Skin Cancer Risk Is Increased by Somatic Mutations Detected Noninvasively in Healthy-Appearing Sun-Exposed Skin.

Skin cancer risk is increased by exposure to ultraviolet radiation (UVR). Because UVR exposure accumulates over time and lighter skin is more susceptible to UVR, age and skin tone are risk factors for skin cancer. However, measurements of somatic mutations in healthy-appearing skin have not been used to calculate skin cancer risk. In this study, we developed a noninvasive test that quantifies somatic mutations in healthy-appearing sun-exposed skin and applied it to a 1038-subject cohort. Somatic mutations were combined with other known skin cancer risk factors to train a model to calculate risk. The final model (DNA-Skin Cancer Assessment of Risk) was trained to predict personal history of skin cancer from age, family history, skin tone, and mutation count. The addition of mutation count significantly improved model performance (OR = 1.3, 95% confidence interval = 1.14-1.48; P = 5.3 × 10-6) and made a more significant contribution than skin tone. Calculations of skin cancer risk matched the known United States population prevalence, indicating that DNA-Skin Cancer Assessment of Risk was well-calibrated. In conclusion, somatic mutations in healthy-appearing sun-exposed skin increase skin cancer risk, and mutations capture risk information that is not accounted for by other risk factors. Clinical utility is supported by the noninvasive nature of skin sample collection through adhesive patches.

Corrigendum: Impact on clinical practice of a non-invasive gene expression melanoma rule-out test: 12-month followup of negative test results and utility data from a large US registry study.

The revised version of the article corrected Figure 2. This change appears in the revised online PDF copy of this article.

Impact on clinical practice of a non-invasive gene expression melanoma rule-out test: 12-month follow-up of negative test results and utility data from a large US registry study.

The Pigmented Lesion Assay (PLA, sensitivity 91-95%, specificity 69-91%, negative predictive value ?99%) is a commercially available, non-invasive gene expression test that helps dermatologists guide pigmented lesion management decisions and rule out melanoma. Earlier studies have demonstrated high clinical utility and no missed melanomas in a 3-6-month follow-up period. We undertook the current investigations to provide 12-month follow-up data on PLA(-) tests, and to further confirm utility. A 12-month chart review follow-up of 734 pigmented lesions that had negative PLA results from 5 US dermatology centers was performed. Thirteen of these lesions (1.8%) were biopsied in the follow-up period and submitted for histopathologic review. None of the lesions biopsied had a histopathologic diagnosis of melanoma. The test's utility was studied further in a registry (N=1575, 40 US dermatology offices, 62 participating providers), which demonstrated that 99.9% of PLA(-) lesions were clinically monitored, thereby avoiding a surgical procedure, and 96.5% of all PLA(+) lesions were appropriately biopsied, most commonly with a tangential shave. This long-term follow-up study confirms the PLA's high negative predictive value and high utility in helping guide the management of pigmented lesions to avoid unnecessary surgical procedures.

Noninvasive Analysis of High-Risk Driver Mutations and Gene Expression Profiles in Primary Cutaneous Melanoma.

Tools that help reduce the number of surgical biopsies performed on benign lesions have the potential to improve patient care. The pigmented lesion assay (PLA) is a noninvasive tool validated against histopathology that helps rule out melanoma and the need for surgical biopsies of atypical pigmented skin lesions. Genetic information is collected using adhesive patches and the expression of two genes, LINC and PRAME, is measured. By using genetic material collected noninvasively and to further validate the PLA, somatic hotspot mutations in genes known to be drivers of early melanoma development (BRAF other than V600E, NRAS, and the TERT promoter) can also be identified. The frequency of these hotspot mutations in samples of early melanoma was 77%, which is higher than the 14% found in nonmelanoma samples (P < 0.0001). TERT promoter mutations were the most prevalent mutation type in PLA-positive melanomas; 82% of PLA-negative lesions had no mutations, and 97% of histopathologically confirmed melanomas were PLA and/or mutation positive (cohort 1, n = 103). Mutation frequencies were similar in prospectively collected real-world PLA samples (cohort 2, n = 519), in which 88% of PLA-negative samples had no mutations. Combining gene expression and mutation analyses enhances the ability to noninvasively detect early cutaneous melanoma.

Gene Expression Analysis Differentiates Melanomas from Spitz Nevi.

INTRODUCTION: Pediatric Spitz nevi can pose significant diagnostic challenges to both clinicians and dermatopathologists when the current image-recognition based gold standard is employed. PRAME (preferentially expressed antigen in melanoma) and/or LINC (long intergeneic non-coding RNA 518) gene expression in adult patients in samples obtained non-invasively via adhesive patches differentiates primary melanomas from atypical nevi and other pigmented lesions with a NPV of over 99%, a sensitivity of 91%, and a specificity of 69%, to help clinicians rule out melanoma and the need for surgical biopsies of atypial pigmented lesions with suspicion for melanoma. Surgically obtained melanomas from adult patients show the same gene expression pattern. METHODS: In this study, we investigate gene expression patterns of pigmented lesions from FFPE tissue block samples (n=23, 9 male, 14 female patients, median age 12) with a focus on differentiating Spitz nevi from melanomas in children and young adults. RESULTS: PRAME levels were significantly (P less than 0.001) increased based on normalized Ct cycle counts (lower cycle counts indicate higher expression levels) in melanomas (mean Ct 33.83 + 0.54, 95% CI 32.85-34.80) when compared to Spitz nevi (mean Ct 37.21 + 0.98, 95% CI 35.41-39.01) or common nevi (mean Ct 36.94 + 0.80, 95% CI 35.47-38.40), respectively. LINC and 4 control genes showed similar expression levels in all 3 pigmented lesion groups investigated. Clinically and histopathologically complex pediatric Spitz nevi demonstrated gene expression signatures almost identical to gene expression signatures of common pediatric nevi but different from melanomas in children and young adults. DISCUSSION: PRAME but not LINC gene expression can be a valuable molecular aid to differentiate melanomas from Spitz nevi, groups of pigmented lesions that can be particularly difficult to assess in children and young adults. J Drugs Dermatol. 2018;17(5):574-576.

An Adhesive Patch-Based Skin Biopsy Device for Molecular Diagnostics and Skin Microbiome Studies.

INTRODUCTION: A number of diagnoses in clinical dermatology are currently histopathologically confirmed and this image recognition-based confirmation generally requires surgical biopsies. The increasing ability of molecular pathology to corroborate or correct a clinical diagnosis based on objective gene expression, mutation analysis, or molecular microbiome data is on the horizon and would be further supported by a tool or procedure to collect samples non-invasively. This study characterizes such a tool in form of a 'bladeless' adhesive patch-based skin biopsy device. METHODS: The performance of this device was evaluated through a variety of complementary technologies including assessment of sample biomass, electron microscopy demonstrating the harvesting of layers of epidermal tissue, and isolation of RNA and DNA from epidermal skin samples. Samples were obtained by application of adhesive patches to the anatomical area of interest. RESULTS: Biomass assessment demonstrated collection of approximately 0.3mg of skin tissue per adhesive patch and electron microscopy confirmed the nature of the harvested epidermal skin tissue. The obtained tissue samples are stored in a stable fashion on adhesive patches over a wide range of temperatures (-80oC to +60oC) and for extended periods of time (7 days or more). Total human RNA, human genomic DNA and microbiome DNA yields were 23.35 + 15.75ng, 27.72 + 20.71ng and 576.2 + 376.8pg, respectively, in skin samples obtained from combining 4 full patches collected non-invasively from the forehead of healthy volunteers. DISCUSSION: The adhesive patch skin sampling procedure is well tolerated and provides robust means to obtain skin tissue, RNA, DNA, and microbiome samples without involving surgical biopsies. The non-invasively obtained skin samples can be shipped cost effectively at ambient temperature by mail or standard courier service, and are suitable for a variety of molecular analyses of the skin microbiome as well as of keratinocytes, T cells, dendritic cells, melanocytes, and other skin cells involved in the pathology of various skin conditions and conditions where the skin can serve as a surrogate target organ.

J Drugs Dermatol. 2017;16(10):979-986.

Utility of a Noninvasive 2-Gene Molecular Assay for Cutaneous Melanoma and Effect on the Decision to Biopsy.

Importance: Expression of long intergenic non-protein coding RNA 518 (LINC00518) and preferentially expressed antigen in melanoma (PRAME) genes, obtained via noninvasive adhesive patch biopsy, is a sensitive and specific method for detection of cutaneous melanoma. However, the utility of this test in biopsy decisions made by dermatologists has not been evaluated. Objective: To determine the utility of the pigmented lesion assay (PLA) for LINC00518/PRAME expression in decisions to biopsy a series of pigmented skin lesions. Design, Setting, and Participants: In this secure web-based, multiple-reader-multiple-case study, 45 board-certified dermatologists each evaluated 60 clinical and dermoscopic images of clinically atypical pigmented lesions, first without and then with PLA gene expression information and were asked whether the lesions should be biopsied. Data were collected from March 24, 2014, through November 13, 2015. Interventions: Participants were given a report for each lesion, which included the results of an assay for expression of LINC00518/PRAME and a PLA score with data on the predictive values of the information provided. Main Outcomes and Measures: Biopsy sensitivity and specificity with vs without PLA data. Results: Forty-five dermatologists (29 male and 16 female) performed the evaluation. After incorporating the PLA into their decision as to whether to biopsy a pigmented lesion suggestive of melanoma, dermatologists improved their mean biopsy sensitivity from 95.0% to 98.6% (P = .01); specificity increased from 32.1% to 56.9% (P < .001) with PLA data. Conclusions and Relevance: The noninvasive PLA enables dermatologists to significantly improve biopsy specificity while maintaining or improving sensitivity. This result may increase the number of early melanomas biopsied and reduce the number of benign lesions biopsied, thereby improving patient outcomes and reducing health care costs.

Development and validation of a noninvasive 2-gene molecular assay for cutaneous melanoma.

BACKGROUND: Clinical and histopathologic assessment of pigmented skin lesions remains challenging even for experts. Differentiated and accurate noninvasive diagnostic modalities are highly desirable. OBJECTIVE: We sought to provide clinicians with such a tool. METHODS: A 2-gene classification method based on LINC00518 and preferentially expressed antigen in melanoma (PRAME) gene expression was evaluated and validated in 555 pigmented lesions (157 training and 398 validation samples) obtained noninvasively via adhesive patch biopsy. Results were compared with standard histopathologic assessment in lesions with a consensus diagnosis among 3 experienced dermatopathologists. RESULTS: In 398 validation samples (87 melanomas and 311 nonmelanomas), LINC00518 and/or PRAME detection appropriately differentiated melanoma from nonmelanoma samples with a sensitivity of 91% and a specificity of 69%. We established LINC00518 and PRAME in both adhesive patch melanoma samples and underlying formalin fixed paraffin embedded (FFPE) samples of surgically excised primary melanomas and in melanoma lymph node metastases. LIMITATIONS: This technology cannot be used on mucous membranes, palms of hands, and soles of feet. CONCLUSIONS: This noninvasive 2-gene pigmented lesion assay classifies pigmented lesions into melanoma and nonmelanoma groups and may serve as a tool to help with diagnostic challenges that may be inherently linked to the visual image and pattern recognition approach.

Analytical Characteristics of a Noninvasive Gene Expression Assay for Pigmented Skin Lesions.

We previously reported clinical performance of a novel noninvasive and quantitative PCR (qPCR)-based molecular diagnostic assay (the pigmented lesion assay; PLA) that differentiates primary cutaneous melanoma from benign pigmented skin lesions through two target gene signatures, LINC00518 (LINC) and preferentially expressed antigen in melanoma (PRAME). This study focuses on analytical characterization of this PLA, including qPCR specificity and sensitivity, optimization of RNA input in qPCR to achieve a desired diagnostic sensitivity and specificity, and analytical performance (repeatability and reproducibility) of this two-gene PLA. All target qPCRs demonstrated a good specificity (100%) and sensitivity (with a limit of detection of 1-2 copies), which allows reliable detection of gene expression changes of LINC and PRAME between melanomas and nonmelanomas. Through normalizing RNA input in qPCR, we converted the traditional gene expression analyses to a binomial detection of gene transcripts (i.e., detected or not detected). By combining the binomial qPCR results of the two genes, an improved diagnostic sensitivity (raised from 52%- 65% to 71% at 1 pg of total RNA input, and to 91% at 3 pg of total RNA input) was achieved. This two-gene PLA demonstrates a high repeatability and reproducibility (coefficient of variation <3%) and all required analytical performance characteristics for the commercial processing of clinical samples.

Characterization of NKIP: a novel, Na+/K+-ATPase interacting protein mediates neural differentiation and apoptosis.

Cellular differentiation and programmed cell death are tightly controlled to maintain tissue homeostasis and proper organ function. In a screen for apoptosis specific gene products, we isolated an immediate early apoptosis response gene from myelomonocytic stem cells that appears to play a key regulatory role in a number of cell types and may be of particular importance in cells of the central nervous system. The gene's 28 kDa protein product interacts with the C-terminal ectodomain of the Na+/K+-ATPase (NKA) beta 1 subunit and was therefore named NKIP (NKA Interacting Protein). NKIP is coexpressed with NKA, localizes to lysosomes and the endoplasmic reticulum and is predominantly expressed in excitable tissues including polarized epithelia and the central nervous system. NKIP has been characterized as an endogenous suppressor of the NKA as reduction of NKIP in PC12 cells significantly increases NKA activity. In pluripotent NT2 progenitor cells, NKIP induced rapidly K+-level-dependent cell death. NKIP overexpression induced growth factor-independent neurite outgrowth, which was associated with MEK-independent phosphorylation of the transcription factor ERK1/2. Thus, we have identified NKIP as an important novel protein that interacts to the NKA complex, influencing cellular ion balance, induction of apoptosis and neuronal differentiation.

The human orthologue of a novel apoptosis response gene induced during rat myelomonocytic stem cell apoptosis maps to 20q13.12.

Stem cell factor (SCF) stimulation of the receptor tyrosine kinase c-kit has effects on the proliferation, differentiation, and apoptotic regulation of hematopoietic progenitor cell populations. Rat bone marrow myelomonocytic stem cells (MSC) isolated in vitro by wheat germ agglutinin culture exclusively undergo self-renewal divisions when stimulated by SCF but bipotentially differentiate in the presence of dexamethasone or 1alpha,25-dihydroxyvitamin D(3) to granulocytes and macrophages, respectively. We show here that withdrawal of SCF from MSC induces rapid apoptosis in all stages of the cell cycle accompanied by development of an ultrastructural apoptotic morphology. To investigate immediate-early gene induction during MSC apoptosis, a differential display polymerase chain reaction (DD-PCR) screen coupled with rapid amplification of cDNA ends (RACE) PCR was performed. An immediate-early apoptosis response gene was isolated from growth factor-deprived MSC that was not expressed during self-renewal or differentiation induction cultures containing SCF. The protein contains a PEST region enriched in proline, glutamic acid, serine, and threonine residues common to proteins with a high turnover and has a cytoplasmic, vesicular localization in apoptotic MSC shown by immunohistochemistry. The human orthologous gene, isolated by RACE PCR, shows 86% homology to the rat protein and high similarity with a human uncharacterized hypothalamus predicted protein (HSMNP1) localized to the long arm of chromosome 20. Because deletions in this region are a common occurrence in a wide range of myeloproliferative disorders characterized by treatment resistance to apoptosis, HSMNP1 expression may play a role in normal and pathological myeloid development.

Mcl-1 blocks radiation-induced apoptosis and inhibits clonogenic cell death.

BACKGROUND: Anti-apoptotic Bcl-2 family proteins, such as Bcl-2 and Bcl-x, can modulate radio- and/or chemosensitivity of human malignancies. Since no information is available on the role Mcl-1 may play in the radioresponse of tumor cells, the relationship between Mcl-1 expression and response to ionizing radiation (IR) was investigated using an antisense strategy. MATERIALS AND METHODS: Human melanoma cells were treated with Mcl-1 antisense oligonucleotides (ASOs) and IR. The effects of antisense treatment alone or in combination with IR on proliferation, induction of apoptosis and clonogenic cell death were evaluated. RESULTS: ASO treatment in combination with IR reduced the mean cell numbers 9.5-fold compared to a 2.6-fold reduction after ASO treatment alone and a 1.6- fold reduction after IR alone. The percentages of apoptosis measured (means +/- SD) were 49% +/- 3.0 in antisense/IR-treated cultures compared to 1.3% +/- 0.5, 14.3% +/- 0.5, 7.3% +/- 1.1 and 10.3% +/- 0.6 in ASO controls, in antisense-treated, in IR-treated and in antisense control plus IR-treated cells, respectively. Colony formation assays demonstrated a synergistic effect of Mcl-1 down-regulation with IR. CONCLUSION: Mcl-1 expression affects the radioresistance of human melanoma cells.

Thalidomide enhances the anti-tumor activity of standard chemotherapy in a human melanoma xenotransplatation model.

It has been demonstrated that thalidomide's anti-angiogenic properties result in clear anti-tumor activity in a number of human malignancies. We studied thalidomide in a human melanoma severe combined immunodeficiency mouse xenotransplantation model. Thalidomide as a single agent showed a significant tumor reduction of 46% compared with the control group. Thalidomide combined with dacarbazine treatment markedly enhanced the anti-tumor effect of chemotherapy and showed a significant tumor reduction relative to the dacarbazine-only group (61%) and even more tumor reduction (74%) compared with the control group. We also measured clearly reduced levels of tumor necrosis factor-alpha in the thalidomide-treated group. A significantly lower microvessel density was encountered in the thalidomide treatment groups (thalidomide alone or combined with DTIC), underscoring the anti-angiogenic effect of thalidomide as a single agent as well as in combination with chemotherapy in this model. In line with these results, we observed a nearly 3-fold increase of apoptosis for the combination of thalidomide and DTIC compared with the rate of apoptotic cells in DTIC-only-treated melanoma xenotransplants. These data underline the rationale for combining dacarbazine--a cytotoxic agent--and thalidomide--an anti-angiogenic cytostatic agent--as a promising strategy for the treatment of melanoma.

Clusterin regulates drug-resistance in melanoma cells.

Clusterin has recently been shown to act as an antiapoptotic protein that confers drug-resistance in models of epithelial tumors. The aim of our work was to provide an insight into a possible role of clusterin in the regulation of drug-resistance in melanoma. In tissue samples, clusterin expression was low in nevi, but high in primary melanoma and melanoma metastases. Clusterin was also strongly expressed in melanoma cell lines, but was barely detectable in cultured melanocytes. To elucidate a possible role of clusterin in drug-resistance of melanoma, clusterin expression was regulated by either plasmid-driven overexpression or by antisense-mediated downregulation. Clusterin overexpression was associated with an increase in drug-resistance, i.e., with an increased survival of melanoma cells in the presence of cytotoxic drugs. In contrast, downregulation of clusterin by 2'-O-(2-methoxy)ethyl (2'MOE)-modified antisense oligonucleotides (AS-ODN) directed against clusterin mRNA significantly reduced drug-resistance, i.e., decreased survival of melanoma cells in the presence of cytotoxic drugs. To evaluate the effects of clusterin-antisense treatment in vivo, we applied an SCID-mouse/human-melanoma xenotransplantation model. Pre-treatment of mice with the 2'MOE-modified clusterin AS-ODN was associated with a significantly improved tumor response to dacarbazine as compared with animals pretreated with a scrambled control oligonucleotide. Taken together, we show that clusterin is strongly expressed in melanoma. Downregulation of clusterin reduces drug-resistance, i.e., reduces melanoma cell survival in response to cytotoxic drugs in vitro and in vivo. Thus, reducing clusterin expression may provide a novel tool to overcome drug-resistance in melanoma.

Antisense clusterin oligodeoxynucleotides increase the response of HER-2 gene amplified breast cancer cells to Trastuzumab.

Clusterin (CLU) is a heterodimeric secreted glycoprotein implicated in several physiological and pathological processes including cancer. Although recent data showed that overexpression of CLU is closely associated with disease progression in patients with breast tumor, the functional role of CLU expression in this tumor hystotype remains to be determined. The objectives in this study were to evaluate CLU expression levels after treatment with Trastuzumab, a HER2-targeted monoclonal antibody used in the clinical management of advanced breast cancer patients, and to test the usefulness of combined treatment with OGX-011, the second generation 2'-methoxyethyl gapmer oligonucleotides targeting the CLU gene, and Trastuzumab in this tumor hystotype. By using the HER-2 gene amplified-BT474 human breast cancer cells, we found Trastuzumab decreased HER-2 expression and inhibited cell proliferation without affecting apoptosis. Interestingly, Trastuzumab treatment up-regulated CLU protein expression in a dose-dependent fashion. We therefore hypothesized that the treatment with OGX-011, by blocking Trastuzumab-induced CLU expression, might potentiate the growth-inhibitory effect of Trastuzumab alone. Although OGX-011 had no effect on the behavior of the BT474 cells when used alone, it significantly enhanced the sensitivity of cells to Trastuzumab. A significant increase in the percentage of apoptotic cells, analyzed in terms of annexin V positivity and cleavage of poly(ADP-ribose) polymerase, was observed after combined treatment with OGX-011 plus Trastuzumab but not with either agent alone. Altogether our findings suggest that combined targeting of HER-2 and CLU may represent a novel, rational approach to breast cancer therapy.

Comparative histopathology of grey-horse-melanoma and human malignant melanoma.

Equine melanoma shows striking features particularly with regard to clinical development in grey horses: in contrast to malignant melanoma in humans and in solid coloured horses that are characterized by early onset of metastasis, pigment cell tumours display almost benign clinical features in ageing grey horses. Through evolution, grey horses appear to be in a favourable position in regard to the biological behaviour of melanomas. Yet unknown factors inhibiting or retarding early melanoma metastasis may be responsible for this phenomenon. In this study, immunostaining profiles and histopathologic patterns of equine vs. human melanotic tumours were compared. In addition, the expression of melanoma markers currently used in human melanoma detection and characterization were evaluated for their applicability in equine melanoma diagnosis. Immunohistopathologic investigations revealed that benign grey horse melanomas share common features with human blue nevi and with human malignant desmoplastic melanomas, whereas their resemblance to other types of human cutaneous malignant melanomas is less pronounced. Our data equally underline that S-100, proliferating cell nuclear antigen (PCNA), HMB-45, Ki-67, T-311 and CD44 can serve as reliable markers for horse melanomas. Further investigations aiming at identifying factors retarding metastasis in affected grey horses are needed, as they may contribute to the development of novel treatment strategies for human malignant melanoma.

Pegylated and conventional interferon-alpha induce comparable transcriptional responses and inhibition of tumor growth in a human melanoma SCID mouse xenotransplantation model.

Interferon-alpha (IFN-alpha) is widely used for the treatment of viral infections and primary cancers. In the present study, we investigated whether the anti-proliferative activity of IFN-alpha is capable of inhibiting melanoma tumor development in the absence of protective immune responses in a severe combined immunodeficiency (SCID) mouse model. Mice treated with either regular (100 microg/3 times per week) or pegylated (300 microg/once weekly) human IFN-alpha 2a showed a marked reduction in tumor weight after 4 wk of treatment. Tumor weight in pegylated and conventional IFN-alpha-treated animals was reduced by 61% and 67%, respectively, as compared to saline control (both p< or =0.01). A decrease of proliferation and an increase of apoptotic tumor cells were observed in IFN-treated tumors. DNA microarrays were applied to analyze transcriptional responses in tumors after 4 wk of treatment and a subset of about 90 genes was differentially expressed. Twenty-four novel and five known interferon-inducible genes were up- and 65 genes downregulated. A direct comparison of IFN-alpha and pegylated IFN-alpha did not reveal any significant differences in tumor growth inhibition indicating that this novel and more stable class of IFN is functionally equivalent. Despite the structural difference between pegylated and conventional IFN-alpha, both agents caused similar transcriptional responses in human melanoma xenotransplants.