Knockdown of Simulated-Solar-Radiation-Sensitive miR-205-5p Does Not Induce Progression of Cutaneous Squamous Cell Carcinoma In Vitro.

Solar radiation is the main risk factor for cSCC development, yet it is unclear whether the progression of cSCC is promoted by solar radiation in the same way as initial tumorigenesis. Additionally, the role of miRNAs, which exert crucial functions in various tumors, needs to be further elucidated in the context of cSCC progression and connection to solar radiation. Thus, we chronically irradiated five cSCC cell lines (Met-1, Met-4, SCC-12, SCC-13, SCL-II) with a custom-built irradiation device mimicking the solar spectrum (UVB, UVA, visible light (VIS), and near-infrared (IRA)). Subsequently, miRNA expression of 51 cancer-associated miRNAs was scrutinized using a flow cytometric multiplex quantification assay (FirePlex®, Abcam). In total, nine miRNAs were differentially expressed in cell-type-specific as well as universal manners. miR-205-5p was the only miRNA downregulated after SSR-irradiation in agreement with previously gathered data in tissue samples. However, inhibition of miR-205-5p with an antagomir did not affect cell cycle, cell growth, apoptosis, or migration in vitro despite transient upregulation of oncogenic target genes after miR-205-5p knockdown. These results render miR-205-5p an unlikely intracellular effector in cSCC progression. Thus, effects on intercellular communication in cSCC or the simultaneous examination of complementary miRNA sets should be investigated.

CD74 and CD44 Expression on CTCs in Cancer Patients with Brain Metastasis.

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.

The German SCREEN project--design and evaluation of the communication strategy.

BACKGROUND: Skin cancer is the most common cancer in light-skinned populations worldwide. Primary and secondary preventive activities such as skin cancer screening are intended to reduce skin cancer burden. In 2003, a population-based skin cancer screening project [SCREEN (Skin Cancer Research to Provide Evidence for Effectiveness of Screening in Northern Germany)] was conducted in Northern Germany with more than 360,000 people screened. SCREEN was supported by a communication intervention that was aimed at informing the population about skin cancer, its risk factors and the screening intervention as well as preparing the health professionals for the project. Within SCREEN both physicians and practice nurses were educated in counselling. The aim of the present article is to describe and evaluate the communication strategy accompanying SCREEN. METHODS: Two computer-assisted telephone interview surveys were performed in April/May 2003 and May 2004. Participants had to be members of the statutory health insurance and be aged 20 years. They were asked about knowledge of skin cancer, perception of physicians' performance and skin cancer screening in general. Data are mainly presented in a descriptive manner. For statistical analyses, Mann-Whitney U test and Pearson's chi-square test were used. RESULTS: Knowledge about sunburn in childhood and high ultraviolet exposure as skin cancer risk factors increased during SCREEN. Simultaneously, the awareness for early detection of skin cancer increased significantly from 41.3 to 74.0% (P < 0.001). A total of 21.5% of the interviewees participated in the skin cancer screening project, similar to the population-based participation rate reached. CONCLUSION: A comprehensive communication strategy accompanying a screening intervention improves the knowledge of potential screenees and may additionally increase the participation rate.

Health initiatives for the prevention of skin cancer.

Skin cancer is the most frequent type of cancer in white population worldwide. However, because the most prominent risk factor-solar UV-radiation and/or artificial UV from sunbeds-is known, skin cancer is highly preventable be primary prevention. This prevention needs, that the public is informed by simple and balanced messages about the possible harms and benefits of UV-exposure and how a person should behave under certain conditions of UV-exposure. For this purpose information and recommendations for the public must be age- and target-group specific to cover all periods of life and to reach all sub-groups of a population, continuously. There is a need that political institutions together with Health Institutions and Societies (e.g., European Commission, WHO, EUROSKIN, ICNIRP, etc.), which are responsible for primary prevention of skin cancer, find a common language to inform the public, in order not to confuse it. This is especially important in connection with the ongoing Vitamin D debate, where possible positive effects of UV have to be balanced with the well known skin cancer risk of UV. A continuously ongoing evaluation of interventions and programs in primary prevention is a pre-requisite to assess the effectiveness of strategies. There is surely no "no message fits all" approach, but balanced information in health initiatives for prevention of skin cancer, which use evidence-base strategies, will further be needed in the future to reduce the incidence, morbidity and mortality skin cancer.

Antimicrobial activity of NK cells to Trypanosoma cruzi infected human primary Keratinocytes.

Infection with the protozoan parasite Trypanosoma cruzi is causative for Chagas disease, which is a highly neglected tropical disease prevalent in Latin America. Humans are primary infected through vectorial transmission by blood-sucking triatomine bugs. The parasite enters the human host through mucous membranes or small skin lesions. Since keratinocytes are the predominant cell type in the epidermis, they play a critical role in detecting disruptions in homeostasis and aiding in pathogen elimination by the immune system in the human skin as alternative antigen-presenting cells. Interestingly, keratinocytes also act as a reservoir for T. cruzi, as the skin has been identified as a major site of persistent infection in mice with chronic Chagas disease. Moreover, there are reports of the emergence of T. cruzi amastigote nests in the skin of immunocompromised individuals who are experiencing reactivation of Chagas disease. This observation implies that the skin may serve as a site for persistent parasite presence during chronic human infection too and underscores the significance of investigating the interactions between T. cruzi and skin cells. Consequently, the primary objective of this study was to establish and characterize the infection kinetics in human primary epidermal keratinocytes (hPEK). Our investigation focused on surface molecules that either facilitated or hindered the activation of natural killer (NK) cells, which play a crucial role in controlling the infection. To simulate the in vivo situation in humans, an autologous co-culture model was developed to examine the interactions between T. cruzi infected keratinocytes and NK cells. We evaluated the degranulation, cytokine production, and cytotoxicity of NK cells in response to the infected keratinocytes. We observed a strong activation of NK cells by infected keratinocytes, despite minimal alterations in the expression of activating or inhibitory ligands on NK cell receptors. However, stimulation with recombinant interferon-gamma (IFN-γ), a cytokine known to be present in significant quantities during chronic T. cruzi infections in the host, resulted in a substantial upregulation of these ligands on primary keratinocytes. Overall, our findings suggest the crucial role of NK cells in controlling acute T. cruzi infection in the upper layer of the skin and shed light on keratinocytes as potential initial targets of infection.

Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma.

Circulating tumor DNA (ctDNA) is the cornerstone of liquid biopsy diagnostics, revealing clinically relevant genomic aberrations from blood of cancer patients. Genomic analysis of single circulating tumor cells (CTCs) could provide additional insights into intra-patient heterogeneity, but it requires whole-genome amplification (WGA) of DNA, which might introduce bias. Here, we describe a novel approach based on mass spectrometry for mutation detection from individual CTCs not requiring WGA and complex bioinformatics pipelines. After establishment of our protocol on tumor cell line-derived single cells, it was validated on CTCs of 33 metastatic melanoma patients and the mutations were compared to those obtained from tumor tissue and ctDNA. Although concordance with tumor tissue was superior for ctDNA over CTC analysis, a larger number of mutations were found within CTCs compared to ctDNA (p = 0.039), including mutations in melanoma driver genes, or those associated with resistance to therapy or metastasis. Thus, our results demonstrate proof-of-principle data that CTC analysis can provide clinically relevant genomic information that is not redundant to tumor tissue or ctDNA analysis.

Skin cancers are the most frequent cancers in fair-skinned populations, but we can prevent them.

Cancers of the skin are the most commonly occurring cancers in humans. In fair-skinned populations, up to 95% of keratinocyte skin cancers and 70-95% of cutaneous melanomas are caused by ultraviolet radiation and are thus theoretically preventable. Currently, however, there is no comprehensive global advice on practical steps to be taken to reduce the toll of skin cancer. To address this gap, an expert working group comprising clinicians and researchers from Africa, America, Asia, Australia, and Europe, together with learned societies (European Association of Dermato-Oncology, Euromelanoma, Euroskin, European Union of Medical Specialists, and the Melanoma World Society) reviewed the extant evidence and issued the following evidence-based recommendations for photoprotection as a strategy to prevent skin cancer. Fair skinned people, especially children, should minimise their exposure to ultraviolet radiation, and are advised to use protective measures when the UV index is forecast to reach 3 or higher. Protective measures include a combination of seeking shade, physical protection (e.g. clothing, hat, sunglasses), and applying broad-spectrum, SPF 30 + sunscreens to uncovered skin. Intentional exposure to solar ultraviolet radiation for the purpose of sunbathing and tanning is considered an unhealthy behaviour and should be avoided. Similarly, use of solaria and other artificial sources of ultraviolet radiation to encourage tanning should be strongly discouraged, through regulation if necessary. Primary prevention of skin cancer has a positive return on investment. We encourage policymakers to communicate these messages to the general public and promote their wider implementation.

Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

Dermal stem cells (DSCs), which are progenitor cells of melanocytes, are isolated from human foreskin and cultivated as mixed cultures containing both DSCs and fibroblasts in varying proportions. These contaminating fibroblasts may have an impact on the results of experimental studies and are a serious limitation for certain applications. The aim of the present study was to purify or enrich DSCs-an indispensable step towards future investigations. Applying different methods, we demonstrated that highly enriched DSCs with a good recovery rate can be obtained through positive selection with MACS® immunomagnetic cell sorting. These DSCs remain vital and proliferate constantly in culture, maintaining a high level of purity after enrichment. Other approaches such as treatment with Geneticin or selective detachment were not suitable to purify DSC-fibroblast co-cultures. Overall, enriched DSCs represent a novel and unique model to study the effects of UV radiation on the differentiation of DSCs into melanocytes and their potential relevance in the genesis of malignant melanoma.

Does skin cancer screening save lives?: an observational study comparing trends in melanoma mortality in regions with and without screening.

BACKGROUND: From July 1, 2003 to June 30, 2004, a population-based skin cancer screening project was conducted in Schleswig-Holstein, Germany. In total, 360,288 individuals aged ≥20 years were screened by means of a whole-body examination. In this report, the authors compare trends in melanoma mortality in Schleswig-Holstein with those in all adjacent regions, none of which had population-based skin cancer screening. METHODS: Trends in melanoma mortality rates for Schleswig-Holstein and the adjacent regions (Denmark and the German federal states of Mecklenburg-Vorpommern, Hamburg, and Lower Saxony) and in Germany excluding Schleswig-Holstein were compared. Log-linear regression was used to assess mortality trends. RESULTS: In Schleswig-Holstein during the pre skin cancer screening period (1998-1999), the age-standardized melanoma mortality rate (World standard population) was 1.9 per 100,000 for men and 1.4 per 100,000 for women. Melanoma mortality declined by 47% to 1.0 per 100,000 men and by 49% to 0.7 per 100,000 women by 2008/2009. The annual percentage change in the most recent 10-year period (2000-2009) was -7.5% (95% confidence interval, -14.0, -0.5) for men and -7.1% (95% confidence interval, -10.5, -2.9) for women. In each of the 4 adjacent regions and in the rest of Germany, mortality rates were stable, and the decline in Schleswig-Holstein was significantly different from the changes observed in all of the other areas studied. CONCLUSIONS: The current data represent strong evidence, but not absolute proof, that the skin cancer screening program produced a reduction in melanoma mortality in Schleswig-Holstein.

UVA-induced epigenetic regulation of P16(INK4a) in human epidermal keratinocytes and skin tumor derived cells.

UVA-radiation (315-400 nm) has been demonstrated to be capable of inducing DNA damage and is regarded as a carcinogen. While chromosomal aberrations found in UVA-irradiated cells and skin tumors provided evidence of the genetic involvement in UVA-carcinogenesis, its epigenetic participation is still illusive. We thus analysed the epigenetic patterns of 5 specific genes that are involved in stem cell fate (KLF4, NANOG), telomere maintenance (hTERT) and tumor suppression in cell cycle control (P16(INK4a), P21(WAFI/CIPI)) in chronically UVA-irradiated HaCaT human keratinocytes. A striking reduction of the permissive histone mark H3K4me3 has been detected in the promoter of P16(INK4a) (4-fold and 9-fold reduction for 10 and 15 weeks UVA-irradiated cells, respectively), which has often been found deregulated in skin cancers. This alteration in histone modification together with a severe promoter hypermethylation strongly impaired the transcription of P16(INK4a) (20-fold and 40-fold for 10 weeks and 15 weeks UVA-irradiation, respectively). Analysis of the skin tumor-derived cells revealed the same severe impairment of the P16(INK4a) transcription attributed to promoter hypermethylation and enrichment of the heterochromatin histone mark H3K9me3 and the repressive mark H3K27me3. Less pronounced UVA-induced epigenetic alterations were also detected for the other genes, demonstrating for the first time that UVA is able to modify transcription of skin cancer associated genes by means of epigenetic DNA and histone alterations.

Systematic skin cancer screening in Northern Germany.

BACKGROUND: The incidence of skin cancer is increasing worldwide. For decades, opportunistic melanoma screening has been carried out to respond to this burden. However, despite potential positive effects such as reduced morbidity and mortality, there is still a lack of evidence for feasibility and effectiveness of organized skin cancer screening. OBJECTIVE: The main aim of the project was to evaluate the feasibility of systematic skin cancer screening. METHODS: In 2003, the Association of Dermatological Prevention was contracted to implement the population-based SCREEN project (Skin Cancer Research to Provide Evidence for Effectiveness of Screening in Northern Germany) in the German state of Schleswig-Holstein. A two-step program addressing malignant melanoma and nonmelanocytic skin cancer was implemented. Citizens (aged ≥ 20 years) with statutory health insurance were eligible for a standardized whole-body examination during the 12-month study period. Cancer registry and mortality data were used to assess first effects. RESULTS: Of 1.88 million eligible citizens, 360,288 participated in SCREEN. The overall population-based participation rate was 19%. A total of 3103 malignant skin tumors were found. On the population level, invasive melanoma incidence increased by 34% during SCREEN. Five years after SCREEN a substantial decrease in melanoma mortality was seen (men: observed 0.79/100,000 and expected 2.00/100,000; women: observed 0.66/100,000 and expected 1.30/100,000). LIMITATIONS: Because of political reasons (resistance as well as lack of support from major German health care stakeholders), it was not possible to conduct a randomized controlled trial. CONCLUSIONS: The project showed that large-scale systematic skin cancer screening is feasible and has the potential to reduce skin cancer burden, including mortality. Based on the results of SCREEN, a national statutory skin cancer early detection program was implemented in Germany in 2008.

UVB damage response of dermal stem cells as melanocyte precursors compared to keratinocytes, melanocytes, and fibroblasts from human foreskin.

Ultraviolet B (UVB) radiation induces mutagenic DNA photolesions in skin cells especially in form of cyclobutane pyrimidine dimers (CPDs). Protection mechanisms as DNA repair and apoptosis are of great importance in order to prevent skin carcinogenesis. In human skin, neural crest-derived precursors of melanocytes, the dermal stem cells (DSCs), are discussed to be at the origin of melanoma. Although they are constantly exposed to solar UV radiation, it is still not investigated how DSCs cope with UV-induced DNA damage. Here, we report a comparative study of the DNA damage response after irradiation with a physiological relevant UVB dose in DSCs in comparison to fibroblasts, melanocytes and keratinocytes isolated from human foreskin. Within our experimental settings, DSCs were able to repair DNA photolesions as efficient as the other skin cell types with solely keratinocytes repairing significantly faster. Interestingly, only fibroblasts showed significant alterations in cell cycle distribution in terms of a transient S phase arrest following irradiation. Moreover, with the applied UVB dose none of the examined cell types was prone to UVB-induced apoptosis. This may cause persistent genomic alterations and in case of DSCs it may have severe consequences for their daughter cells, the differentiated melanocytes. Altogether, this is the first study demonstrating a similar UV response in dermal stem cells compared to differentiated skin cells.

Effect of tumour-cell-derived or recombinant keratinocyte growth factor (KGF) on proliferation and radioresponse of human epithelial tumour cells (HNSCC) and normal keratinocytes in vitro.

Purpose of this work was to test the effect of tumour-cell-derived keratinocyte growth factor (KGF) or recombinant KGF (palifermin) on cell proliferation and radiation response of human HNSCC cells and normal keratinocytes in vitro. Four tumour cell cultures derived from head and neck squamous cell carcinomas, primary keratinocytes, and immortalized keratinocytes were analysed. Fibroblasts, the natural source of KGF protein, served as controls. KGF expression was observed in primary and immortalized keratinocytes, fibroblasts, and in tumour cells, while significant KGF receptor expression was only found in keratinocytes. Recombinant KGF as well as tumour-cell-derived KGF caused a significant growth stimulation and radioprotection in keratinocytes, which was abolished by a neutralizing anti-KGF antibody. This indicates that tumour-cell-derived KGF is biologically active. In the tumour cell lines, no significant growth stimulation was induced by recombinant KGF, and the neutralizing antibody did not influence tumour cell growth or radiation response. Our results indicate that the normal, paracrine KGF regulatory mechanisms, which are based on KGF receptor expression, are lost in malignant cells, with the consequence of irresponsiveness of the tumour cells to exogenous KGF. In face of the amelioration of the radiation response of normal epithelia, demonstrated in various clinical and various preclinical animal studies, recombinant KGF represents a candidate for the selective protection of normal epithelia during radio(chemo) therapy of squamous cell carcinoma.

Characterisation of the novel spontaneously immortalized and invasively growing human skin keratinocyte line HaSKpw.

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) "premalignant" keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

UV-type specific alteration of miRNA expression and its association with tumor progression and metastasis in SCC cell lines.

PURPOSE: UV exposure is the main risk factor for development of cutaneous squamous cell carcinoma (cSCC). While early detection greatly improves cSCC prognosis, locally advanced or metastatic cSCC has a severely impaired prognosis. Notably, the mechanisms of progression to metastatic cSCC are not well understood. We hypothesized that UV exposure of already transformed epithelial cSCC cells further induces changes which might be involved in the progression to metastatic cSCCs and that UV-inducible microRNAs (miRNAs) might play an important role. METHODS: Thus, we analyzed the impact of UV radiation of different quality (UVA, UVB, UVA + UVB) on the miRNA expression pattern in established cell lines generated from primary and metastatic cSCCs (Met-1, Met-4) using the NanoString nCounter platform. RESULTS: This analysis revealed that the expression pattern of miRNAs depends on both the cell line used per se and on the quality of UV radiation. Comparison of UV-induced miRNAs in cSCC cell lines established from a primary tumor (Met-1) and the respective (un-irradiated) metastasis (Met-4) suggest that miR-7-5p, miR-29a-3p and miR-183-5p are involved in a UV-driven pathway of progression to metastasis. This notion is supported by the fact that these three miRNAs build up a network of 81 potential target genes involved e.g. in UVA/UVB-induced MAPK signaling and regulation of the epithelial-mesenchymal transition. As an example, PTEN, a target of UV-upregulated miRNAs (miR-29a-3p, miR-183-5p), could be shown to be down-regulated in response to UV radiation. We further identified CNOT8, the transcription complex subunit 8 of the CCR4-NOT complex, a deadenylase removing the poly(A) tail from miRNA-destabilized mRNAs, in the center of this network, targeted by all three miRNAs. CONCLUSION: In summary, our results demonstrate that UV radiation induces an miRNA expression pattern in primary SCC cell line partly resembling those of metastatic cell line, thus suggesting that UV radiation impacts SCC progression beyond initiation.

Pre-analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients.

The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell-free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell-free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next-generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high-quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre-analytical variable.

Oxidative stress impairs the repair of oxidative DNA base modifications in human skin fibroblasts and melanoma cells.

Irradiation of mammalian cells with solar light is associated with the generation of reactive oxygen species (ROS) and oxidative stress, which is mediated in part by endogenous photosensitizers absorbing in the visible range of the solar spectrum. Accordingly, oxidative DNA base modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are the predominant types of DNA damage in cells irradiated at wavelengths >400 nm. We have analysed the repair of oxidative purine modifications in human skin fibroblasts and melanoma cells using an alkaline elution technique, both under normal conditions and after depletion of glutathione. Similar repair rates were observed in fibroblasts and melanoma cells from three different patients (t1/2 approximately 4h). In both cell types, glutathione depletion (increased oxidative stress) caused a pronounced repair retardation even under non-toxic irradiation conditions. Furthermore, the cleavage activity at 8-oxoG residues measured in protein extracts of the cells dropped transiently after irradiation. An addition of dithiothreitol restored normal repair rates. Interestingly, the repair rates of cyclobutane pyrimidine dimers (t1/2 approximately 18 h), AP sites (t1/2 approximately 1h) and single-strand breaks (t1/2 <30 min) were not affected by the light-induced oxidative stress. We conclude that the base excision repair of oxidative purine modifications is surprisingly vulnerable to oxidative stress, while the nucleotide excision repair of pyrimidine dimers is not.

Intra-Patient Heterogeneity of Circulating Tumor Cells and Circulating Tumor DNA in Blood of Melanoma Patients.

Despite remarkable progress in melanoma therapy, the exceptional heterogeneity of the disease has prevented the development of reliable companion biomarkers for the prediction or monitoring of therapy responses. Here, we show that difficulties in detecting blood-based markers, like circulating tumor cells (CTC), might arise from the translation of the mutational heterogeneity of melanoma cells towards their surface marker expression. We provide a unique method, which enables the molecular characterization of clinically relevant CTC subsets, as well as circulating tumor DNA (ctDNA), from a single blood sample. The study demonstrates the benefit of a combined analysis of ctDNA and CTC counts in melanoma patients, revealing that CTC subsets and ctDNA provide synergistic real-time information on the mutational status, RNA and protein expression of melanoma cells in individual patients, in relation to clinical outcome.

Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm.

New imaging techniques using near-infrared (NIR) femtosecond lasers (fs-lasers) in multiphoton laser scanning microscopy (MPLSM) have great potential for in vivo applications, particularly in human skin. However, little is known about possible risks. In order to evaluate the risk, a "biological dosimeter" was used. We irradiated fresh human skin samples with both an fs-laser and a solar simulator UV source (SSU). DNA damage introduced in the epidermis was evaluated using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immunofluorescence image analysis. Four fs-irradiation regimes (at 800-nm wavelength) were evaluated differing in laser power and step width of horizontal scans. Fs-irradiation did not give CPDs at 15-mW or 30-mW irradiation power using 10 horizontal scans every 5 microns. CPDs could be seen at 60-mW laser power and 5-microm step size and at 35-mW using 1-micron step width. Quantitative comparison of SSU-induced CPDs showed that the 60-mW laser irradiation regime is comparable to UV-irradiation, giving 0.6 minimal erythemal dose (MED). The 1-micron irradiation regime was comparable to 0.45 MED. Under these experimental conditions, the risk of DNA damage due to fs-laser irradiation on skin is in the range of natural UV-exposure.

Association between risk factors and detection of cutaneous melanoma in the setting of a population-based skin cancer screening.

Early detection is considered to improve the prognosis of cutaneous melanoma. The value of population-based screening for melanoma, however, is still controversial. The aim of this study was to evaluate the predictive power of established risk factors in the setting of a population-based screening and to provide empirical evidence for potential risk stratifications. We reanalyzed data (including age, sex, risk factors, and screening results) of 354 635 participants in the Skin Cancer Research to provide Evidence for Effectiveness of Screening in Northern Germany (SCREEN)project conducted in the German state of Schleswig-Holstein (2003-2004). In multivariable analysis, atypical nevi [odds ratio (OR): 17.4; 95% confidence interval (CI): 14.4-20.1], personal history of melanoma (OR: 5.3; 95% CI: 3.6-7.6), and multiple (≥40) common nevi (OR: 1.3; 95% CI: 1.1-1.6) were associated with an increased risk of melanoma detection. Family history and congenital nevi were not significantly associated with melanoma detection in the SCREEN. The effects of several risk-adapted screening strategies were evaluated. Hypothesizing a screening of individuals aged more than or equal to 35 years, irrespective of risk factors (age approach), the number needed to screen is 559 (95% CI: 514-612), whereas a screening of adults (aged ≥20) with at least one risk factor (risk approach) leads to a number needed to screen of 178 (95% CI: 163-196). Converted into one screen-detected melanoma, the number of missed melanomas is 0.15 (95% CI: 0.12-0.18) with the age approach and 0.22 (95% CI: 0.19-0.26) with the risk approach. The results indicate that focusing on individuals at high risk for melanoma may improve the cost-effectiveness and the benefit-to-harm balance of melanoma screening programs.