In an attempt to diagnose a suspected amelanotic melanoma tumour, we examined a variety of tissue and cell samples from one patient at the ultrastructural level, which consisted of single cell suspensions of tumour cells with and without DOPA treatment, tumour cells after culture in agar with and without DOPA treatment, and single tumour cells hetero-transplanted into a nude mouse. Premalanosomes were not observed in sections of the amelanotic tumour with routine electron microscopy. Osmophilic-dense bodies, suggestive of melanosomes, were noted in the single cells in suspension treated with DOPA and cells grown in agar without DOPA treatment. Definitive premelanosomes, with an identifiable striated matrix, were only observed in cells grown into colonies in agar and treated with DOPA. Positive L-DOPA reaction products were noted in the golgi complex, endoplasmic reticulum closely related to the golgi (GERL), and in vacuoles from cells grown in agar. As controls, Cloudman S91 53.1 melanoma cells were evaluated as single cells in suspension or as colonies after culture in agar, both with and without DOPA treatment. Premelanosomes were always observed in this established melanoma cell line while DOPA-treated cells contained positive L-DOPA reaction products. The overall findings identified the tumour as amelanotic melanoma and indicated that both DOPA treatment and culture in agar were needed for the demonstration of premelanosomes.

Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson's Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best's carmine, Page-Green method for inclusion bodies, and Snook's reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis.

Advanced melanoma is considered the most aggressive and deadly form of skin cancer whose incidence has been rising over the past three decades. In the absence of treatment, the median overall survival for advanced-stage metastatic disease is less than 6 months. Although most melanomas detected at an early stage can be cured with surgery, a subset of these eventually metastasize. Therefore, a critical need exists to identify unique molecular features that would be predictive of long-term outcome and response to specific therapies. Recent promising therapeutic regimens have included the use of immune checkpoint inhibitors, such as anti-PD1 antibodies. However, the ability to identify responders and non-responders to this therapy remains elusive. To address this challenge at the molecular level, previously our laboratory identified the emergence of a stem cell phenotype associated with advanced melanoma and other aggressive forms of cancer. Underlying this phenotype is the aberrant re-expression of the embryonic morphogen Nodal. Particularly noteworthy, we have observed Nodal to remain in advanced tumors of non-responders to standard-of-care therapies (i.e., BRAFi). This pilot study is the first proof-of-principle attempt to predict treatment response survival outcome in a small cohort of melanoma patients receiving anti-PD1 immune checkpoint inhibitor therapy - based on their Nodal expression profile. Using advanced multiplex immunohistochemistry-based digital pathology, the major finding of this preliminary study indicates that higher Nodal expression is often associated with poorer overall survival after anti-PD1 therapy, reaching nearly statistical relevance.

Abstract Progress in understanding the molecular basis of melanoma has made possible the identification of molecular targets with important implications in clinical practice. In fact, new therapeutic approaches are emerging from basic science and it will be important to implement their rapid translation into clinical practice by active clinical investigation. The first meeting of Melanoma Research: a bridge Naples-USA, organized by Paolo A. Ascierto (INT, Naples, Italy) and Francesco Marincola (NIH, Bethesda, USA) took place in Naples, on 6-7 December 2010. This international congress gathered more than 30 international and Italian faculty members and was focused on recent advances in melanoma molecular biology, immunology and therapy, and created an interactive discussion across Institutions belonging to Government, Academy and Pharmaceutical Industry, in order to stimulate new approaches in basic, translational and clinical research. Four topics of discussion were identified: New pathways in Melanoma, Biomarkers, Clinical Trials and New Molecules and Strategies.