W.A.M. Blokx

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.

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.

Activating mutations of the GNAQ gene: a frequent event in primary melanocytic neoplasms of the central nervous system

Primary melanocytic neoplasms of the central nervous system (CNS) are uncommon neoplasms derived from melanocytes that normally can be found in the leptomeninges. They cover a spectrum of malignancy grades ranging from low-grade melanocytomas to lesions of intermediate malignancy and overtly malignant melanomas. Characteristic genetic alterations in this group of neoplasms have not yet been identified. Using direct sequencing, we investigated 19 primary melanocytic lesions of the CNS (12 melanocytomas, 3 intermediate-grade melanocytomas, and 4 melanomas) for hotspot oncogenic mutations commonly found in melanocytic tumors of the skin (BRAF, NRAS, and HRAS genes) and uvea (GNAQ gene). Somatic mutations in the GNAQ gene at codon 209, resulting in constitutive activation of GNAQ, were detected in 7/19 (37%) tumors, including 6/12 melanocytomas, 0/3 intermediate-grade melanocytomas, and 1/4 melanomas. These GNAQ-mutated tumors were predominantly located around the spinal cord (6/7). One melanoma carried a BRAF point mutation that is frequently found in cutaneous melanomas (c.1799 T>A, p.V600E), raising the question whether this is a metastatic rather than a primary tumor. No HRAS or NRAS mutations were detected. We conclude that somatic mutations in the GNAQ gene at codon 209 are a frequent event in primary melanocytic neoplasms of the CNS. This finding provides new insight in the pathogenesis of these lesions and suggests that GNAQ-dependent mitogen-activated kinase signaling is a promising therapeutic target in these tumors. The prognostic and predictive value of GNAQ mutations in primary melanocytic lesions of the CNS needs to be determined in future studies.

HRAS-mutated Spitz tumors: A subtype of Spitz tumors with distinct features.

It is often very difficult to confidently distinguish benign and malignant Spitz lesions, and a diagnosis of Spitz tumor of unknown malignant potential (STUMP) is rendered. To address this problem, we performed molecular genetic analysis in a large group of Spitz tumors (93 Spitz nevi and 77 STUMPs) and identified a subgroup of 24 lesions harboring a HRAS mutation. This subgroup lay predominantly in the dermis, had a relatively low cellularity, showed desmoplasia (with single cells interspersed between the collagen bundles), and had an infiltrating base. In 7 of these 24 cases (29%) melanoma had been the initial diagnosis, or an important differential diagnostic consideration, mainly based on the presence of multiple or deeply located mitotic figures, especially in adult patients. In our series none of the patients with the HRAS-mutated lesions developed recurrences or metastases (mean and median follow-up: 10.5 y). This was in accordance with the literature: review showed that no HRAS mutations had so far been reported in Spitzoid melanomas. We therefore conclude that HRAS mutation analysis may be a useful diagnostic tool to help differentiate between Spitz nevus and Spitzoid melanoma, thereby reducing the frequency of overdiagnosis of melanoma, and to help predict the biological behavior of a STUMP. Moreover, this might be a first step toward a more reproducible classification of Spitz tumors combining histological and genetic data.

Molecular cytogenetics of cutaneous melanocytic lesions - diagnostic, prognostic and therapeutic aspects.

Blokx W A M, van Dijk M C R F & Ruiter D J
(2010) Histopathology56, 121–132
Molecular cytogenetics of cutaneous melanocytic lesions – diagnostic, prognostic and therapeutic aspects

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.

Ultrastructural Proof of Polyomavirus in Merkel Cell Carcinoma Tumour Cells and Its Absence in Small Cell Carcinoma of the Lung

Background A new virus called the Merkel Cell Polyomavirus (MCPyV) has recently been found in Merkel Cell Carcinoma (MCC). MCC is a rare aggressive small cell neuroendocrine carcinoma primarily derived from the skin, morphologically indistinguishable from small cell lung carcinoma (SCLC). So far the actual presence of the virus in MCC tumour cells on a morphological level has not been demonstrated, and the presence of MCPyV in other small cell neuroendocrine carcinomas has not been studied yet. Methodology/Principal Findings We investigated MCC tissue samples from five patients and SCLCs from ten patients for the presence of MCPyV-DNA by PCR and sequencing. Electron microscopy was used to search ultrastructurally for morphological presence of the virus in MCPyV-DNA positive samples. MCPyV was detected in two out of five primary MCCs. In one MCC patient MCPyV-DNA was detected in the primary tumour as well as in the metastasis, strongly suggesting integration of MCPyV in the cellular DNA of the tumour in this patient. In the primary MCC of another patient viral particles in tumour cell nuclei and cytoplasm were identified by electron microscopy, indicating active viral replication in the tumour cells. In none of the SCLCs MCPyV-DNA was detected. Conclusions/Significance Our results strongly suggest that MCPyV is an oncogenic polyomavirus in humans, and is potentially causally related to the development of MCC but not to the morphological similar SCLC.

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.

In situ Expression of Tumor Antigens by Messenger RNA―Electroporated Dendritic Cells in Lymph Nodes of Melanoma Patients

Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) for cancer immunotherapy has been proved efficient and clinically safe. It obviates prior knowledge of CTL and Th epitopes in the antigen and leads to the presentation of multiple epitopes for several HLA alleles. Here we studied the migration capacity and the antigen expression of mRNA-electroporated DC (mRNA-DC) in lymph nodes after vaccination in melanoma patients. DC were electroporated with mRNA encoding gp100 or tyrosinase, labeled with indium-111 and superparamagnetic iron oxide particles, and injected intranodally in melanoma patients 24 to 48 hours before scheduled dissection of regional lymph nodes. Immunohistochemical analysis of the lymph nodes after surgery revealed that mRNA-DC migrated from the injection site into the T-cell areas of the same and subsequent lymph nodes, where they expressed the antigen encoded by the electroporated mRNA. Furthermore, vaccine-related CD8(+) T-cell responses could be detected in 7 of 11 patients vaccinated with mRNA-DC. Together these data show that mature DC electroporated with mRNA encoding TAA migrate and express antigens in the lymph nodes and induce specific immune responses.

Expert review remains important in the histopathological diagnosis of cutaneous melanocytic lesions

Aims:  To assess the type of problems encountered in diagnosing melanocytic lesions and to evaluate the contribution of expert review.