Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma.

BACKGROUND: Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity. Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness. As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression. RESULTS: Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters. SOX9 is methylated and lowly expressed in the highly proliferative group. SOX9 overexpression results in decreased proliferation but increased invasion in vitro. In a B16 mouse model, sox9 overexpression increases the number of lung metastases. Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways. Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates. Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients. CONCLUSIONS: Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups. One of the genes regulated by DNA methylation between the two groups is SOX9. SOX9 induces melanoma cell invasion and metastasis and decreases patient survival. A number of genes downregulated by SOX9 have a negative impact on patient survival. In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.

Clinical implications of distinct metastasizing preferences of different melanoma subtypes.

BACKGROUND: The incidence and mortality of malignant melanoma have been rising during the past decades, the latter being due to the high invasion capacity and the metastatic potential of melanoma cells to distant organs. OBJECTIVE: We investigated the distribution pattern of melanoma metastases taking into account different clinicopathological subtypes of melanoma. METHODS: We studied 310 stage IV (AJCC 2009) melanoma patients retrospectively with regard to potential correlations between frequency and occurrence of metastasis and the genetic background and pathological/clinical melanoma subtypes. For all patients, the time to distant metastasis (TTDM) and the distribution patterns of metastases were analyzed and correlated to the median survival time. RESULTS: Superficially Spreading (SSM) and Nodular melanomas (NMM) spread to the brain more frequently than Acrolentiginous (ALM) and Mucosal (MM) melanomas (p = 0.0012). The preference to affect the skeleton was significantly higher for ALM and MM in comparison to SSM and NMM (p = 0.0049). Lentigo maligna (LMM) tumors showed a significantly lower metastatic spread to distant lymph nodes (p = 0.0159). BRAF mutant versus wildtype tumors showed no significant differences concerning localization of metastasis but patients with BRAF mutant tumors were significantly younger at primary diagnosis and had a significantly shorter stage IV survival (p = 0.0106). CONCLUSION: This study shows a clear distinction of melanoma subtypes with regard to metastasizing preferences. Further knowledge about melanoma subtype specific characteristics, including molecular markers predictive of homing preferences, may help to understand and manage this heterogeneous disease in terms of prognosis and follow-up procedures.

RASopathic skin eruptions during vemurafenib therapy.

PURPOSE: Vemurafenib is a potent inhibitor of V600 mutant BRAF with significant impact on progression-free and overall survival in advanced melanoma. Cutaneous side effects are frequent. This single-center observational study investigates clinical and histological features of these class-specific cutaneous adverse reactions. PATIENTS AND METHODS: Patients were all treated with Vemurafenib 960 mg b.i.d. within local ethic committees approved clinical trials. All skin reactions were collected and documented prospectively. Cutaneous reactions were classified by reaction pattern as phototoxic and inflammatory, hair and nail changes, keratinocytic proliferations and melanocytic disorders. RESULTS: Vemurafenib was well tolerated, only in two patients the dose had to be reduced to 720 mg due to arthralgia. 26/28 patients (93%) experienced cutaneous side effects. Observed side effects included UVA dependent photosensitivity (n = 16), maculopapular exanthema (n = 14), pruritus (n = 8), folliculitis (n = 5), burning feet (n = 3), hair thinning (mild alopecia) (n = 8), curly hair (n = 2) and nail changes (n = 2). Keratosis pilaris and acanthopapilloma were common skin reactions (n = 12/n = 13), as well as plantar hyperkeratosis (n = 4), keratoacanthoma (n = 5) and invasive squamous cell carcinoma (n = 4). One patient developed a second primary melanoma after more than 4 months of therapy (BRAF and RAS wild type). CONCLUSION: Vemurafenib has a broad and peculiar cutaneous side effect profile involving epidermis and adnexa overlapping with the cutaneous manifestations of genetic diseases characterized by activating germ line mutations of RAS (RASopathy). They must be distinguished from allergic drug reaction. Regular skin examination and management by experienced dermatologists as well as continuous prophylactic photo protection including an UVA optimized sun screen is mandatory.

Aldara activates TLR7-independent immune defence.

Aldara is a cream used for topical treatment of non-melanoma skin cancer, and is thought to act through stimulation of anti-tumour immunity. The active ingredient, imiquimod, has been shown to stimulate toll-like receptor 7. Aldara also induces psoriasis-like lesions when applied to naive murine skin, and as such is used as a mouse model for psoriasis. Here we find that in naive murine skin, Aldara induces inflammation largely independently of toll-like receptor 7. Surprisingly, inflammasome activation, keratinocyte death and interleukin 1 release also occur in response to the vehicle cream in the absence of imiquimod. We show that isostearic acid, a major component of the vehicle, promotes inflammasome activation in cultured keratinocytes, and so may contribute to the observed effects of Aldara on murine skin. Aldara therefore stimulates at least two immune pathways independently, and both imiquimod and vehicle are required for a full inflammatory response. Although it remains to be tested, it is possible that imiquimod-independent effects also contribute to the therapeutic efficacy of Aldara.

Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system.

UNLABELLED: Formalin fixation and paraffin embedding present the standard procedures for conserving clinical tissues for histological analysis. However, molecular analysis is impaired by the cross linking properties of formalin. The PAXgene tissue system (PreAnalytix, Switzerland) is a new formalin-free tissue collection device. AIMS: In this study we aimed to evaluate this new tissue preservation technique in comparison with formalin fixation and fresh frozen tissue samples. METHODS: 12 melanoma biopsy samples were divided and fixed simultaneously with formalin, PAXgene or fresh frozen in liquid nitrogen and analysed with regard to morphology, immunohistochemistry,  DNA and RNA content and quality. Markers of melanocytic differentiation and tumour cell proliferation were used. RESULTS: Morphology was well preserved in PAXPE samples. However, 5 out of 11 immunohistochemical markers showed significantly lower overall staining and staining intensity with PAXPE tissues in comparison with formalin-fixed, paraffin-embedded (FFPE). Increasing membrane permeability through adding a detergent did proportionally increase staining intensity in PAXPE samples. Amplification of different mRNA amplicons showed a direct relationship with the size of the amplicon with greater template integrity observed in PAXPE samples. Sequencing and mutational analysis of DNA samples were comparable for all the different fixation methods, while the level of DNA fragmentation seemed to be lower in PAXPE compared with FFPE tissues. CONCLUSIONS: The switch from formalin to PAXgene fixation would require a re-evaluation of immunohistochemical markers and staining procedures originally developed for FFPE tissues. Our data demonstrate that PAXPE fixation offers some advantages concerning molecular analysis. However, these advantages would not justify substituting formalin fixation in any routine pathology laboratory.

Cutaneous drug eruptions associated with the use of new oncological drugs.

There is a variety of adverse effects and toxicities of newer and older chemotherapeutic agents which emerge in the skin, mucosa and adnexa. Common skin reactions while undergoing chemotherapy include alopecia, changes in skin pigmentation, palmoplantar erythrodysesthesia, nail dystrophies and stomatitis. Extravasation injuries or hypersensitivity reactions may be severe. New oncologic agents have led to the development of different, class-specific cutaneous side effects. Epidermal growth factor receptor (EGFR) inhibitors induce papulopustular rashes in a high percentage of patients as well as, to a smaller degree, xerosis cutis, hair and nail changes, hyper pigmentation and enhancement of radiation dermatitis. Multikinase inhibitors will often cause hand-foot syndrome, but may also induce facial erythema, subungual splinter hemorrhages and other less frequent skin changes. BRAF inhibitors can lead to rash and development of cutaneous keratinocytic neoplasias for which patients should be closely monitored. Finally, MEK/ERK inhibitors induce similar skin toxicities to EGFR inhibitors such as papulopustular rashes, xerosis cutis and paronychia. Our chapter will focus on the clinical picture, histopathology and treatment options of these new class-specific cutaneous side effects.

Sinonasal, genital and acrolentiginous melanomas show distinct characteristics of KIT expression and mutations.

KIT aberrations predict the outcome of targeted therapies in acrolentiginous (ALM) and mucosal (MM) melanoma patients. KIT immunoreactivity and mutation status was assessed in 41 ALM and 25 MM patients. Of these, 19 ALM and 15 MM patients had matched primary and metastatic lesions. P-ERK was investigated in a subset of 9 ALM and 7 MM matched primary/metastatic pairs by immunohistochemistry. Heterogeneous KIT immunoreactivity was observed in both primary and metastatic lesions. Mutations were present in four of 41 ALM (10%) and five of 25 MM (20%) patients. Only vulvar mucosal samples carried KIT mutations in contrast to sinonasal lesions (p = 0.0109). In KIT-mutated tumours, the mutations were present in KIT expressing as well as KIT negative cells, as shown by Laser Capture Microdissection (LCM). P-ERK expression was preferentially found in metastases. KIT mutations predict treatment outcome with KIT inhibitors. Therefore, especially vulvar melanoma patients should be screened for activating KIT mutations.

Recent advances in the management of cutaneous lymphomas.

The treatment options for patients with primary cutaneous B- and T-cell lymphomas are as diverse as the diseases themselves, including both skin-directed and systemic therapies. Long-term remission can be attained in many cases; however, no treatment is curative with the possible exception of allogeneic stem cell transplant. Improved insight into the molecular biology and pathophysiology of these diseases has led to the development of novel drugs and targeted therapies, including monoclonal antibodies and antimetabolites, as well as improved radiotherapeutic techniques. We aim to summarize these new and emerging treatment modalities, and to outline how they may be integrated into the clinical management of patients with primary cutaneous lymphoma (CL).

Systematic classification of melanoma cells by phenotype-specific gene expression mapping.

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.

Atypical diaper dermatitis: contact allergy to mercapto compounds.

We present a case of allergic contact dermatitis in an 18-month-old boy caused by type-IV allergy to mercapto mix and mercaptobenzothiazole as components of the elastic border of diapers. Allergic contact dermatitis should be included in the differential diagnosis of diaper dermatitis, especially in difficult-to-treat cases or atypical clinical presentation.

Prognostic value of [18F]-fluoro-deoxy-glucose PET/CT, S100 or MIA for assessment of cancer-associated mortality in patients with high risk melanoma.

PURPOSE: To assess the prognostic value of FDG PET/CT compared to the tumor markers S100B and melanoma inhibitory activity (MIA) in patients with high risk melanoma. METHODS: Retrospective study in 125 consecutive patients with high risk melanoma that underwent FDG PET/CT for re-staging. Diagnostic accuracy and prognostic value was determined for FDG PET/CT as well as for S100B and MIA. As standard of reference, cytological, histological, PET/CT or MRI follow-up findings as well as clinical follow-up were used. RESULTS: Of 125 patients, FDG PET/CT was positive in 62 patients. 37 (29.6%) patients had elevated S100B (>100 pg/ml) and 24 (20.2%) had elevated MIA (>10 pg/ml) values. Overall specificities for FDG PET/CT, S100B and MIA were 96.8% (95% CI, 89.1% to 99.1%), 85.7% (75.0% to 92.3%), and 95.2% (86.9% to 98.4%), corresponding sensitivities were 96.8% (89.0% to 99.1%), 45.2% (33.4% to 55.5%), and 36.1% (25.2% to 48.6%), respectively. The negative predictive values (NPV) for PET/CT, S100B, and MIA were 96.8% (89.1% to 99.1%), 61.4% (50.9% to 70.9%), and 60.6% (50.8% to 69.7%). The positive predictive values (PPV) were 96.7% (89.0% to 99.1%), 75.7% (59.9% to 86.6%), and 88.0% (70.0% to 95.8%). Patients with elevated S100B- or MIA values or PET/CT positive findings showed a significantly (p<0.001 each, univariate Cox regression models) higher risk of melanoma associated death which was increased 4.2-, 6.5- or 17.2-fold, respectively. CONCLUSION: PET/CT has a higher prognostic power in the assessment of cancer-associated mortality in melanoma patients compared with S100 and MIA.

Different patterns of fibronectin and tenascin-C splice variants expression in primary and metastatic melanoma lesions.

We have investigated the staining patterns of primary and metastatic melanoma lesions using F8, L19 and F16. These three clinical-stage antibodies are currently being studied in clinical trials for the pharmacodelivery of cytokines or therapeutic radionuclides to neoplastic sites in patients with cancer. Frozen sections of 24 primary and 29 metastatic melanoma lesions were stained, using immunofluorescence procedures, with biotinylated preparations of the F8, L19 and F16 antibodies, which are specific to the alternatively spliced extra domain A and extra domain B domains of fibronectin and A1 domain of tenascin-C, respectively. Blood vessels were costained using von Willebrand factor-specific antibodies. In primary cutaneous melanoma lesions, F16 and F8 (but not L19) strongly stained the basal lamina at the interface between epidermis and dermis, with a strikingly complementary pattern. By contrast, metastatic melanoma lesions displayed a strong and diffuse pattern of immunoreactivity with all three antibodies. It was found that the extracellular matrix in melanoma undergoes extensive remodelling during the transition from primary to metastatic lesions. The intense staining of metastatic melanoma lesions by the F8, L19 and F16 antibodies provides a strong rationale for the use of these antibodies and their derivatives for the treatment of melanoma patients and possibly for the personalized choice of the best performing antibody in individual patients.

Human CD271-positive melanoma stem cells associated with metastasis establish tumor heterogeneity and long-term growth.

Human melanoma is composed of distinct cell types reminiscent of neural crest derivatives and contains multipotent cells that express the neural crest stem cell markers CD271(p75(NTR)) and Sox10. When isolated from solid tumors by using a method that leaves intact cell surface epitopes, CD271-positive, but not CD271-negative, cells formed tumors on transplantation into nude or nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. These tumors fully mirrored the heterogeneity of the parental melanoma and could be passaged more than 5 times. In contrast, in more immunocompromised NOD/SCID/IL2rγ(null) mice, or in natural killer cell-depleted nude or NOD/SCID mice, both CD271-positive and CD271-negative tumor cell fractions established tumors. However, tumors resulting from either fraction did not phenocopy the parental tumors, and tumors derived from the CD271-negative cell fraction could not be passaged multiple times. Together, our findings identify CD271-positive cells as melanoma stem cells. Our observation that a relatively high frequency of CD271/Sox10-positive cells correlates with higher metastatic potential and worse prognosis further supports that CD271-positive cells within human melanoma represent genuine cancer stem cells.

Prevalence of MCPyV in Merkel cell carcinoma and non-MCC tumors.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is the likely causative agent of Merkel cell carcinoma (MCC). However, the prevalence of MCPyV in non-MCC population and its possible role in the pathogenesis of other skin cancers are not known yet. METHODS: A molecular pathology study was performed in 33 MCC samples and 33 age- and sex-matched samples of sun exposed non-MCC tumors [12 seborrheic keratoses (SK), 11 basal cell carcinomas (BCC) and 10 lentigo maligna melanomas (LMM)]. All tumors were analyzed for presence of MCPyV-DNA by polymerase chain reaction (PCR) and Southern-Blot hybridization of PCR products. RESULTS: MCPyV sequences were detected in 21 MCC samples (64%) and in 2 non-MCC tumors of sun exposed skin (6%; both SK-patients). Neither the tissue samples from BCC nor LMM proved positive for MCPyV sequences. CONCLUSION: We were able to confirm prior data on prevalence of MCPyV-DNA in MCC. Furthermore, a female predominance of MCPyV-positive MCC-patients was detected. There was no relevant association of MCPyV with SK, BCC and LMM. Speculative, prevalence of MCPyV in an age- and sex-matched non-MCC population could average up to 6%.