Safety and immunologic correlates of Melanoma GVAX, a GM-CSF secreting allogeneic melanoma cell vaccine administered in the adjuvant setting.

BACKGROUND: Limited adjuvant treatment options exist for patients with high-risk surgically resected melanoma. This first-in-human study investigated the safety, tolerability and immunologic correlates of Melanoma GVAX, a lethally irradiated granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting allogeneic whole-cell melanoma vaccine, administered in the adjuvant setting. METHODS: Patients with stage IIB-IV melanoma were enrolled following complete surgical resection. Melanoma GVAX was administered intradermally once every 28 days for four cycles, at 5E7 cells/cycle (n = 3), 2E8 cells/cycle (n = 9), or 2E8 cells/cycle preceded by cyclophosphamide 200 mg/m(2) to deplete T regulatory cells (Tregs; n = 8). Blood was collected before each vaccination and at 4 and 6 months after treatment initiation for immunologic studies. Vaccine injection site biopsies and additional blood samples were obtained 2 days after the 1st and 4th vaccines. RESULTS: Among 20 treated patients, 18 completed 4 vaccinations. Minimal treatment-related toxicity was observed. One patient developed vitiligo and patches of white hair during the treatment and follow-up period. Vaccine site biopsies demonstrated complex inflammatory infiltrates, including significant increases in eosinophils and PD-1+ lymphocytes from cycle 1 to cycle 4 (P < 0.05). Serum GM-CSF concentrations increased significantly in a dose-dependent manner 48 h after vaccination (P = 0.0086), accompanied by increased numbers of activated circulating monocytes (P < 0.0001) and decreased percentages of myeloid-derived suppressor cells among monocytes (CD14+ , CD11b+ , HLA-DR low or negative; P = 0.002). Cyclophosphamide did not affect numbers of circulating Tregs. No significant changes in anti-melanoma immunity were observed in peripheral T cells by interferon-gamma ELIPSOT, or immunoglobulins by serum Western blotting. CONCLUSION: Melanoma GVAX was safe and tolerable in the adjuvant setting. Pharmacodynamic testing revealed complex vaccine site immune infiltrates and an immune-reactive profile in circulating monocytic cell subsets. These findings support the optimization of Melanoma GVAX with additional monocyte and dendritic cell activators, and the potential development of combinatorial treatment regimens with synergistic agents. TRIAL REGISTRATION: NCT01435499.

Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma.

BACKGROUND: Specific populations of highly tumorigenic cells are thought to exist in many human tumors, including pancreatic adenocarcinoma. However, the clinical significance of these tumor-initiating (ie, cancer stem) cells remains unclear. Aldehyde dehydrogenase (ALDH) activity can identify tumor-initiating cells and normal stem cells from several human tissues. We examined the prognostic significance and functional features of ALDH expression in pancreatic adenocarcinoma. METHODS: ALDH expression was analyzed by immunohistochemistry in 269 primary surgical specimens of pancreatic adenocarcinoma and examined for association with clinical outcomes and in paired primary tumors and metastatic lesions from eight pancreatic cancer patients who had participated in a rapid autopsy program. The clonogenic growth potential of ALDH-positive pancreatic adenocarcinoma cells was assessed in vitro by a colony formation assay and by tumor growth in immunodeficient mice (10-14 mice per group). Mesenchymal features of ALDH-positive pancreatic tumor cells were examined by using quantitative reverse transcription-polymerase chain reaction and an in vitro cell invasion assay. Gene expression levels and the invasive potential of ADLH-positive pancreatic cancer cells relative to the bulk cell population were examined by reverse transcription-polymerase chain reaction and an in vitro invasion assays, respectively. All statistical tests were two-sided. RESULTS: ALDH-positive tumor cells were detected in 90 of the 269 primary surgical specimens, and their presence was associated with worse survival (median survival for patients with ALDH-positive vs ALDH-negative tumors: 14 vs 18 months, hazard ratio of death = 1.28, 95% confidence interval = 1.02 to 1.68, P = .05). Six (75%) of the eight patients with matched primary and metastatic tumor samples had ALDH-negative primary tumors, and in four (67%) of these six patients, the matched metastatic lesions (located in liver and lung) contained ALDH-positive cells. ALDH-positive cells were approximately five- to 11-fold more clonogenic in vitro and in vivo compared with unsorted or ALHD-negative cells, expressed genes consistent with a mesenchymal state, and had in vitro migratory and invasive potentials that were threefold greater than those of unsorted cells. CONCLUSIONS: ALDH expression marks pancreatic cancer cells that have stem cell and mesenchymal features. The enhanced clonogenic growth and migratory properties of ALDH-positive pancreatic cancer cells suggest that they play a key role in the development of metastatic disease that negatively affects the overall survival of patients with pancreatic adenocarcinoma.

Microarray profiles of human basal cell carcinoma: insights into tumor growth and behavior.

PURPOSE: Basal cell carcinoma (BCC) is the most common human neoplasm. Much interest lies in determining the genetic basis of BCC to explain the unique locally invasive phenotype and infrequent metastatic behavior of these skin tumors. OBJECTIVE: We sought to examine a gene expression profile for BCC to elucidate new molecules responsible for its unique growth characteristics. METHODS: We analyzed gene expression patterns of 50 BCC tumors using spotted cDNA microarrays of 1718 characterized human genes related to cancer and immunity. This is the largest and most comprehensive gene expression study ever performed for BCC. Nodular and sclerosing histological subtypes of BCC were examined and compared to normal control skin. After statistical filtering, 374 significantly dysregulated genes were sorted by hierarchical clustering to determine trends of gene expression and similarities between patient gene expression profiles. RESULTS: A total of 165 upregulated genes and 115 downregulated genes were identified. These covered a range of categories, including extracellular matrix, cell junctions, motility, metastasis, oncogenes, tumor suppressors, DNA repair, cell cycle, immune regulation and angiogenesis. Clusters of genes were either commonly dysregulated across the 50 patient sample, or selectively affected in subsets of tumors. Histological subtypes were not distinguishable by hierarchical clustering. Many of the genes elucidated, including collagen type IV subunits and other novel candidates, possess functions related to extracellular matrix remodeling and metastasis. CONCLUSION: These results suggest a gene profile which may explain the invasive growth yet rarely metastatic behavior of BCC. The genes identified may also be potential targets for therapeutics aimed at further controlling invasiveness and local destruction of BCC.

Anti-vascular endothelial growth factor receptor-2 (Flk-1/KDR) antibody suppresses contact hypersensitivity.

The angiogenic mediator vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) have been studied extensively in neoplastic disease and some inflammatory conditions. Contact hypersensitivity (CHS) is a prototypic Langerhans' cell-dependent, T-helper (Th) 1 cell-mediated inflammatory skin disease that is now also thought to involve angiogenic mediators. The purpose of our study was to examine the role of angiogenesis and VEGF in CHS. We demonstrated that VEGF production is up-regulated in murine skin after challenge with dinitrofluorobenzene. Administration of a monoclonal antibody directed against the VEGFR-2 (DC101) resulted in a 28.8% decrease in CHS response (P < 0.001). Examination of the DC101-treated mouse skin 24 h after challenge revealed decreases in dermal inflammatory cellular infiltrates and total vessel area. Furthermore, mRNA and protein of the Th1-type cytokine interferon (IFN)-gamma was significantly down-regulated in skin of DC101-treated animals 24 h after challenge. The results of the study demonstrate that VEGFR-2 blockade significantly reduces vascular enlargement and edema formation and effects IFN-gamma expression in the skin during challenge in CHS. Our findings suggest that DC101 could function by reducing inflammatory cell migration and hence IFN-gamma expression during the CHS response.

Cytokine knockouts in contact hypersensitivity research.

Contact hypersensitivity (CHS) is a Langerhans cell (LC)-dependent, T cell-mediated cutaneous immune response. CHS reflects a culmination of LC activities in vivo: uptake of epicutaneous antigens, migration into lymph nodes, and presentation of antigens to naïve T cells. Although studies have suggested involvement of the cytokine network in LC migration and CHS initiation, the in vivo function of individual cytokines remains largely unknown. Gene targeting technology has made it possible to study in vivo functions of cytokines through gene-targeted knockout (KO) mice deficient in a given cytokine or its receptor. A variety of cytokine knockouts have been used to assign biological functions to specific cytokines in CHS. These studies have contributed significantly to our understanding of molecular mechanisms underlying CHS.

Low-dose ultraviolet B rays alter the mRNA expression of the circadian clock genes in cultured human keratinocytes.

Current understanding of mammalian circadian rhythms suggests that they are regulated by light targeting signaling pathways in the hypothalamic suprachiasmatic nuclei. Recently, investigators have identified the existence of extraretinal photoreceptors and a potential role for the skin in this regulatory process has been implied. We demonstrated that mRNA of the circadian clock genes Per1, Clock, and bmal1/mop3 are expressed in normal human cultured keratinocytes. Low-dose ultraviolet B rays initially downregulate all circadian clock genes and then induce altered expression of the genes in keratinocyte cell cultures. Ultraviolet light targeting superficial layers of skin (keratinocytes) may therefore contribute to circadian rhythm modulation.

Contribution of Langerhans cell-derived IL-18 to contact hypersensitivity.

The epidermal Langerhans cells (LC), a member of the dendritic cell family, and the LC-derived cytokine IL-12 play a pivotal role in the initiation of contact hypersensitivity (CHS), a Th1 immune response in the skin. Because IL-18, another LC-derived cytokine, shares functional and biological properties with IL-12, we examined a potential role for IL-18 in CHS initiation. Our studies demonstrated that during the induction phase of murine CHS, IL-18 mRNA was significantly up-regulated in the skin-draining lymph nodes (LN). Migratory hapten-modified LC in LN expressed high levels of IL-18 mRNA and secreted functional IL-18 protein. LN cells produced significant amounts of IFN-gamma following in vitro IL-12 stimulation, which could be partially blocked by anti-IL-18 Ab, suggesting a synergistic role for endogenous IL-18 in IFN-gamma production by LN cells. Because mature IL-18 requires cleavage of immature precursors by caspase-1, we further examined IL-12-induced IFN-gamma production in caspase-1(-/-) LN cells. An impaired IFN-gamma production was seen in caspase-1(-/-) LN cells, which could be restored by addition of exogenous IL-18, supporting a role for caspase-1-cleaved, mature IL-18 in IFN-gamma production. Finally, in vivo studies showed that CHS responses were significantly inhibited in mice treated with neutralizing IL-18 Ab as well as in caspase-1(-/-) mice deficient in mature IL-18, indicating functional relevance for IL-18 in CHS. Taken together, our studies demonstrate that LC-derived IL-18 significantly contributes to CHS initiation.