Michelle M. van Rossum

New common variants affecting susceptibility to basal cell carcinoma

In a follow-up to our previously reported genome-wide association study of cutaneous basal cell carcinoma (BCC), we describe here several new susceptibility variants. SNP rs11170164, encoding a G138E substitution in the keratin 5 (KRT5) gene, affects risk of BCC (OR = 1.35, P = 2.1 × 10−9). A variant at 9p21 near CDKN2A and CDKN2B also confers susceptibility to BCC (rs2151280[C]; OR = 1.19, P = 6.9 × 10−9), as does rs157935[T] at 7q32 near the imprinted gene KLF14 (OR = 1.23, P = 5.7 × 10−10). The effect of rs157935[T] is dependent on the parental origin of the risk allele. None of these variants were found to be associated with melanoma or fair-pigmentation traits. A melanoma- and pigmentation-associated variant in the SLC45A2 gene, L374F, is associated with risk of both BCC and squamous cell carcinoma. Finally, we report conclusive evidence that rs401681[C] in the TERT-CLPTM1L locus confers susceptibility to BCC but protects against melanoma.

Limited amounts of dendritic cells migrate into the T-cell area of lymph nodes but have high immune activating potential in melanoma patients.

Purpose: The success of immunotherapy with dendritic cells (DC) to treat cancer is dependent on effective migration to the lymph nodes and subsequent activation of antigen-specific T cells. In this study, we investigated the fate of DC after intradermal (i.d.) or intranodal (i.n.) administration and the consequences for the immune activating potential of DC vaccines in melanoma patients. Experimental Design: DC were i.d. or i.n. administered to 25 patients with metastatic melanoma scheduled for regional lymph node resection. To track DC in vivo with scintigraphic imaging and in lymph nodes by immunohistochemistry, cells were labeled with both [111In]-indium and superparamagnetic iron oxide. Results: After i.d. injection, maximally 4% of the DC reached the draining lymph nodes. When correctly delivered, all DC were delivered to one or more lymph nodes after i.n. injection. Independent of the route of administration, large numbers of DC remained at the injection site, lost viability, and were cleared by infiltrating CD163+ macrophages within 48 hours. Interestingly, 87 ± 10% of the surviving DC preferentially migrated into the T-cell areas, where they induced antigen-specific T-cell responses. Even though more DC reached the T-cell areas, i.n. injection of DC induced similar antigen-specific immune responses as i.d. injection. Immune responses were already induced with <5 × 105 DC migrating into the T-cell areas. Conclusions: Monocyte-derived DC have high immune activating potential irrespective of the route of vaccination. Limited numbers of DC in the draining lymph nodes are sufficient to induce antigen-specific immunologic responses.

β-Defensin-2 Protein Is a Serum Biomarker for Disease Activity in Psoriasis and Reaches Biologically Relevant Concentrations in Lesional Skin

Background Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2) is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis. Methodology/Principal Findings We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects. Conclusions/Significance Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases.

Route of Administration Modulates the Induction of Dendritic Cell Vaccine–Induced Antigen-Specific T Cells in Advanced Melanoma Patients

Purpose: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine. Experimental Design: HLA-A2.1+ melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 × 106 to 17 × 106 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with 51Cr release assays or IFNγ release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells. Results: In 19 of 43 vaccinated patients, functional tumor antigen–specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen–specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies. Conclusion: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown. Clin Cancer Res; 17(17); 5725–35. ©2011 AACR.

Targeting CD4+ T-Helper Cells Improves the Induction of Antitumor Responses in Dendritic Cell―Based Vaccination

To evaluate the relevance of directing antigen-specific CD4(+) T helper cells as part of effective anticancer immunotherapy, we investigated the immunologic and clinical responses to vaccination with dendritic cells (DC) pulsed with either MHC class I (MHC-I)-restricted epitopes alone or both MHC class I and II (MHC-I/II)-restricted epitopes. We enrolled 33 stage III and IV HLA-A*02:01-positive patients with melanoma in this study, of whom 29 were evaluable for immunologic response. Patients received intranodal vaccinations with cytokine-matured DCs loaded with keyhole limpet hemocyanin and MHC-I alone or MHC-I/II-restricted tumor-associated antigens (TAA) of tyrosinase and gp100, depending on their HLA-DR4 status. In 4 of 15 patients vaccinated with MHC-I/II-loaded DCs and 1 of 14 patients vaccinated with MHC-I-loaded DCs, we detected TAA-specific CD8(+) T cells with maintained IFN-γ production in skin test infiltrating lymphocyte (SKIL) cultures and circulating TAA-specific CD8(+) T cells. If TAA-specific CD4(+) T-cell responses were detected in SKIL cultures, it coincided with TAA-specific CD8(+) T-cell responses. In 3 of 13 patients tested, we detected TAA-specific CD4(+)CD25(+)FoxP3(-) T cells with high proliferative capacity and IFN-γ production, indicating that these were not regulatory T cells. Vaccination with MHC-I/II-loaded DCs resulted in improved clinical outcome compared with matched control patients treated with dacarbazine (DTIC), median overall survival of 15.0 versus 8.3 months (P = 0.089), and median progression-free survival of 5.0 versus 2.8 months (P = 0.0089). In conclusion, coactivating TAA-specific CD4(+) T-helper cells with DCs pulsed with both MHC class I and II-restricted epitopes augments TAA-specific CD8(+) T-cell responses, contributing to improved clinical responses.

Vaccination with mRNA-electroporated dendritic cells induces robust tumor antigen-specific CD4+ and CD8+ T cells responses in stage III and IV melanoma patients

Purpose: Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) has multiple advantages compared to peptide loading. We investigated the immunologic and clinical responses to vaccination with mRNA-electroporated DC in stage III and IV melanoma patients. Experimental design: Twenty-six stage III HLA*02:01 melanoma patients scheduled for radical lymph node dissection (stage III) and 19 melanoma patients with irresectable locoregional or distant metastatic disease (referred to as stage IV) were included. Monocyte-derived DC, electroporated with mRNA encoding gp100 and tyrosinase, were pulsed with keyhole limpet hemocyanin and administered intranodally. TAA-specific T-cell responses were monitored in blood and skin-test infiltrating lymphocyte (SKIL) cultures. Results: Comparable numbers of vaccine-induced CD8+ and/or CD4+ TAA-specific T-cell responses were detected in SKIL cultures; 17/26 stage III patients and 11/19 stage IV patients. Strikingly, in this population, TAA-specific CD8+ T cells that recognize multiple epitopes and produce elevated levels of IFNγ upon antigenic challenge in vitro, were significantly more often observed in stage III patients; 15/17 versus 3/11 stage IV patients, P = 0.0033. In stage IV patients, one mixed and one partial response were documented. The presence or absence of IFNγ-producing TAA-specific CD8+ T cells in stage IV patients was associated with marked difference in median overall survival of 24.1 months versus 11.0 months, respectively. Conclusion: Vaccination with mRNA-electroporated DC induces a broad repertoire of IFNγ producing TAA-specific CD8+ and CD4+ T-cell responses, particularly in stage III melanoma patients. Clin Cancer Res; 18(19); 5460–70. ©2012 AACR.

A variant in FTO shows association with melanoma risk not due to BMI

We report the results of an association study of melanoma that is based on the genome-wide imputation of the genotypes of 1,353 cases and 3,566 controls of European origin conducted by the GenoMEL consortium. This revealed an association between several SNPs in intron 8 of the FTO gene, including rs16953002, which replicated using 12,313 cases and 55,667 controls of European ancestry from Europe, the USA and Australia (combined P = 3.6 × 10−12, per-allele odds ratio for allele A = 1.16). In addition to identifying a new melanoma-susceptibility locus, this is to our knowledge the first study to identify and replicate an association with SNPs in FTO not related to body mass index (BMI). These SNPs are not in intron 1 (the BMI-related region) and exhibit no association with BMI. This suggests FTOs function may be broader than the existing paradigm that FTO variants influence multiple traits only through their associations with BMI and obesity.

Trends in Basal Cell Carcinoma Incidence Rates: A 37-Year Dutch Observational Study

Basal cell carcinoma (BCC) incidence rates are increasing. From 1973 to 2009, data on all first histologically confirmed BCCs were gained from the Eindhoven Cancer Registry to estimate trends in patient-based BCC incidence rates by sex, age group, and site in the southeast Netherlands. Trends in European age-standardized rates and age- and site-specific incidence rates were assessed by calculating the estimated annual percentage change (EAPC). Between 1973 and 2009, the European standardized rate quadrupled from 40 to 165 per 100,000 person-years for men and from 34 to 157 for women, significantly increasing since 1973 in both sexes, but accelerating from 2002 until 2009 with an EAPC of 6.8% (95% confidence interval (CI), 5.3-8.3) for men and 7.9% (95% CI, 6.2-9.7) for women. Women below the age of 40 years exhibited a constant linear increase of 6.3% since 1973. The head and neck region was most often affected in both sexes, but the steepest increase was seen for the trunk (EAPC men 13%, women 15%). In the absence of reliable tumor-based rates, these alarming patient-based rates are probably an interesting indicator for the impact of more intensive UV exposure in a prosperous European population.

Effective Clinical Responses in Metastatic Melanoma Patients after Vaccination with Primary Myeloid Dendritic Cells

Purpose: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro–generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c+ myeloid DCs, naturally circulating in the blood. Experimental Design: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c+ myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100. Results: Our results show that therapeutic vaccination against melanoma with small amounts (3–10 × 106) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12–35 months), which directly correlated with the development of multifunctional CD8+ T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNγ as well as TNFα and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth. Conclusions: We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155–66. ©2015 AACR.

Genetically Programmed Differences in Epidermal Host Defense between Psoriasis and Atopic Dermatitis Patients

In the past decades, chronic inflammatory diseases such as psoriasis, atopic dermatitis, asthma, Crohn’s disease and celiac disease were generally regarded as immune-mediated conditions involving activated T-cells and proinflammatory cytokines produced by these cells. This paradigm has recently been challenged by the finding that mutations and polymorphisms in epithelium-expressed genes involved in physical barrier function or innate immunity, are risk factors of these conditions. We used a functional genomics approach to analyze cultured keratinocytes from patients with psoriasis or atopic dermatitis and healthy controls. First passage primary cells derived from non-lesional skin were stimulated with pro-inflammatory cytokines, and expression of a panel of 55 genes associated with epidermal differentiation and cutaneous inflammation was measured by quantitative PCR. A subset of these genes was analyzed at the protein level. Using cluster analysis and multivariate analysis of variance we identified groups of genes that were differentially expressed, and could, depending on the stimulus, provide a disease-specific gene expression signature. We found particularly large differences in expression levels of innate immunity genes between keratinocytes from psoriasis patients and atopic dermatitis patients. Our findings indicate that cell-autonomous differences exist between cultured keratinocytes of psoriasis and atopic dermatitis patients, which we interpret to be genetically determined. We hypothesize that polymorphisms of innate immunity genes both with signaling and effector functions are coadapted, each with balancing advantages and disadvantages. In the case of psoriasis, high expression levels of antimicrobial proteins genes putatively confer increased protection against microbial infection, but the biological cost could be a beneficial system gone awry, leading to overt inflammatory disease.