cytotoxicity assay. Gene editing was implemented to knock out or knock in TNF receptors in melanoma cell lines. Knockout and knock-in cell line variants were employed to assess the intrinsic roles of these receptors in TNF-induced resistance to MAPKi. Multicolor immunofluorescence microscopy was utilized to test TNFR2 expression by melanoma in patients receiving MAPKi therapy. Results TNFR1 and TNFR2 are co-expressed at various levels on 4/7 BRAFV600E+ melanoma cell lines evaluated in this study. In vitro treatments with solTNF induce MAPKi resistance solely in TNFR2-expressing BRAFV600E+ melanoma cell lines. TNFR1 and TNFR2 knockout and knock-in studies indicate that solTNF-mediated MAPKi resistance in BRAFV600E+ melanomas is predicated on TNFR1 and TNFR2 co-expression, where TNFR1 is the central mediator of NF-kB signaling, while TNFR2 plays an auxiliary role. solTNF-mediated effects are transient and can be abrogated with biologics. Evaluation of patient specimens indicates that TNFR2 is expressed on 50% of primary BRAFV600E+ melanoma cells and that MAPKi therapy may lead to the enrichment of TNFR2-expressing tumor cells. Conclusions Our data suggest that TNFR2 is essential to solTNF-induced MAPKi resistance and a possible biomarker to identify melanoma patients that can benefit from solTNF-targeting therapies.

Abstract Background We hypothesized that a gender difference in clinical response may exist to adjuvant CTLA4 blockade with ipilimumab versus high-dose IFNα (HDI). We investigated differences in candidate immune biomarkers in the circulation and tumor microenvironment (TME). Patients and methods This gender-based analysis was nested within the E1609 trial that tested adjuvant therapy with ipilimumab 3 mg/kg (ipi3) and 10 mg/kg (ipi10) versus HDI in high risk resected melanoma. We investigated gender differences in treatment efficacy with ipi3 and ipi10 versus HDI while adjusting for age, stage, ECOG performance (PS), ulceration, primary tumor status and lymph node number. Forest plots were created to compare overall survival (OS) and relapse free survival (RFS) between ipi and HDI. Gene expression profiling (GEP) was performed on tumors of 718 (454 male, 264 female) patients. Similarly, serum and peripheral blood mononuclear cells (PBMC) samples were tested for soluble and cellular biomarkers (N = 321 patients; 109 female and 212 male). Results The subgroups of female, stage IIIC, PS = 1, ulcerated primary, in-transit metastasis demonstrated significant improvement in RFS and/or OS with ipi3 versus HDI. Female gender was significant for both OS and RFS and was further explored. In the RFS comparison, a multivariate Cox regression model including significant variables indicated a significant interaction between gender and treatment (P = 0.024). In peripheral blood, percentages of CD3+ T cells (P = 0.024) and CD3+ CD4+ helper T cells (P = 0.0001) were higher in females compared to males. Trends toward higher circulating levels of IL1β (P = 0.07) and IL6 (P = 0.06) were also found in females. Males had higher percentages of monocytes (P = 0.03) with trends toward higher percentages of regulatory T cells (T-reg). Tumor GEP analysis supported enhanced infiltration with immune cells including gammadelta T cells (P = 0.005), NK cells (P = 0.01), dendritic cells (P = 0.01), CD4+ T cells (P = 0.03), CD8+ T cells (P = 0.03) and T-reg (P = 0.008) in the tumors of females compared to males and a higher T-effector and IFNγ gene signature score (P = 0.0244). Conclusion Female gender was associated with adjuvant CTLA4 blockade clinical benefits and female patients were more likely to have evidence of type1 immune activation within the TME and the circulation. Trial registration ClinicalTrials.gov NCT01274338. Registered 11 January 2011, https://www.clinicaltrials.gov/ct2/show/NCT01274338

The documentation of the change in the number and appearance of pigmented cutaneous lesions over time is critical to the early detection of skin cancers and may provide preliminary signals of efficacy in early-phase therapeutic prevention trials for melanoma. Despite substantial progress in computer-aided diagnosis of melanoma, automated methods to assess the evolution of lesions are relatively undeveloped. This report describes the development and narrow validation of mathematical algorithms to register nevi between sequential digital photographs of large areas of skin and to align images for improved detection and quantification of changes. Serial posterior truncal photographs from a pre-existing database were processed and analyzed by the software, and the results were evaluated by a panel of clinicians using a separate Extensible Markup Language‒based application. The software had a high sensitivity for the detection of cutaneous lesions as small as 2 mm. The software registered lesions accurately, with occasional errors at the edges of the images. In one pilot study with 17 patients, the use of the software enabled clinicians to identify new and/or enlarged lesions in 3‒11 additional patients versus the unregistered images. Automated quantification of size change performed similarly to that of human raters. These results support the further development and broader validation of this technique.

BackgroundPreclinical and translational evidence suggest BRAF/MEK inhibitors modulate the tumor microenvironment (TME), providing rationale for combination with immunotherapy.MethodsThis investigator-initiated, phase I trial evaluated pembrolizumab, vemurafenib, and cobimetinib in patients with untreated, BRAFV600E/K mutant advanced melanoma. The first 4 patients received vemurafenib with pembrolizumab, and the next 5 patients received vemurafenib and cobimetinib with pembrolizumab. Primary endpoints: safety and maximum tolerated dose of the triplet.Secondary endpointsobjective response rate (ORR), progression-free survival (PFS), overall survival (OS), and quality of life (QoL). The trial was closed after enrollment of 9 (planned 30) patients due to dose-limiting toxicity (DLT). Study NCT02818023 was approved by the IRB, and all patients provided informed consent.ResultsPatients received a median of 6 cycles of therapy. 8 of 9 experienced drug-related grade 3/4 AEs. DLTs included dermatitis (n=8), hepatitis (n=1), QTc prolongation (n=1), and arthralgias (n=1 each). QoL assessments identified a clinically significant decrease in self assessed QoL at 1 year compared to baseline (0.38 v 0.43). Median PFS was 20.7 months and median OS was 23.8 months for vemurafenib with pembrolizumab. Median PFS and OS were not reached for patients receiving triple therapy. ORR in the overall cohort was 78% (7/9). 2 patients experienced a complete response, 5 had a partial response, 1 had stable disease, and 1 had progressive disease. 4 patients had ongoing responses at data analysis. Peripheral blood flow cytometry identified significantly decreased PD1 expression on CD4+ T-cells at 3 and 9 weeks compared to baseline, not corresponding to clinical response.ConclusionsTriple therapy with vemurafenib, cobimetinib and pembrolizumab is associated with high response rates but significant adverse events, leading to early study closure.

ABSTRACT Elevated serum lactate dehydrogenase (sLDH) is associated with poor clinical outcomes in patients with stage IV metastatic melanoma (MM). It is currently unknown if sLDH elevation correlates with distinct molecular, metabolic, or immune features of melanoma metastases. The identification of such features may identify rational therapeutic strategies for patients with elevated sLDH. Thus, we obtained sLDH levels for melanoma patients with metastases who had undergone molecular and/or immune profiling. Our analysis of multi‐omics data from independent cohorts of melanoma metastases showed that elevated sLDH was not significantly associated with differences in immune cell infiltrate, point mutations, DNA copy number variations, promoter methylation, RNA expression, or protein expression in melanoma metastases. The only significant association observed for elevated sLDH was with the number of metastatic sites of disease. Our data support that sLDH correlates with disease burden, but not specific molecular or immunological phenotypes, in metastatic melanoma.

T cell immunoglobulin mucin domain-containing protein 3 (Tim-3) negatively regulates innate and adaptive immunity in cancer. To identify the mechanisms of Tim-3 in cancer immunity, we evaluated the effects of Tim-3 blockade in human and mouse melanoma. Here, we show that human programmed cell death 1–positive (PD-1+) Tim-3+CD8+ tumor-infiltrating lymphocytes (TILs) upregulate phosphatidylserine (PS), a receptor for Tim-3, and acquire cell surface myeloid markers from antigen-presenting cells (APCs) through transfer of membrane fragments called trogocytosis. Tim-3 blockade acted on Tim-3+ APCs in a PS-dependent fashion to disrupt the trogocytosis of activated tumor antigen–specific CD8+ T cells and PD-1+Tim-3+ CD8+ TILs isolated from patients with melanoma. Tim-3 and PD-1 blockades cooperated to disrupt trogocytosis of CD8+ TILs in 2 melanoma mouse models, decreasing tumor burden and prolonging survival. Deleting Tim-3 in dendritic cells but not in CD8+ T cells impeded the trogocytosis of CD8+ TILs in vivo. Trogocytosed CD8+ T cells presented tumor peptide–major histocompatibility complexes and became the target of fratricide T cell killing, which was reversed by Tim-3 blockade. Our findings have uncovered a mechanism Tim-3 uses to limit antitumor immunity.

BackgroundAnti-PD-1 immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment of melanoma by producing durable long-term responses in a subset of patients. ICI-treated patients develop unique toxicities - immune related adverse events (irAEs) – that arise from unrestrained immune activation. The link between irAE development and clinical outcome in melanoma and other cancers is inconsistent; and little data exists on the occurrence of multiple irAEs. We sought to characterize development of single and multiple irAEs, and association of irAE(s) development with clinical variables and impact upon outcomes in advanced melanoma patients treated with anti-PD-1 ICIs.MethodsWe conducted a retrospective study of 190 patients with metastatic melanoma treated with single-agent anti-PD-1 ICI therapy between June 2014 and August 2020 at a large integrated network cancer center identified through retrospective review of pharmacy records. irAEs were graded based on the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0.Results190 patients were evaluated of whom 114 patients (60.0%) experienced ≥1 irAE, including 30 (15.8%) with grade 3/4 irAEs. The occurrence of any irAE was strongly associated with the development of investigator-assessed response to anti-PD-1 therapy (p < 0.0001); whether evaluated by current (p=0.0082) or best (p=0.0001) response. In patients with ≥2 irAEs, distinct patterns were observed. Median progression-free survival (PFS) and overall survival (OS) were greater in those with any irAE compared to those without (PFS, 28 months vs. 5 months, p < 0.0001; OS, not reached vs. 9 months, p < 0.0001). Development of ≥2 irAEs had a trend towards improved PFS and OS compared to those who developed a single irAE, although this did not reach statistical significance (p=0.2555, PFS; p=0.0583, OS). Obesity but not age or gender was distinctly associated with irAE development.ConclusionsIn this study, we demonstrated that irAE occurrence was significantly associated with response to anti-PD-1 therapy and improved PFS/OS. Those who developed multiple irAEs had a trend towards improved PFS and OS compared to those who developed only a single irAE. Increased BMI but neither age nor gender were associated with irAE development. Distinct patterns of irAEs observed suggest shared etiopathogenetic mechanisms.

Abstract Immune checkpoint inhibitors (ICBs) have revolutionized cancer treatment producing remarkable and durable responses for a range of malignancies. However, the additional modulation of immune response by ICBs may rarely cause immune-related infectious complications, including re-activation of latent tuberculosis infection (LTBC) with detrimental effects on those patients’ outcome. Here, we present two “real-world” melanoma cases that were treated in our department with blockade of PD-1/PD-L1 and developed active Mycobacterium tuberculosis (MTB) during immunotherapy. In view of these cases, we review the literature for ICB-associated MTB reactivation and discuss our considerations about the possible interactions of immunotherapy and the underlying co-existent mycobacterial infection. Based on the current evidence from preclinical findings prior to this experience, we raise questions regarding cancer patients who are at higher risk for developing MTB infection, whether ICB-treated patients should be considered immunocompromised, and how they should be managed for latent and/or active tuberculosis. Aside from the well-established clinical benefit of immunotherapy, the blockade of PD-1/PD-L1 axis may concurrently disrupt the immune control of specific opportunistic infections such as tuberculosis that should be carefully and expectantly managed in order to avoid compromising the outcome of cancer treatment and the affected patient’s survival.

Abstract Background Cancer vaccines are designed to promote systemic antitumor immunity and tumor eradication. Cancer vaccination may be more efficacious in combination with additional interventions that may build on or amplify their effects. Methods Based on our previous clinical and in vitro studies, we designed an antigen-engineered DC vaccine trial to promote a polyclonal CD8+ and CD4+ T cell response against three shared melanoma antigens. The 35 vaccine recipients were then randomized to receive one month of high-dose IFNα or observation. Results The resulting clinical outcomes were 2 partial responses, 8 stable disease and 14 progressive disease among patients with measurable disease using RECIST 1.1, and, of 11 surgically treated patients with no evidence of disease (NED), 4 remain NED at a median follow-up of 3 years. The majority of vaccinated patients showed an increase in vaccine antigen-specific CD8+ and CD4+ T cell responses. The addition of IFNα did not appear to improve immune or clinical responses in this trial. Examination of the DC vaccine profiles showed that IL-12p70 secretion did not correlate with immune or clinical responses. In depth immune biomarker studies support the importance of circulating Treg and MDSC for development of antigen-specific T cell responses, and of circulating CD8+ and CD4+ T cell subsets in clinical responses. Conclusions DC vaccines are a safe and reliable platform for promoting antitumor immunity. This combination with one month of high dose IFNα did not improve outcomes. Immune biomarker analysis in the blood identified several predictive and prognostic biomarkers for further analysis, including MDSC. Trial registration NCT01622933.

We defined the lethal interaction between the novel therapeutic GZ17-6.02 and the standard of care combination of the MEK1/2 inhibitor trametinib and the B-RAF inhibitor dabrafenib in PDX isolates of cutaneous melanoma expressing a mutant B-RAF V600E protein. GZ17-6.02 interacted with trametinib/dabrafenib in an additive fashion to kill melanoma cells. Regardless of prior vemurafenib resistance, the drugs when combined interacted to prolong ATM S1981/AMPK T172 and eIF2α S51 phosphorylation and prolong the reduced phosphorylation of JAK2 Y1007, STAT3 Y705 and STAT5 Y694. In vemurafenib-resistant cells GZ17-6.02 caused a prolonged reduction in mTORC1 S2448, mTORC2 S2481 and ULK1 S757 phosphorylation; regardless of vemurafenib resistance, GZ17-6.02 caused a prolonged elevation in CD95 and FAS-L expression. Knock down of eIF2α, Beclin1, ATG5, ATM, AMPKα, CD95 or FADD significantly reduced the ability of GZ17-6.02 to kill as a single agent or when combined with the kinase inhibitors. Expression of activated mTOR, activated STAT3, activated MEK1 or activated AKT significantly reduced the ability of GZ17-6.02 to kill as a single agent or when combined with kinase inhibitors; protective effects that were significantly less pronounced in cells treated with trametinib/dabrafenib. Regardless of vemurafenib resistance, the drugs alone or in combination all reduced the expression of PD-L1 and increased the levels of MHCA, which was linked to degradation of multiple HDAC proteins. Our findings support the use of GZ17-6.02 in combination with trametinib/dabrafenib in the treatment of melanomas expressing mutant B-RAF V600E proteins.

The advent of first and second-generation immune checkpoint blockade (ICI) has resulted in improved survival of patients with metastatic melanoma over the past decade. However, the majority of patients ultimately progress despite these treatments, which has served as an impetus to consider a range of subsequent therapies. Many of the next generation of immunotherapeutic agents focus on modifying the immune system to overcome resistance to checkpoint blockade. ICI resistance can be understood as primary, or acquired—where the latter is the most common scenario. While there are several postulated mechanisms by which resistance, particularly acquired resistance, occurs, the predominant escape mechanisms include T cell exhaustion, upregulation of alternative inhibitory checkpoint receptors, and alteration of the tumor microenvironment (TME) into a more suppressive, anti-inflammatory state. Therapeutic agents in development are designed to work by combating one or more of these resistance mechanisms. These strategies face the added challenge of minimizing immune-related toxicities, while improving antitumor efficacy. This review focuses upon the following categories of novel therapeutics: 1) alternative inhibitory receptor pathways; 2) damage- or pathogen-associated molecular patterns (DAMPs/PAMPs); and 3) immune cell signaling mediators. We present the current state of these therapies, including preclinical and clinical data available for these targets under development.

Abstract Molecular profiling of small extracellular vesicles (sEV) isolated from plasma of cancer patients emerges as promising strategy for biomarkers discovery. We investigated the proteomic profiles of sEV immunoselected using anti‐CSPG4 antibodies from 15 melanoma patients’ plasma. The proteomes of sEV separated into melanoma cell‐derived (MTEX) and non‐malignant cell‐derived (NMTEX) were compared using high‐resolution mass spectrometry. Paired analysis identified the MTEX‐associated profile of 16 proteins that discriminated MTEX from NMETEX. We also identified the MTEX profile that discriminated between seven patients with no evidence of melanoma (NED) after therapy and eight with progressive disease (PD). Among 75 MTEX proteins overexpressed in PD patients, PDCD6IP (ALIX) had the highest discriminating value, while CNTN1 (contactin‐1) was upregulated only in MTEX of NED patients. This is the first report documenting that proteomes of tumour‐derived sEV in patients’ plasma discriminate cancer from non‐cancer and identify proteins with potential to serve as prognostic biomarkers in melanoma.

BackgroundHigh tumor-infiltrating lymphocytes (TILs) and hemorrhage are important prognostic factors in patients who have undergone craniotomy for melanoma brain metastases (MBM) before 2011 at the University of Pittsburgh Medical Center (UPMC). We have investigated the prognostic or predictive role of these histopathologic factors in a more contemporary craniotomy cohort from the University of North Carolina at Chapel Hill (UNC-CH). We have also sought to understand better how various immune cell subsets, angiogenic factors, and blood vessels may be associated with clinical and radiographic features in MBM.MethodsBrain tumors from the UPMC and UNC-CH patient cohorts were (re)analyzed by standard histopathology, tumor tissue imaging, and gene expression profiling. Variables were associated with overall survival (OS) and radiographic features.ResultsThe patient subgroup with high TILs in craniotomy specimens and subsequent treatment with immune checkpoint inhibitors (ICIs, n=7) trended to have longer OS compared to the subgroup with high TILs and no treatment with ICIs (n=11, p=0.059). Bleeding was significantly associated with tumor volume before craniotomy, high melanoma-specific expression of basic fibroblast growth factor (bFGF), and high density of CD31+αSMA- blood vessels. Brain tumors with high versus low peritumoral edema before craniotomy had low (17%) versus high (41%) incidence of brisk TILs. Melanoma-specific expression of the vascular endothelial growth factor (VEGF) was comparable to VEGF expression by TILs and was not associated with any particular prognostic, radiographic, or histopathologic features. A gene signature associated with gamma delta (gd) T cells was significantly higher in intracranial than same-patient extracranial metastases and primary melanoma. However, gdT cell density in MBM was not prognostic.ConclusionsICIs may provide greater clinical benefit in patients with brisk TILs in MBM. Intratumoral hemorrhage in brain metastases, a significant clinical problem, is not merely associated with tumor volume but also with underlying biology. bFGF may be an essential pathway to target. VEGF, a factor principally associated with peritumoral edema, is not only produced by melanoma cells but also by TILs. Therefore, suppressing low-grade peritumoral edema using corticosteroids may harm TIL function in 41% of cases. Ongoing clinical trials targeting VEGF in MBM may predict a lack of unfavorable impacts on TIL density and/or intratumoral hemorrhage.

Ipilimumab (IPI) can enhance immunity to the cancer-testis antigen NY-ESO-1. A clinical trial was designed to assess safety, immunogenicity, and clinical responses with IPI + NY-ESO-1 vaccines and effects on the tumor microenvironment (TME). Patients with measurable NY-ESO-1+ tumors were enrolled among three arms: A) IPI + NY-ESO-1 protein + poly-ICLC (pICLC) + incomplete Freund’s adjuvant (IFA); B) IPI + NY-ESO-1 overlapping long peptides (OLP) + pICLC + IFA; and C) IPI + NY-ESO-1 OLP + pICLC. Clinical responses were assessed by irRC. T cell and Ab responses were assessed by ex vivo IFN-gamma ELIspot and ELISA. Tumor biopsies pre- and post-treatment were evaluated for immune infiltrates. Eight patients were enrolled: 5, 2, and 1 in Arms A-C, respectively. There were no DLTs. Best clinical responses were SD (4) and PD (4). T-cell and antibody (Ab) responses to NY-ESO-1 were detected in 6 (75%) and 7 (88%) patients, respectively, and were associated with SD. The breadth of Ab responses was greater for patients with SD than PD (p = .036). For five patients evaluable in the TME, treatment was associated with increases in proliferating (Ki67+) CD8+ T cells and decreases in RORγt+ CD4+ T cells. T cell densities increased for those with SD. Detection of T cell responses to NY-ESO-1 ex vivo in most patients suggests that IPI may have enhanced those responses. Proliferating intratumoral CD8+ T cells increased after vaccination plus IPI suggesting favorable impact of IPI plus NY-ESO-1 vaccines on the TME. List of Abbreviations: Ab = antibody; CTCAE = NCI Common Terminology Criteria for Adverse Events; DHFR/DHRP = dihydrofolate reductase; DLT = Dose-limiting toxicity; ELISA = enzyme-linked immunosorbent assay; IFA = incomplete Freund’s adjuvant (Montanide ISA-51); IFNγ = Interferon gamma; IPI = Ipilimumab; irRC = immune-related response criteria; mIFH = multispectral immunofluorescence histology; OLP = NY-ESO-1 overlapping long peptides; PBMC = peripheral blood mononuclear cells; PD = Progressive disease; pICLC = poly-ICLC (Hiltonol), a TLR3/MDA-5 agonist; RLT = Regimen-limiting Toxicity; ROI = regions of interest; RT = room temperature; SAE = serious adverse event; SD = stable disease; TEAE = treatment-emergent adverse events; TLR = toll-like receptor; TME = tumor microenvironment; TRAE = treatment-related adverse events.

Abstract Purpose To develop recommendations for clinical trial reporting that address the unique efficacy, toxicity, and combination and sequencing aspects of immuno-oncology (IO) treatments. Methods ASCO and the Society for Immunotherapy of Cancer (SITC) convened a working group that consisted of practicing medical oncologists, immunologists, clinical researchers, biostatisticians, and representatives from industry and government to develop Trial Reporting in Immuno-Oncology (TRIO) recommendations. These recommendations are based on expert consensus, given that existing data to support evidence-based recommendations are limited. Conclusion The TRIO recommendations are intended to improve the reporting of IO clinical trials and thus provide more complete evidence on the relative benefits and risks of an IO therapeutic approach. Given the rapid expansion of the number of IO clinical trials and ongoing improvements to the evidence base supporting the use of IO treatments in clinical care, these recommendations will likely need regular revision as the IO field develops.

Abstract Background Neoadjuvant immunotherapy utilizing novel combinations has the potential to transform the standard of care for locally/regionally advanced melanoma. We hypothesized that neoadjuvant ipilimumab in combination with high dose IFNα2b (HDI) is safe and associated with durable pathologic complete responses (pCR). Methods Patients with locally/regionally advanced melanoma were randomized to ipilimumab 3 or 10 mg/kg × 4 doses bracketing definitive surgery, then every 12 weeks × 4. HDI was given concurrently. We evaluated the safety and efficacy of the combination with ipilimumab 3 or 10 mg/kg. The impact on T-cell fraction and clonality were investigated in tumor and blood. Results Thirty patients (age 37–76), 15 each at 3 and 10 mg/kg, 18 male and 12 female were treated. Considering immune related adverse events (irAEs) of interest, more grade 3/4 irAEs were seen with ipilimumab 10 mg/kg versus 3 mg/kg (p = 0.042). Among 28 evaluable patients, 11 relapsed, of whom 5 died. Median follow-up for 17 patients who have not relapsed was 32 months. The radiologic preoperative response rate was 36% (95% CI, 21–54); 4 patients at ipilimumab 3 mg/kg and 6 at 10 mg/kg and 2 (at 10 mg/kg) later relapsed. The pCR was 32% (95% CI, 18–51); 5 patients at ipilimumab 3 mg/kg and 4 at 10 mg/kg and one (at 3 mg/kg) had a late relapse. In patients with pCR, T-cell fraction was significantly higher when measured in primary melanoma tumors (p = 0.033). Higher tumor T-cell clonality in primary tumor and more so following neoadjuvant therapy was significantly associated with improved relapse free survival. Conclusions Neoadjuvant ipilimumab-HDI was relatively safe and exhibited promising tumor response rates with an associated measurable impact on T-cell fraction and clonality. Most pCRs were durable supporting the value of pCR as a primary endpoint in neoadjuvant immunotherapy trials. Trial registration ClinicalTrials.gov, NCT01608594. Registered 31 May 2012.

Screening for melanoma may save lives, but may also cause patient distress. One key reason that preventative visual skin examinations for skin cancer are not currently recommended is the inadequate available evidence to assess potential harm to psychosocial wellbeing. We investigated potential psychological harms and benefits of skin examinations by conducting telephone surveys in 2015 of 187 screened participants; all were ≥35 years old. Participants had their skin examined by practitioners who had completed INFORMED, a validated web-based training for detection of skin cancers, particularly melanoma. Participants underwent the Spielberger State-Trait Anxiety Inventory (STAI), Psychological Consequences of Screening (PCQ), Hospital Anxiety and Depression (HAD) scale, and the 12-Item Short Form Health Survey (SF-12). Analyses were conducted in 2017. Of the entire study sample, 40% were thoroughly screened as determined by patient-reported level of undress and skin areas examined. Participants who were thoroughly screened: did not differ on negative psychosocial measures; scored higher on measures of positive psychosocial wellbeing (PCQ); and were more motivated to conduct monthly self-examinations and seek annual clinician skin examinations, compared to other participants (p

Abstract The U.S. Food and Drug Administration (FDA) recently approved pembrolizumab, an anti- programmed cell death protein 1 cancer immunotherapeutic, for use in advanced solid tumors in patients with the microsatellite-high/DNA mismatch repair-deficient biomarker. This is the first example of a tissue-agnostic FDA approval of a treatment based on a patient’s tumor biomarker status, rather than on tumor histology. Here we discuss key issues and implications arising from the biomarker-based disease classification implied by this historic approval.

Abstract Background Cancer immunotherapy has been firmly established as a standard of care for patients with advanced and metastatic melanoma. Therapeutic outcomes in clinical trials have resulted in the approval of 11 new drugs and/or combination regimens for patients with melanoma. However, prospective data to support evidence-based clinical decisions with respect to the optimal schedule and sequencing of immunotherapy and targeted agents, how best to manage emerging toxicities and when to stop treatment are not yet available. Methods To address this knowledge gap, the Society for Immunotherapy of Cancer (SITC) Melanoma Task Force developed a process for consensus recommendations for physicians treating patients with melanoma integrating evidence-based data, where available, with best expert consensus opinion. The initial consensus statement was published in 2013, and version 2.0 of this report is an update based on a recent meeting of the Task Force and extensive subsequent discussions on new agents, contemporary peer-reviewed literature and emerging clinical data. The Academy of Medicine (formerly Institute of Medicine) clinical practice guidelines were used as a basis for consensus development with an updated literature search for important studies published between 1992 and 2017 and supplemented, as appropriate, by recommendations from Task Force participants. Results The Task Force considered patients with stage II-IV melanoma and here provide consensus recommendations for how they would incorporate the many immunotherapy options into clinical pathways for patients with cutaneous melanoma. Conclusion These clinical guidleines provide physicians and healthcare providers with consensus recommendations for managing melanoma patients electing treatment with tumor immunotherapy.

Abstract Background Durable remissions are observed in a fraction of metastatic melanoma patients treated with high-dose interleukin-2 (HD IL-2). Early studies reported overall (OR) and complete response (CR) rates of 16% and 8% respectively. Toxicity limited use to specialized centers with standardized protocols. We report on 243 patients treated at the University of Pittsburgh in a non-intensive care unit (ICU) oncology specialty setting. Methods Clinical and radiological data were collected on 243 patients treated between 1992 and 2015. Each HD IL-2 cycle was given over 5 days, cycles were repeated after 9 days and courses (2 cycles) were repeated every 6–9 weeks in patients with stable or responding disease, for up to 3 courses total. Influence of baseline characteristics on outcomes was assessed using Kaplan-Meier estimates and Cox proportional hazards analysis. Results Two hundred forty-three patients received 692 cycles (5270 doses) between 1992 and 2015. Two hundred thirty-seven patients were evaluable for response: OR rate 18.1% with CR rate 8.0%. Median overall survival (OS) 9.6 months in the entire cohort but 64.9 months in responders. Median number of cycles delivered was 2,and median number of doses per cycle was 8. Toxicity was consistent with prior reports. HD IL-2 required ICU transfers in 11 patients and 1 death was attributed to HD IL-2. Pre-treatment lactate dehydrogenase (LDH) levels correlated significantly with progression-free survival [1-2× upper limit normal (ULN) HR 1.95; >2× ULN HR 2.32] and overall survival (1-2× ULN HR 1.67; >2× ULN 2.49). Response to HD IL-2 and site of metastatic disease also correlated significantly with progression-free and overall survival. Conclusions In this large series of patients spanning more than two decades, OR/CR rates with HD IL-2 were 18.1%/8.0% respectively. Toxicity data was consistent with prior reports. Pre-treatment LDH values and site(s) of metastatic disease may be useful markers to select patients at greater likelihood of benefit to HD IL-2 therapy.

Abstract Background Tremelimumab is an antibody that blocks CTLA-4 and demonstrates clinical efficacy in a subset of advanced melanoma patients. An unmet clinical need exists for blood-based response-predictive gene signatures to facilitate clinically effective and cost-efficient use of such immunotherapeutic interventions. Methods Peripheral blood samples were collected in PAXgene® tubes from 210 treatment-naïve melanoma patients receiving tremelimumab in a worldwide, multicenter phase III study (discovery dataset). A central panel of radiologists determined objective response using RECIST criteria. Gene expression for 169 mRNA transcripts was measured using quantitative PCR. A 15-gene pre-treatment response-predictive classifier model was identified. An independent population (N = 150) of refractory melanoma patients receiving tremelimumab after chemotherapy enrolled in a worldwide phase II study (validation dataset). The classifier model, using the same genes, coefficients and constants for objective response and one-year survival after treatment, was applied to the validation dataset. Results A 15-gene pre-treatment classifier model (containing ADAM17, CDK2, CDKN2A, DPP4, ERBB2, HLA-DRA, ICOS, ITGA4, LARGE, MYC, NAB2, NRAS, RHOC, TGFB1, and TIMP1) achieved an area under the curve (AUC) of 0.86 (95% confidence interval 0.81 to 0.91, p

Abstract As cancer strikes, individuals vary not only in terms of factors that contribute to its occurrence and development, but as importantly, in their capacity to respond to treatment. While exciting new therapeutic options that mobilize the immune system against cancer have led to breakthroughs for a variety of malignancies, success is limited to a subset of patients. Pre-existing immunological features of both the host and the tumor may contribute to how patients will eventually fare with immunotherapy. A broad understanding of baseline immunity, both in the periphery and in the tumor microenvironment, is needed in order to fully realize the potential of cancer immunotherapy. Such interrogation of the tumor, blood, and host immune parameters prior to treatment is expected to identify biomarkers predictive of clinical outcome as well as to elucidate why some patients fail to respond to immunotherapy. To approach these opportunities for progress, the Society for Immunotherapy of Cancer (SITC) reconvened the Immune Biomarkers Task Force. Comprised of an international multidisciplinary panel of experts, Working Group 4 sought to make recommendations that focus on the complexity of the tumor microenvironment, with its diversity of immune genes, proteins, cells, and pathways naturally present at baseline and in circulation, and novel tools to aid in such broad analyses.

Oncogene activation induces DNA damage responses and cell senescence. We report a key role of type I interferons (IFNs) in oncogene-induced senescence. IFN signaling-deficient melanocytes expressing activated Braf do not exhibit senescence and develop aggressive melanomas. Restoration of IFN signaling in IFN-deficient melanoma cells induces senescence and suppresses melanoma progression. Additional data from human melanoma patients and mouse transplanted tumor models suggest the importance of non-cell-autonomous IFN signaling. Inactivation of the IFN pathway is mediated by the IFN receptor IFNAR1 downregulation that invariably occurs during melanoma development. Mice harboring an IFNAR1 mutant, which is partially resistant to downregulation, delay melanoma development, suppress metastatic disease, and better respond to BRAF or PD-1 inhibitors. These results suggest that IFN signaling is an important tumor-suppressive pathway that inhibits melanoma development and progression and argue for targeting IFNAR1 downregulation to prevent metastatic disease and improve the efficacy of molecularly target and immune-targeted melanoma therapies.

Natural killer (NK) cells are innate cytotoxic and immunoregulatory lymphocytes that have a central role in anti-tumor immunity and play a critical role in mediating cellular immunity in advanced cancer immunotherapies, such as dendritic cell (DC) vaccines. Our group recently tested a novel recombinant adenovirus-transduced autologous DC-based vaccine that simultaneously induces T cell responses against three melanoma-associated antigens for advanced melanoma patients. Here, we examine the impact of this vaccine as well as the subsequent systemic delivery of high-dose interferon-α2b (HDI) on the circulatory NK cell profile in melanoma patients. At baseline, patient NK cells, particularly those isolated from high-risk patients with no measurable disease, showed altered distribution of CD56dim CD16+ and CD56dim CD16− NK cell subsets, as well as elevated serum levels of immune suppressive MICA, TN5E/CD73 and tactile/CD96, and perforin. Surprisingly, patient NK cells displayed a higher level of activation than those from healthy donors as measured by elevated CD69, NKp44 and CCR7 levels, and enhanced K562 killing. Elevated cytolytic ability strongly correlated with increased representation of CD56dim CD16+ NK cells and amplified CD69 expression on CD56dim CD16+ NK cells. While intradermal DC immunizations did not significantly impact circulatory NK cell activation and distribution profiles, subsequent HDI injections enhanced CD56bright CD16− NK cell numbers when compared to patients that did not receive HDI. Phenotypic analysis of tumor-infiltrating NK cells showed that CD56dim CD16− NK cells are the dominant subset in melanoma tumors. NanoString transcriptomic analysis of melanomas resected at baseline indicated that there was a trend of increased CD56dim NK cell gene signature expression in patients with better clinical response. These data indicate that melanoma patient blood NK cells display elevated activation levels, that intra-dermal DC immunizations did not effectively promote systemic NK cell responses, that systemic HDI administration can modulate NK cell subset distributions and suggest that CD56dim CD16− NK cells are a unique non-cytolytic subset in melanoma patients that may associate with better patient outcome.

IntroductionThis pilot study aimed at exploring the utility of the proliferation tracer F-18 fluorothymidine (FLT) and positron-emission tomography (PET)/magnetic resonance imaging (MRI) (FLT–PET/MRI) for early treatment monitoring in patients with melanoma brain metastasis (MBM) who undergo targeted therapy or immunotherapy.Material and MethodsPatients with newly diagnosed MBM underwent baseline and follow-up FLT–PET/MRI scans at 3–4 weeks of targeted therapy or immunotherapy. Up to six measurable brain lesions ≥1.0 cm per subject, as identified on T1-weighted post-gadolinium images, were included for quantitative analyses. The maximum SUV of each lesion was divided by the mean SUV of the pons to obtain the SUV ratio (SUVR).ResultsFive enrolled subjects underwent the baseline FLT–PET/MRI study in which the MBM showed a median size of 1.7 cm (range 1.0–2.9) and increased metabolic activity with SUVR of 9.9 (range 3.2–18.4). However, only two subjects (cases #1 and #2) returned for a follow-up scan. At baseline, a total of 22 lesions were analyzed in all five subjects, which showed a median size of 1.7 cm (range 1.0–2.9) and median SUVR of 9.9 (range 3.2–18.4). At follow-up, case #1 was a 55-year-old man who received targeted BRAF inhibitor and MEK inhibitor therapy with dabrafenib and trametinib. Fused PET/MRI data of six measured lesions demonstrated a significant reduction in MBM proliferative activity (median −68%; range −38 to −77%) and size (median −23%; range −4 to −55%) at three weeks of therapy. Nevertheless, the subject eventually progressed and died 13 months after therapy initiation. Case #2 was a 36-year-old man who received immunotherapy with nivolumab and ipilimumab. The five measured MBM lesions showed a mixed response at both proliferative and morphologic imaging at 1-month follow-up. Some lesions demonstrated interval decrease while others interval increase in proliferative activity with a median −44% (range −77 to +68%). On MRI, the size change was +7% (range −64 to +50%). The therapy was switched to dabrafenib and trametinib, which led to a partial response. The patient is still alive 16 months following therapy initiation.ConclusionThe five cases presented show the potential benefit of hybrid FLT–PET/MRI for the diagnosis of MBM and treatment monitoring of targeted therapy and immunotherapy. However, further studies are required to assess their complementary role in distinguishing true progression from pseudoprogression.

Background High dose interleukin-2 (HD IL-2) can induce durable responses in a subset of patients leading to long-term survival. Immune checkpoint blockade (ICB) has demonstrated similarly durable responses in a larger proportion of patients. However, not all patients respond to immune checkpoint blockade and subsequent therapeutic options need to be explored.Methods The PROCLAIM database was queried for patients with metastatic melanoma who had received HD IL-2 after treatment with ipilimumab or without prior ICB. Patient characteristics, toxicity and efficacy were analyzed.Results A total of 52 metastatic melanoma patients were treated with high dose IL-2 after ipilimumab and 276 patients were treated with high dose IL-2 without prior ICB. The overall response rate in the prior ipilimumab group was 21 % as compared to 12 % in the group that had not received prior ipilimumab. The median overall survival, measured from the initiation of HD IL-2 therapy, was 19.3 months in the prior ipilimumab group and 19.4 months in the no prior ICB group. Toxicities observed on HD IL-2 were relatively equivalent between the groups although there were cases of CTLA4 antibody-induced colitis reported after HD IL-2 treatment and a CTLA4 antibody-induced colitis related death.Conclusion In this retrospective analysis HD IL-2 therapy displayed antitumor activity in melanoma patients who progressed following treatment with ipilimumab. Most HD IL-2 toxicity was not worsened by prior ipilimumab therapy except for one treatment related death from colitis. Care should be taken to avoid reactivation of CTLA4 antibody-induced colitis.

On the basis of remarkable antitumor activity, programmed death receptor-1 (PD-1) inhibitors pembrolizumab and nivolumab were approved for the treatment of advanced melanoma in the second-line setting following progression on either CTLA-4 inhibitor ipilimumab or BRAF/MEK inhibitors (for BRAF mutated melanoma). Given hypothesized risk of triggering exacerbations of autoimmune diseases and/or chronic viral infections, clinical trials (including regulatory studies) evaluating checkpoint blocking antibodies PD-1 and CTLA-4 have excluded patients with autoimmune diseases, chronic hepatitis B/C virus (HBV/HCV), and/or human immunodeficiency virus (HIV) infections. Herein, we describe two patients with advanced melanoma and concomitant HCV/HIV infections treated with PD-1 inhibitor pembrolizumab. Patient 2 with HIV/HCV coinfection progressed after 2 doses of pembrolizumab. Patient 1 who had HCV alone was treated with pembrolizumab with initial partial response. HCV viral load remained stable after 9 cycles of pembrolizumab following which 12-week course of HCV-directed therapy was commenced, resulting in prompt reduction of HCV viral load below detectable levels. Response is ongoing and HCV viral load remains undetectable. In both patients, no significant toxicities were observed when pembrolizumab was initiated. We argue for the further investigation of checkpoint inhibition in cancer patients with underlying chronic viral infections in the context of carefully designed clinical trials.

Melanoma patients frequently exhibit a deficiency in Type-1 (but not Type-2 or regulatory) CD4+ T cell responses against tumor-associated antigens (TAA), which may limit protection against cancer progression or responsiveness to immunotherapy in these individuals. Since such deficiency was acutely evident in patients with active disease, where chronic stimulation of anti-tumor CD4+ T cells would be expected and activation-induced cell death may be prevalent, we employed MHC Class II-peptide tetramers to characterize the frequency and apoptotic status of TAA- vs. influenza (FluM1) virus-specific CD4+ T cells in the peripheral blood of HLA-DR*0401+ patients with melanoma or renal cell carcinoma (RCC). We observed that Flu-specific CD4+ T cells ranged from 0.17 to 3.89%, while up to approximately 1% of CD4+ T cells reacted against individual TAA epitopes derived from the EphA2 or MAGE-6 proteins. The frequencies of EphA2 and MAGE-6-specific CD4+ T cells in patients were significantly correlated with active disease and patient gender (i.e. females > males), while frequencies of Flu-specific CD4+ T cells were distributed within a normal range in all patients. Notably, patient CD4+ T cells reactive with MHC class II-TAA (but not MHC class II-Flu) tetramers were significantly enriched for a pro-apoptotic (Annexin-V+) phenotype, particularly amongst the Th1 (T-bet+) subset. These results suggest that the preferential sensitivity of TAA (but not viral)-specific CD4+ Th1 cells to apoptosis in melanoma patients with active disease will need to be overcome for optimal clinical benefit of immunotherapeutic approaches to be realized.

With an incidence that is increasing at 2–5% per year, cutaneous melanoma is an international scourge that disproportionately targets young individuals. Despite much research, the treatment of advanced disease is still quite challenging. Immunotherapy with high-dose interferon-α2b or interleukin-2 benefits a select group of patients in the adjuvant and metastatic settings, respectively, with significant attendant toxicity. Advances in the biology of malignant melanoma and the role of immunomodulatory therapy have produced advances that have stunned the field. In this paper, we review the data for the use of interferon-α2b in various dosing ranges, vaccine therapy, and the role of radiotherapy in the adjuvant setting for malignant melanoma. Recent trials in the metastatic setting using anticytoxic T-lymphocyte antigen-4 (anti-CTLA-4) monoclonal antibody therapy and BRAF inhibitor therapy have demonstrated clear benefit with prolongation of survival. Trials investigating combinations of these novel agents with existing immunomodulators are at present underway.

Atypical nevi are the precursors and risk markers of melanoma. Apart from persistently monitoring these nevocytic lesions and resecting them at the earliest signs of clinical changes, there is as yet no systemic clinical treatment available to interfere with their progression to melanoma. To explore clinical treatments that might interfere with and possibly prevent atypical nevus progression, a previous study documented that 3 months systemic low-dose interferon-α (IFN-α) treatment of patients with a clinical history of melanoma and numerous atypical nevi, led to inactivation of the STAT1 and STAT3 transcription factors in atypical nevi. Based upon this finding, we initiated a second study to determine whether systemic low-dose IFN-α treatment also impairs the expression of upstream regulators and downstream targets of STAT1 and STAT3 in atypical nevi. Using cyanine dye-conjugated antibodies, fluorescence imaging analysis revealed expression of JAK2, JNK1, AKT1, NF-κB, and IFN-αβ receptor in benign and atypical nevi, and early- and advanced-stage melanomas. To determine possible changes in the level of expression of these molecules in atypical nevi, excised before and after 3 months of systemic low-dose IFN-α treatment, newly designed optical imaging software was used to quantitate the captured fluorescent hybridization signals on a cell-by-cell basis and across an entire nevus section. The results of this analysis did not provide evidence that systemic low-dose IFN-α treatment alters the level of expression of upstream regulators or downstream targets of STAT1 and STAT3.