Immune cells infiltrating the microenvironment of melanoma metastases may either limit or promote tumor progression, but the characteristics that distinguish these effects are obscure. In this study, we systematically evaluated the composition and organization of immune cells that infiltrated melanoma metastases in human patients. Three histologic patterns of immune cell infiltration were identified, designated immunotypes A, B and C. Immunotype A was characterized by no immune cell infiltrate. Immunotype B was characterized by infiltration of immune cells limited only to regions proximal to intratumoral blood vessels. Immunotype C was characterized by a diffuse immune cell infiltrate throughout a metastatic tumor. These immunotypes represented 29%, 63%, and 8% of metastases with estimated median survival periods of 15, 23, and 130 months, respectively. Notably, all three immunotypes showed increasing proportions of B cells and decreasing proportions of macrophages. Overall, the predominant immune cells were T cells (53%), B cell lineage cells (33%), and macrophages (13%), with NK and mature dendritic cells only rarely present. While higher densities of CD8+ T cells correlated best with survival, a higher density of CD45+ leukocytes, T cells, and B cells also correlated with increased survival. Together, our findings reveal striking differences in the immune infiltrate in melanoma metastases in patients, suggesting microenvironmental differences in immune homing receptors and ligands that affect immune cell recruitment. These findings are important, not only by revealing how the immune microenvironment can affect outcomes but also because they reveal characteristics that may help improve individualized therapy for patients with metastatic melanoma.

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BackgroundMelanoma vaccines have not been optimized. Adjuvants are added to activate dendritic cells (DCs) and to induce a favourable immunologic milieu, however, little is known about their cellular and molecular effects in human skin. We hypothesized that a vaccine in incomplete Freund's adjuvant (IFA) would increase dermal Th1 and Tc1-lymphocytes and mature DCs, but that repeated vaccination may increase regulatory cells.MethodsDuring and after 6 weekly immunizations with a multipeptide vaccine, immunization sites were biopsied at weeks 0, 1, 3, 7, or 12. In 36 participants, we enumerated DCs and lymphocyte subsets by immunohistochemistry and characterized their location within skin compartments.ResultsMature DCs aggregated with lymphocytes around superficial vessels, however, immature DCs were randomly distributed. Over time, there was no change in mature DCs. Increases in T and B-cells were noted. Th2 cells outnumbered Th1 lymphocytes after 1 vaccine 6.6:1. Eosinophils and FoxP3+ cells accumulated, especially after 3 vaccinations, the former cell population most abundantly in deeper layers.ConclusionsA multipeptide/IFA vaccine may induce a Th2-dominant microenvironment, which is reversed with repeat vaccination. However, repeat vaccination may increase FoxP3+T-cells and eosinophils. These data suggest multiple opportunities to optimize vaccine regimens and potential endpoints for monitoring the effects of new adjuvants.Trail RegistrationClinicalTrials.gov Identifier: NCT00705640

Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in two clinical trials of melanoma patients. In one study (Mel38), patients received one injection with an adjuvant mixture alone, comprised of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature dendritic cells (DC) and vessels resembling high endothelial venules (HEV). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DC, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4+ and CD8+ T lymphocytes, as well as FoxP3+CD4+ lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination.

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Dermatomyositis, a connective tissue disease syndrome where antibodies to the endothelium of the microvasculature of the skin, muscle and lung are implicated in lesional propagation, is characterized by photodistributed erythema, heliotrope rash, Gottron's papules, muscle weakness and interstitial pulmonary fibrosis. Endotheliotropic viruses and underlying neoplasia are among the inciting triggers. Uncommon drugs, namely the lipid-lowering agents, have been implicated in dermatomyositis. The patient, a 57-year-old man, developed a photodistributed rash and muscle weakness following treatment with the antifungal medication, terbinafine. A skin biopsy was performed, showing an atrophying interface dermatitis with pandermal mucinosis and striking vasculopathic changes including endothelial cell necrosis with denudement and basement membrane zone reduplication. Ultrastructural studies confirmed the presence of endothelial cell injury. Direct immunofluorescent testing showed prominent staining of C5b-9 along the dermal-epidermal junction and within the vasculature. Western blot studies showed strong seroreactivity of his serum to an endothelial-based protein weighing 45,000, a common target described in other microvascular injury-based syndromes. We have shown a temporal association between use of terbinafine and the development of dermatomyositis. The exact basis remains speculative. One potential hypothesis is based on the fact that terbinafine, the active agent in terbinafine, triggers apoptosis of human endothelial cells in culture. Enhanced endothelial cell apoptosis results in the displacement of various cellular antigens creating a state of neoantigenicity; its attendant sequelae is held to be one of anti-endothelial cell antibody formation, a defining pathogenetic event in the evolution of dermatomyositis. The second may be because of the effects of the drug on the promotion of an interferon-rich T-helper-1-dominant cytokine milieu.

ALLP represents a form of cutaneous lymphoid dyscrasia given the relatively self-limited nature of the eruption, albeit in the context of clinical recurrence.

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