Noël E.C. Schartz

Malignant effusions and immunogenic tumour-derived exosomes.

BACKGROUND Exosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supernatants. To assess the role of these exosomes in vivo, we examined malignant effusions for their presence. We also investigated whether these exosomes could induce production of tumour-specific T cells when pulsed with dendritic cells. METHODS We isolated exosomes by ultracentrifugation on sucrose and D(2)O gradients of 11 malignant effusions. We characterised exosomes with Western blot analyses, immunoelectron microscopy, and in-vitro stimulations of autologous T lymphocytes. FINDINGS Malignant effusions accumulate high numbers of membrane vesicles that have a mean diameter of 80 nm (SD 30). These vesicles have antigen-presenting molecules (MHC class-I heat-shock proteins), tetraspanins (CD81), and tumour antigens (Her2/Neu, Mart1, TRP, gp100). These criteria, including their morphological characteristics, indicate the similarities between these vesicles and exosomes. Exosomes from patients with melanoma deliver Mart1 tumour antigens to dendritic cells derived from monocytes (MD-DCs) for cross presentation to clones of cytotoxic T lymphocytes specific to Mart1. In seven of nine patients with cancer, lymphocytes specific to the tumour could be efficiently expanded from peripheral blood cells by pulsing autologous MD-DCs with autologous ascitis exosomes. In one patient tested, we successfully expanded a restricted T-cell repertoire, which could not be recovered carcinomatosis nodules. INTERPRETATION Exosomes derived from tumours accumulate in ascites from patients with cancer. Ascitis exosomes are a natural and new source of tumour-rejection antigens, opening up new avenues for immunisation against cancers.

Tumor-derived exosomes: a new source of tumor rejection antigens

Exosomes are small vesicles released by a broad array of hematopoietic cells. Previous studies showed that exosomes released by antigen loaded dendritic cells induce immune-mediated anti-tumor response in mice. Here, we will describe the biochemical properties of tumor-derived exosomes and, their pre-clinical activity as cancer vaccines.

Exosome-based immunotherapy

Exosomes are small membrane vesicles originating from late endosomes and secreted by hematopoietic and epithelial cells in culture. Exosome proteic and lipid composition is unique and might shed some light into exosome biogenesis and function. Exosomes secreted from professional antigen-presenting cells (i.e., B lymphocytes and dendritic cells) are enriched in MHC class I and II complexes, costimulatory molecules, and hsp70–90 chaperones, and have therefore been more extensively studied for their immunomodulatory capacities in vitro and in vivo. This review will present the main biological features pertaining to tumor or DC-derived exosomes, will emphasize their immunostimulatory function, and will discuss their implementation in cancer immunotherapy.

Complete regression of a previously untreated melanoma brain metastasis with ipilimumab.

Anecdotal cases of partial or complete responses have been reported after ipilimumab therapy for stage IV melanoma with brain metastasis treated earlier by surgery or radiosurgery. We report the first case of ipilimumab monotherapy resulting in durable complete remission of untreated, progressive brain metastases in a patient with stage IV melanoma. This case and earlier reports provide support for the further evaluation of ipilimumab in melanoma patients with brain metastasis.

Kaposi's sarcoma in HIV-negative men having sex with men.

Background:Four epidemiologic forms of Kaposis sarcoma have been described, all of which are associated with the human herpesvirus-8. In western countries, human herpesvirus-8 is more prevalent in homosexual men than in the general population, and anecdotal cases of Kaposis sarcoma in HIV-negative homosexual men have been reported. Patients and methods:We included HIV-negative homosexual and bisexual male patients with histologically proven Kaposis sarcoma in a retrospective study. Clinical data were collected using a standardized form. Risk factors for human herpesvirus-8 infection and for the development of Kaposis sarcoma were systematically recorded. Results:Between 1995 and 2007, 28 men met the defined inclusion criteria. Mean age at first symptoms of Kaposis sarcoma was 53 years. Clinical presentation resembled classical Kaposis sarcoma, with limited disease in most patients. No cellular or humoral immunodeficiency was observed. Serologic tests for human herpesvirus-8 (latent immunofluorescence assay) were positive in 88% of patients, and only two patients displayed human herpesvirus-8 viremia at the time of Kaposis sarcoma diagnosis. Three patients developed lymphoproliferative disorders (Castleman disease, follicular lymphoma and Burkitt lymphoma). In this population, α-interferon was well tolerated and gave a complete response, but most patients require only local treatment, if any. Conclusion:Kaposis sarcoma may develop in homosexual or bisexual men without HIV infection. This type of Kaposis sarcoma has clinical features in common with classical Kaposis sarcoma but occurs in younger patients. Its prognosis is good, as Kaposis sarcoma is generally limited, but clinicians should be aware of the association with lymphoproliferative diseases, which may affect prognosis.

Highly Sensitive Multivariable Assay Detection of Melanocytic Differentiation Antigens and Angiogenesis Biomarkers in Sentinel Lymph Nodes With Melanoma Micrometastases

OBJECTIVES To evaluate the prognostic value of melanocytic differentiation antigens and angiogenesis biomarkers in sentinel lymph nodes (SLNs) with melanoma micrometastases. DESIGN Prognostic study of an inception cohort. SETTING Academic research. Patients Between July 1, 1999, and July 31, 2002, all patients who had primary cutaneous or mucosal melanomas that have a Breslow depth of 1.5 mm or greater, ulceration, or Clark level IV or V, or had SLN biopsies. MAIN OUTCOME MEASURES By the use of quantitative reverse transcription-polymerase chain reaction, the expression of the following was analyzed in SLNs: 2 melanocytic differentiation antigens (tyrosinase [P17646] and melanoma antigen recognized by T cells [MART-1; Q16655]) and genes involved in angiogenesis (VEGF [NM_001025366] and VEGFR2 [AF035121]), lymphangiogenesis (VEGFC [NM_005429], VEGFR3 [X68203], LYVE1 [NM_016164], and PROX1 [002763]), and invasion (uPA [NM_002658], PAI1 [NM_00602], and EMMPRIN [L10240]). Outcome measures were the association of these melanocytic differentiation antigens and angiogenesis biomarkers with clinicopathologic characteristics of patients, and an evaluation of the prognostic value for relapse-free survival and overall survival. RESULTS Ninety-one patients were included, with a median follow-up period of 41 months. Micrometastases were present in 15% (14 of 91) of patients. Tyrosinase (P < .001), MART-1 (P < .001), vascular endothelial growth factor 121 (VEGF(121)) (P = .007), and PAI1 (P = .02) expression was significantly associated with micrometastasis. In univariate analysis, histologic findings and tyrosinase and MART-1 expression were significantly associated with relapse-free survival. Tyrosinase and MART-1 expression was associated with overall survival. A multiple Cox proportional hazards regression model identified negative histologic findings and tyrosinase expression that exceeded 27 copies/copy of TATA box-binding protein (third quartile) as significantly associated with an increased risk of relapse or death. CONCLUSIONS Quantitative assessment of melanocytic differentiation antigens in SLNs, which has prognostic value, is more specific than qualitative assessment. Prognosis may be more effectively predicted by the combination of quantitative assessment of melanocytic differentiation antigens in SLNs with histologic assessment. A significant association was found between the presence of micrometastases and the expression of angiogenesis biomarkers.

IL-2 production by dendritic cells is not critical for the activation of cognate and innate effectors in draining lymph nodes

Dendritic cells (DC) are unique antigen‐presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL‐2 production by mouse DC. Previous reports demonstrated that IL‐2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC‐derived IL‐2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone morrow‐derived DC results in early IL‐2 production unless IL‐4 is introduced in DC cultures. Here we show that IL‐2 produced by LPS‐activated DC is dispensable for cognate T cell responses since IL‐2 loss of function DC elicit OVA‐specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL‐2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL‐2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.