The common heat shock protein receptor CD91 is up-regulated on monocytes of advanced melanoma slow progressors

Despite advances in our understanding of tumour immunology there is no therapy of proven survival benefit for advanced melanoma. Nevertheless, disease progression is slow in a small proportion of patients with metastatic melanoma, suggesting a contribution to outcome from host factors. Recent data have indicated the importance of the heat shock protein receptor CD91 in immune responses to, and progression of, infectious disease. Here we investigate the relationship between CD91 expression and outcome in malignancy. Rare melanoma patients were recruited with advanced disease that was progressing unusually slowly. CD91 expression on their monocytes was compared with control patients with more typical rapidly advancing metastatic disease. Th1 and Th2 cytokines, as well as innate and adaptive immune subsets, were also measured in the two groups. A significant increase in median CD91 expression levels was observed in slow progressors (P = 0.006). There were no differences in other immune subset markers or inflammatory cytokines. The ability of CD91 to internalize and cross-present tumour antigens through the major histocompatibility complex class I pathway may maintain CD8-positive cytotoxic T cell responses and contribute to slow progression of advanced melanoma.     

How morphometric analysis of metastatic load predicts the (un)usefulness of PET scanning: the case of lymph node staging in melanoma

BACKGROUND: In primary cutaneous melanoma, the sentinel node (SN) biopsy is an accurate method for the staging of the lymph nodes. Positron emission tomography (PET) has been suggested as a useful alternative. However, the sensitivity of PET may be too low to detect SN metastases, which are often small. AIM: To predict the value of PET for initial lymph node staging in melanoma based on morphometric analysis of SN metastatic load, without exposing patients to PET. MATERIALS AND METHODS: In 59 SN positive patients with melanoma, the sizes of tumour deposits in the SNs and subsequent dissection specimens were measured by morphometry and correlated with the detection limits of current and future PET scanners. RESULTS: The median tumour volume within the basin was 0.15 mm(3) (range, 0.0001-118.86). Seventy per cent of these deposits were smaller than 1 mm(3). State of the art PET scanners that have a resolution of about 5 mm would detect only 15-49% of positive basins. Logistic regression analysis revealed no pretest indicators identifying patients expected to have a positive PET. However, the SN tumour load was a significant and single predictor of the presence of PET detectable residual tumour. CONCLUSION: Morphometric analysis of metastatic load predicts that PET scanning is unable to detect most metastatic deposits in sentinel lymph nodes of patients with melanoma because the metastases are often small. Therefore, the SN biopsy remains the preferred method for initial regional staging.     

Cutaneous pH landscape as a facilitator of melanoma initiation and progression

Melanoma incidence is on the rise and currently causes the majority of skin cancer‐related deaths. Yet, therapies for metastatic melanoma are still insufficient so that new concepts are essential. Malignant transformation of melanocytes and melanoma progression are intimately linked to the cutaneous pH landscape and its dysregulation in tumour lesions. The pH landscape of normal skin is characterized by a large pH gradient of up to 3 pH units between surface and dermis. The Na⁺/H⁺ exchanger NHE1 is one of the major contributors of acidity in superficial skin layers. It is also activated by the most frequent mutation in melanoma, BRAF^V600E, thereby causing pH dysregulation during melanoma initiation. Melanoma progression is supported by an extracellular acidification and/or NHE1 activity which promote the escape of single melanoma cells from the primary tumour, migration and metastatic spreading. We propose that viewing melanoma against the background of the acid‐base physiology of the skin provides a better understanding of the pathophysiology of this disease and allows the development of novel therapeutic concepts.     

Main roads to melanoma

The characterization of the molecular mechanisms involved in development and progression of melanoma could be helpful to identify the molecular profiles underlying aggressiveness, clinical behavior, and response to therapy as well as to better classify the subsets of melanoma patients with different prognosis and/or clinical outcome. Actually, some aspects regarding the main molecular changes responsible for the onset as well as the progression of melanoma toward a more aggressive phenotype have been described. Genes and molecules which control either cell proliferation, apoptosis, or cell senescence have been implicated. Here we provided an overview of the main molecular changes underlying the pathogenesis of melanoma. All evidence clearly indicates the existence of a complex molecular machinery that provides checks and balances in normal melanocytes. Progression from normal melanocytes to malignant metastatic cells in melanoma patients is the result of a combination of down- or up-regulation of various effectors acting on different molecular pathways.     

Immunohistochemistry in melanocytic proliferative lesions

Melanoma incidence is rising worldwide. Early diagnosis is very important, as the most effective treatment for melanoma still consists of excision of the tumour before onset of the metastatic growth phase. Immunohistochemistry is a valuable tool for (dermato)pathologists to aid establishing diagnosis. Melanoma markers can be classified into two main categories: melanocytic differentiation markers and melanoma progression markers. Melanocytic differentiation markers are mostly used to distinguish poorly differentiated melanomas from non-melanocytic tumours and for staging of melanocytic proliferative lesions. Melanoma progression markers are most suitable to determine the level of malignancy and/or aggressiveness of tumour cells. This review describes the classification of melanoma markers, including commonly used and recently identified antigens with potential marker function. We characterize their expression profile in melanocytic proliferative lesions and their potential usefulness for diagnosis, prognosis, microstaging, immunotherapeutic purposes and evaluation of therapies.     

Tenascin-C in primary malignant melanoma of the skin

AIMS: To investigate the expression and the prognostic role of glycoprotein Tenascin-C (Tn-C) in primary melanoma of the skin. METHODS AND RESULTS: The immunohistochemical expression of Tn-C was studied in 98 primary melanomas and related to inflammation, invasion, and patient outcome. Patients were followed up for disease recurrence for 0.04-7.4 years (median 3.9) and for survival for 0.5 to 12.1 years (median 9.3). The expression of Tn-C was evaluated for each tumour invasion border; the stromal and intracytoplasmic Tn-C of the melanoma islets were also recorded. Tn-C is widely expressed in primary melanoma samples, the staining pattern varying from focal to diffuse in different parts of the tumour. No correlation existed between intensity of Tn-C staining and inflammation. No stromal Tn-C was detected at the upper dermal lateral border in 12 patients, nor at the deep, dermal or subcutaneous border in 14 patients. These patients showed better disease-free survival (DFS) than did those cases with focal or diffuse staining (P = 0.06, P = 0.05). Also, absence of intracytoplasmic Tn-C was a beneficial prognostic factor for DFS (P = 0.04). In multivariate analysis, tumour ulceration and intracytoplasmic Tn-C expression of melanoma cells were independent adverse prognostic factors for DFS. CONCLUSIONS: In primary melanoma of the skin, absence of Tn-C in the stroma of invasion fronts and within tumour cells seems to be related to a more benign disease behaviour with a lower risk of developing metastases.     

In-transit metastatic cutaneous melanoma: current management and future directions

Management of in-transit melanoma encompasses a variety of possible treatment pathways and modalities. Depending on the location of disease, number of lesions, burden of disease and patient preference and characteristics, some treatments may be more beneficial than others. After full body radiographic staging is performed to rule out metastatic disease, curative therapy may be performed through surgical excision, intraarterial regional perfusion and infusion therapies, intralesional injections, systemic therapies or various combinations of any of these. While wide excision is limited in indication to superficial lesions that are few in number, the other listed therapies may be effective in treating unresectable disease. Where intraarterial perfusion based therapies have been shown to successfully treat extremity disease, injectable therapies can be used in lesions of the head and neck. Although systemic therapies for in-transit melanoma have limited specific data to support their primary use for in-transit disease, there are patients who may not be eligible for any of the other options, and current clinical trials are exploring the use of concurrent and sequential use of regional and systemic therapies with early results suggesting a synergistic benefit for oncologic response and outcomes.

     

Expression of interleukin-8 detected by in situ hybridization correlates with worse prognosis in primary cutaneous melanoma

Previous in vitro studies have demonstrated that endogenously produced human interleukin-8 (IL-8) can act as an important growth factor for human melanoma cells in vitro. The present study, has investigated whether IL-8 mRNA expression in primary melanomas may be of prognostic relevance with regard to melanoma progression and metastatic spread. In order to evaluate the clinical significance of IL-8 mRNA expression of melanoma cells in vivo, 59 melanocytic tissue specimens (37 primary melanomas and 22 melanocytic naevi) were studied using a semiquantitative in situ hybridization technique. Significant mRNA expression of IL-8 was found in 59 per cent (22/37) of melanomas. In 19 per cent (7/37) of the malignant melanomas, additional hybridization signals were noted within keratinocytes of the overlying epidermis. In contrast, paralesional normal-appearing epidermis and melanocytes in non-malignant lesions (melanocytic naevi) showed no IL-8 mRNA. Analysis of the relationship between IL-8 expression and clinico-histopathological features showed a significant association between IL-8 mRNA expression and the histological melanoma subtype (IL-8 mRNA: 14/19 in superficial spreading melanoma versus 4/12 in nodular melanoma, p< 0.05). Furthermore, IL-8 expression in primary tumours could be correlated with the patients' clinical course, with time to progression being significantly reduced in primary tumours expressing IL-8 in either the tumour cells or keratinocytes of the overlying epidermis. These results demonstrate for the first time that IL-8 expression, as detected by in situ hybridization in primary tumours, may serve as a significant prognostic factor for tumour progression in human malignant melanoma.     

A putative marker for human melanoma. A monoclonal antibody derived from the melanoma gene in the Xiphophorus melanoma model.

A MoAb was raised against a peptide corresponding to an exposed domain of the putative tyrosine kinase receptor protein encoded by Xmrk, a gene involved in melanoma formation and/or progression in the Xiphophorus fish melanoma model. The antibody reacts specifically with cells from human melanocytic lesions, ie, common acquired nevi, primary and metastatic melanoma biopsies. No reactivity with other cells, including normal melanocytes, was observed in the biopsies or with cells in biopsies from normal tissue (skin, liver, lung, spleen) and from other malignancies including those of neuroectodermal origin. The reactivity was very weak and variable in metastatic melanomas but very strong and characteristic of a receptor-type antigen in primary melanomas, a stage in melanoma progression in which cells have acquired metastasizing potential. It is suggested that the antigen recognized may be involved in growth promotion and represents the human equivalent of the fish melanoma gene product.     

Malignant peripheral nerve sheath tumour-like primary cutaneous malignant melanoma

Malignant melanoma is known for its protean cytomorphological features, architectural patterns, and stromal changes, in addition to its ability to mimic various benign and malignant non-melanocytic tumours. Anecdotal cases of metastatic malignant melanoma simulating soft tissue sarcomas have been reported. Interestingly, this mimicry is more often seen in recurrent lesions and metastatic deposits. This report describes a case of a primary spindle cell cutaneous malignant melanoma with a prominent neural-like fascicular pattern and nuclear palisading, simulating a conventional malignant peripheral nerve sheath tumour (MPNST). Clinical, microscopic, and immunohistochemical features of the different entities included in the differential diagnosis of cutaneous spindle cell malignant tumours, such as MPNST, atypical fibroxanthoma, and spindle cell squamous cell carcinoma are discussed. Of note, the presence of an atypical epidermal or junctional component, cell pigmentation, and cell nesting, in addition to diffuse and strong reactivity for S-100 protein and other melanocytic markers, are helpful in the diagnosis of these troublesome lesions.     

The morphologic characteristics of tumor blood vessels as a marker of tumor progression in primary human uveal melanoma: a matched case-control study.

Nine morphologic patterns of tumor vessels were identified in eyes removed for ciliary body or choroidal melanoma by the examination of tissue sections stained with fluorescein-conjugated Ulex europaeus I using laser scanning confocal microscopy. This technique also highlights intravascular tumor invasion. Each of these nine morphologic patterns of tumor vessels also may be demonstrated by a modification of the periodic acid-Schiff reaction, viewed with a green narrow band pass filter, but this modified histochemical technique does not accurately identify intravascular tumor invasion. Most tumors have a heterogeneous distribution of vascular patterns. Melanomas in two groups of 20 tumors each were matched by tumor size and location (one group of tumors from patients who survived at least 15 years free of metastatic melanoma after enucleation and one group of tumors from patients who died of metastatic melanoma). A matched case-control analysis indicates that the presence of at least one closed vascular loop in a uveal melanoma is the most significant vascular pattern associated with death from metastatic melanoma after enucleation. Closed loops are associated with other histologic features that are predictive of an unfavorable outcome after enucleation: epithelioid cells and mitotic figures. In this preliminary study the formation of closed vascular loops is a marker of tumor progression in ciliary body and choroidal melanomas.     

New avenues for melanoma immunotherapy: Natural Killer cells?

Human solid malignant tumours may be particularly resistant to conventional therapies. Among solid tumours, immunological features of cutaneous melanoma have been well characterized in the past and today melanoma patients are routinely treated with the anti-immune checkpoints immunotherapy that has completely changed metastatic melanoma treatment and prognosis. Two cytotoxic cell populations may lead to the physical elimination of tumour cell targets: cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Tumour recognition by CTLs depends on major histocompatibility complex (MHC) class I molecules, while NK cells recognize tumours expressing low or null levels of MHC class I molecules. Despite this well-established complementarity, NK cells are still left behind in the optimization of innovative immunotherapy approaches. NK cells are members of innate lymphoid cells (ILCs) that play a critical role in early host defence against invading pathogens and transformed cells. Recent findings suggest that NK cell frequencies directly correlate with the overall survival of ipilimumab-treated melanoma patients. Furthermore, in vitro and in vivo evidences indicate that NK cells can selectively kill cancer stem cells, reducing tumour size and delaying metastatic progression. The aim of this review is to provide a survey of the evidences indicating NK cells as an excellent candidate to complement the newest solid tumour immunotherapy approaches.     

Malignant melanoma

CONTEXT: The rapidly developing fields of melanoma research are revolutionizing the current concepts on melanoma etiology and pathogenesis and are introducing newer diagnostic techniques and potential therapeutic approaches. OBJECTIVES: To present the most current concepts on the etiology and pathogenesis of melanoma and to introduce the recent diagnostic techniques and the potential therapeutic approaches. METHODS: Data sources were reports on melanoma published in the English language literature and observations made using specimens available at Harvard University, Johns Hopkins Medical Center, Albany Medical College, Loyola University Medical Center, and University of Tennessee Health Science Center. RESULTS: Studies on melanoma containing chromosomal or genetic evaluation were selected for further analysis. Current clinical and pathologic categories with the reported genetic abnormalities were related to the latest information on pigment biology. The data extracted were used to develop a conceptual framework on the pathogenesis of melanoma; the generated model was then evaluated and used to suggest potential therapeutic approaches. CONCLUSIONS: (1) Melanoma is not genetically homogeneous, and the existing differences between the pathologic categories, particularly in areas such as type of growth phase (radial vs vertical growth), total vertical dimension, ulceration of primary tumor, and metastatic process, have profound prognostic and therapeutic implications. (2) Chromosomal aberrations and gene mutations are found in sporadic and familial melanomas; among the most important are those affecting the 9p21, which contains the p16 locus, a site known to be critical for normal progression of the cell cycle. Aberrant p16 expression is associated with more aggressive behavior. (3) Melanoma cells possess a remarkable repertoire of biosynthetic capacities represented by the production of hormones, growth factors, and their receptors that may sustain and accelerate tumor development and progression. For example, expression of the tumoral products alpha-melanocyte-stimulating hormone and adrenocorticotropic hormone is regulated in vitro by ultraviolet light, a known carcinogen. (4) Melanomas differ from other tumors in their intrinsic capability to express melanogenic enzymes with the corresponding structural proteins to actually synthesize melanin. Melanogenesis-related proteins are rapidly entering the clinical arena, being used not only as diagnostic markers, but also as potential targets for melanoma therapy.     

Nestin and vimentin colocalization affects the subcellular location of glucocorticoid receptor in cutaneous melanoma

Aims: Nestin (a neuronal stem cell/progenitor cell marker of central nervous system development), vimentin (which is ubiquitously expressed in mesenchymal cells), and the glucocorticoid receptor (GR, which is involved in the immune response, cell proliferation, and apoptosis) have been shown to interact in embryonic and undifferentiated tissues in modulating cell proliferation. The aim of this study was to analyse nestin, vimentin and GR expression in tumour tissue (melanoma), and their association with clinicopathological variables, to evaluate any effect on tumour progression. Methods and results: Immunohistochemistry, double‐label immunofluorescence and confocal laser scanning microscopy were performed on biopsy specimens of cutaneous melanoma from 81 patients. Fisher’s and Pearson’s tests showed a correlation between nestin, vimentin and subcellular GR location (P = 0.008). Their concomitant expression also correlated with Clark level and thickness (P = 0.02 and P = 0.029, respectively). Kaplan–Meier analysis revealed a poorer outcome for stage III and IV patients with associated expression of nestin, vimentin and cytoplasmic GR in tumour tissue (P = 0.02). Conclusions: These results suggest the presence in melanoma of growth mechanisms involving nestin, vimentin, and GR, similarly to that occurring in embryonic and undifferentiated cells, and may help in understanding tumour biology to provide a molecular basis for clinical therapies.     

Relationship between responsiveness of cancer cells to Oncostatin M and/or IL-6 and survival of stage III melanoma patients treated with tumour-infiltrating lymphocytes

Immunotherapy by adoptive transfer of autologous tumour-infiltrating lymphocytes (TIL) shows promising clinical results for stage III (lymph nodes metastasis) melanoma patients, but some of them remain unresponsive. Here we analysed retrospectively the impact of resistance of melanoma cells to anti-proliferative cytokines on the clinical outcome of 24 TIL-treated metastatic melanoma patients. Patient relapse-free survival correlated significantly with Oncostatin M (OSM) and/or IL-6 sensitivity of melanoma cells, but not with interferon (IFN) gamma or tumour necrosis factor (TNF) alpha sensitivity. However, OSM/IL-6 sensitivity did not correlate with other known prognostic factors. Moreover, OSM and IL-6 were produced by TIL just before their injection to patients. In immunodeficient mice, OSM reduced human melanoma xenograft tumour growth, this effect being directly through inhibition of tumour cell proliferation rather than induction of apoptosis or necrosis. Thus, OSM/IL-6 resistance of melanoma cells appears to be a new escape mechanism to TIL treatment that could be added to the existing prognostic factors for early stage melanoma patients. This mechanism of action could be also relevant in other immunotherapy protocols, and could lead to better prognosis and anti-cancer treatments.     

HLA allele frequency and clinical outcome in Italian patients with cutaneous melanoma

The current study focuses the analysis on the possible relationship between HLA allele frequency and clinical outcome of melanoma in a population of 382 Italian patients, as compared with 203 ethnically matched controls. In a 3-year follow-up study, results showed significant differences between groups of patients selected according to clinical stage, histology, and progression of the disease. A*01 seems to be correlated with a less aggressive variant of the disease, whereas DRB1*01-DQB1*0501 seems to be associated with metastatic progression of melanoma. Moreover, a negative association with B*13, B*44, as well as with DRB1*04-DQB1*0302 was found. A multivariate logistic regression model showed HLA-DRB1*04 to behave as an independent favorable prognostic marker of melanoma in our population (OR = 2.34, CI = 1.15-4.74).     

Targeted therapy in melanoma: the era of personalized medicine

Malignant melanoma is the most aggressive of all cutaneous tumours, with over 76, 000 new cases and 9700 deaths estimated for 2014 in the United States.¹ In Canada, both the incidence and mortality of melanoma are increasing, with a risk of developing melanoma being 1 in 59 for men and 1 in 73 for women.² The incidence of melanoma is higher in Australia, with a risk of 1 in 14 for males and 1 in 23 for females to age 85 reported for 2009.³ Although early melanoma can be managed surgically, until recently there have been few advances in the treatment of advanced melanoma. However, with the introduction of molecular targeted therapies, the landscape of melanoma treatment has changed dramatically in the past five years, resulting in improved survival rates for patients with metastatic disease. In this review, we will discuss the molecular basis and implementation for some of these novel treatments with particular emphasis on BRAF and BRAF inhibitors.     

Ocular melanoma: an overview of the current status

Ocular melanoma is the second most common type of melanoma after cutaneous and the most common primary intraocular malignant tumor in adults. Large majority of ocular melanomas originate from uvea, while conjunctival melanomas are far less frequent. Incidence of uveal melanoma has remained stable over last three decades. Diagnosis is in most cases established by clinical examination with great accuracy. Local treatment of uveal melanoma has improved, with increased use of conservative methods and preservation of the eye, but survival rates have remained unchanged. Recent advances in cytogenetics and genetics enhanced prognostication and enabled to determine tumors with high metastatic potential. However, due to lack of effective systemic therapy, prognosis of patients with metastasis remains poor and metastatic disease remains the leading cause of death among patients with uveal melanoma. Conjunctival melanoma is rare, but its incidence is increasing. It mostly occurs among white adults. In majority of cases it originates from preceding primary acquired melanosis. Current standard treatment for conjunctival melanoma is wide local excision with adjuvant therapy, including brachytherapy, cryotherapy and topical application of chemotherapeutic agent. Rarity of this tumor limits conduction of controlled trials to define the best treatment modality. As well as for uveal melanoma, prognosis of patients with metastasis is poor because there is no effective systemic therapy. Better understanding of underlying genetic and molecular abnormalities implicated in development and progression of ocular melanomas provides a great opportunity for development of targeted therapy, which will hopefully improve prognosis of patients with metastatic disease.