Detection of DNA-Psoralen Photoadducts in Mammalian Skin

An immunofluorescence (IF) method for the detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA has been developed to assess nuclear damage in keratinocytes and melanocytes after psoralen plus UVA (PUVA) treatment, both under in vitro and in vivo conditions. Cryostat sections of the albino and pigmented guinea pig and human skin were used for in vitro studies to establish minimal and maximal drug concentration and UVA dosimetry for the detection of DNA-8-MOP photoadducts. Limits of detection were as low as 10 ng/cm2 8-MOP and 1 J/cm2 UVA for skin sections and sodium bromide-split epidermal sheets. Guinea pigs treated with topical PUVA revealed positive IF stain in epidermal cell nuclei at a threshold dose of 100 μg/cm2: 8-MOP and 13 J/cm2 UVA. Pretreatments of cryostat cuts with ethanol and alkali before IF test enhanced the sensitivity of detection in vivo about 10-fold and enabled us to follow the repair of DNA damage after treating normal guinea pig skin with a dose of 50 μg/cm2 8-MOP plus 6 J/cm2 UVA. The most interesting findings were as follows: (1) A sensitive method to detect PUVA-induced nuclear damage in epidermal and dermal cells was developed. (2) PUVA treatment induced nuclear DNA damage to melanocytes as well as to adjacent keratinocytes, and melanocytes appeared to be 10 times less vulnerable to photo-damage than keratinocytes. (3) There was a greater propensity for the proliferative cells to be damaged by PUVA. (4) PUVA induced nuclear damage up to 700 μm depth in the dermis. (5) The usefulness of the IF test in detecting DNA damage in μg and ng amounts in vivo and in following the repair of damaged DNA induced by PUVA

Isolation, characterization, and in vitro propagation of infantile hemangioma stem cells and an in vivo mouse model

<p>Abstract</p> <p>Background</p> <p>Infantile hemangiomas (IH) are the most common benign tumors of infancy. The typical clinical course consists of rapid growth during the first year of life, followed by natural and gradual involution over a multi-year time span through unknown cellular mechanisms. Some tumors respond to medical treatment with corticosteroids or beta-blockers, however, when this therapy fails or is incomplete, surgical extirpation may be necessary. Noninvasive therapies to debulk or eliminate these tumors would be an important advance. The development of an <it>in vitro </it>cell culture system and an animal model would allow new insights into the biological processes involved in the development and pathogenesis of IH.</p> <p>Results</p> <p>We observed that proliferative stage IH specimens contain significantly more SALL4+ and CD133+ cells than involuting tumors, suggesting a possible stem cell origin. A tumor sphere formation assay was adapted to culture IH cells <it>in vitro</it>. Cells in IH tumor spheres express GLUT1, indicative of an IH cell of origin, elevated levels of VEGF, and various stem/progenitor cell markers such as SALL4, KDR, Oct4, Nanog and CD133. These cells were able to self-renew and differentiate to endothelial lineages, both hallmarks of tumor stem cells. Treatment with Rapamycin, a potent mTOR/VEGF inhibitor, dramatically suppressed IH cell growth <it>in vitro</it>. Subcutaneous injection of cells from IH tumor spheres into immunodeficient NOD-SCID mice produced GLUT1 and CD31 positive tumors with the same cellular proliferation, differentiation and involution patterns as human hemangiomas.</p> <p>Conclusions</p> <p>The ability to propagate large numbers of IH stem cells <it>in vitro </it>and the generation of an <it>in vivo </it>mouse model provides novel avenues for testing IH therapeutic agents in the future.</p

Loss of 5-hydroxymethylcytosine correlates with increasing morphologic dysplasia in melanocytic tumors

DNA methylation is the most well studied epigenetic modification in cancer biology. 5-hydroxymethylcytosine is an epigenetic mark that can be converted from 5-methylcytosine by the ten-eleven translocation gene family. We recently reported the loss of 5-hydroxymethylcytosine in melanoma compared to benign nevi and suggested that loss of this epigenetic marker is correlated with tumor virulence based on its association with a worse prognosis. In this study we further characterize the immunoreactivity patterns of 5-hydroxymethylcytosine in the full spectrum of melanocytic lesions to further validate the potential practical application of this epigenetic marker. 175 cases were evaluated: 18 benign nevi, 20 dysplastic nevi (10 low-grade and 10 high-grade lesions), 10 atypical Spitz nevi, 20 borderline tumors, 5 melanomas arising within nevi, and 102 primary melanomas. Progressive loss of 5-hydroxymethylcytosine from benign dermal nevi to high-grade dysplastic nevi to borderline melanocytic neoplasms to melanoma was observed. In addition, an analysis of the relationship of nuclear diameter to 5-hydroxymethylcytosine staining intensity within lesional cells revealed a significant correlation between larger nuclear diameter and decreased levels of 5-hydroxymethylcytosine. Furthermore, borderline lesions uniquely exhibited a diverse spectrum of staining of each individual case. This study further substantiates the association of 5-hydroxymethylcytosine loss with dysplastic cytomorphologic features and tumor progression and supports the classification of borderline lesions as a biologically distinct category of melanocytic lesions

Apocrine-Eccrine Carcinomas: Molecular and Immunohistochemical Analyses

Apocrine-eccrine carcinomas are rare and associated with poor prognosis. Currently there is no uniform treatment guideline. Chemotherapeutic drugs that selectively target cancer-promoting pathways may complement conventional therapeutic approaches. However, studies on genetic alterations and EGFR and Her2 status of apocrine-eccrine carcinomas are few in number. In addition, hormonal studies have not been comprehensive and performed only on certain subsets of apocrine-eccrine carcinomas. To investigate whether apocrine-eccrine carcinomas express hormonal receptors or possess activation of oncogenic pathways that can be targeted by available chemotherapeutic agent we performed immunohistochemistry for AR, PR, ER, EGFR, and HER2 expression; fluorescence in situ hybridization (FISH) for EGFR and ERBB2 gene amplification; and molecular analyses for recurrent mutations in 15 cancer genes including AKT-1, EGFR, PIK3CA, and TP53 on 54 cases of apocrine-eccrine carcinomas. They include 10 apocrine carcinomas, 7 eccrine carcinomas, 9 aggressive digital papillary adenocarcinomas, 10 hidradenocarcinomas, 11 porocarcinomas, 1 adenoid cystic carcinoma, 4 malignant chondroid syringomas, 1 malignant spiradenoma, and 1 malignant cylindroma. AR, ER, PR, EGFR and HER2 expression was seen in 36% (19/53), 27% (14/51), 16% (8/51), 85% (44/52) and 12% (6/52), respectively. Polysomy or trisomy of EGFR was detected by FISH in 30% (14/46). Mutations of AKT-1, PIK3CA, and TP53 were detected in 1, 3, and 7 cases, respectively (11/47, 23%). Additional investigation regarding the potential treatment of rare cases of apocrine-eccrine carcinomas with PI3K/Akt/mTOR pathway inhibitors, currently in clinical testing, may be of clinical interest

Sustained activation of c-Jun N-terminal and extracellular signal-regulated kinases in port-wine stain blood vessels

BACKGROUND: Port-wine stain (PWS) is a congenital, progressive vascular malformation but the pathogenesis remains incompletely understood. OBJECTIVE: We sought to investigate the activation status of various kinases, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, AKT, phosphatidylinositol 3-kinase, P70 ribosomal S6 kinase, and phosphoinositide phospholipase C γ subunit, in PWS biopsy tissues. METHODS: Immunohistochemistry was performed on 19 skin biopsy samples from 11 patients with PWS. RESULTS: c-Jun N-terminal kinase, extracellular signal-regulated kinase, and P70 ribosomal S6 kinase in pediatric and adult PWS blood vessels were consecutively activated. Activation of AKT and phosphatidylinositol 3-kinase was found in many adult hypertrophic PWS blood vessels but not in infants. Phosphoinositide phospholipase C γ subunit showed strong activation in nodular PWS blood vessels. LIMITATION: Infantile PWS sample size was small. CONCLUSION: Our data suggest a subsequent activation profile of various kinases during different stages of PWS: (1) c-Jun N-terminal and extracellular signal-regulated kinases are firstly and consecutively activated in all PWS tissues, which may contribute to both the pathogenesis and progressive development of PWS; (2) AKT and phosphatidylinositol 3-kinase are subsequently activated, and are involved in the hypertrophic development of PWS blood vessels; and (3) phosphoinositide phospholipase C γ subunit is activated in the most advanced stage of PWS and may participate in nodular formation

Evaluation of stromal HGF immunoreactivity as a biomarker for melanoma response to RAF inhibitors

Of more than 150,000 published studies evaluating new biomarkers, fewer than 100 biomarkers have been implemented for patient care[1]. One reason for this is lack of rigorous testing by the medical community to validate claims for biomarker clinical relevance, and potential reluctance to publish negative results when confirmation is not obtained. Here we sought to determine the utility and reproducibility of immunohistochemical detection of hepatocyte growth factor (HGF) in melanoma tissue, an approach of potential assistance in defining patients with innate resistance to BRAF inhibitor therapy[2]. To this end, a published and a revised method that retained sensitivity but with greater specificity for HGF detection, were evaluated in cells known to endogenously express HGF, models where HGF is upregulated via cytokine induction, and via overexpression by gene transfection. Consequent patient evaluation in collaboration with the Melanoma Institute Australia of a cohort of 41 melanoma specimens with extensive clinical annotation failed to validate HGF immunohistochemistry as a predictor of response to BRAF inhibitors. Targeted therapies for advanced melanoma[3–5] and other cancers show great promise, and rigorous validation studies are thus indicated for approaches that seek to personalize such therapies in order to maximize therapeutic efficacy

Targeted next-generation sequencing reveals high frequency of mutations in epigenetic regulators across treatment-naïve patient melanomas

Background: Recent developments in genomic sequencing have advanced our understanding of the mutations underlying human malignancy. Melanoma is a prototype of an aggressive, genetically heterogeneous cancer notorious for its biologic plasticity and predilection towards developing resistance to targeted therapies. Evidence is rapidly accumulating that dysregulated epigenetic mechanisms (DNA methylation/demethylation, histone modification, non-coding RNAs) may play a central role in the pathogenesis of melanoma. Therefore, we sought to characterize the frequency and nature of mutations in epigenetic regulators in clinical, treatment-naïve, patient melanoma specimens obtained from one academic institution. Results: Targeted next-generation sequencing for 275 known and investigative cancer genes (of which 41 genes, or 14.9 %, encoded an epigenetic regulator) of 38 treatment-naïve patient melanoma samples revealed that 22.3 % (165 of 740) of all non-silent mutations affected an epigenetic regulator. The most frequently mutated genes were BRAF, MECOM, NRAS, TP53, MLL2, and CDKN2A. Of the 40 most commonly mutated genes, 12 (30.0 %) encoded epigenetic regulators, including genes encoding enzymes involved in histone modification (MECOM, MLL2, SETD2), chromatin remodeling (ARID1B, ARID2), and DNA methylation and demethylation (TET2, IDH1). Among the 38 patient melanoma samples, 35 (92.1 %) harbored at least one mutation in an epigenetic regulator. The genes with the highest number of total UVB-signature mutations encoded epigenetic regulators, including MLL2 (100 %, 16 of 16) and MECOM (82.6 %, 19 of 23). Moreover, on average, epigenetic genes harbored a significantly greater number of UVB-signature mutations per gene than non-epigenetic genes (3.7 versus 2.4, respectively; p = 0.01). Bioinformatics analysis of The Cancer Genome Atlas (TCGA) melanoma mutation dataset also revealed a frequency of mutations in the 41 epigenetic genes comparable to that found within our cohort of patient melanoma samples. Conclusions: Our study identified a high prevalence of somatic mutations in genes encoding epigenetic regulators, including those involved in DNA demethylation, histone modification, chromatin remodeling, and microRNA processing. Moreover, UVB-signature mutations were found more commonly among epigenetic genes than in non-epigenetic genes. Taken together, these findings further implicate epigenetic mechanisms, particularly those involving the chromatin-remodeling enzyme MECOM/EVI1 and histone-modifying enzyme MLL2, in the pathobiology of melanoma. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0091-3) contains supplementary material, which is available to authorized users

On the biological relevance of MHC class II and B7 expression by tumour cells in melanoma metastases

A large number of studies have indicated that specific immune reactivity plays a crucial role in the control of malignant melanoma. In this context, expression of MHC I, MHC II and B7 molecules by melanoma cells is seen as relevant for the immune response against the tumour. For a better understanding of the biological relevance of MHC II and B7 expression by tumour cells in metastatic melanoma, we studied the expression of these molecules in melanoma metastases in relation to the inflammatory response, regression of the tumour and survival from 27 patients treated with biochemotherapy (30 mg m−2 Cisplatin and 250 mg m−2 decarbazine (dimethyl-triazene-imidazole-carboxamide, DTIC) on days 1–3 i.v., and 107 IU IFN-α2b 3 days a week s.c., q. 28d). In 19 out of 27 lesions studied, we found expression of MHC II by the tumour cells, while only in one out of 11 tumour biopsies obtained from untreated metastatic melanoma patients, MHC II expression was detected. Expression of B7.1 and B7.2 by tumour cells was found in nine out of 24 and 19 out of 24 lesions, respectively. In all cases where B7.1 expression was found, expression of B7.2 by the tumour cells was also seen. In general, no or only few inflammatory cells positive for B7 were found. Expression of MHC II by tumour cells was positively correlated with the presence of tumour-infiltrating lymphocytes, regression of the lesion, and with time to progression (TTP) and overall survival (OS) of the patient. However, no significant correlation between B7.1 or B7.2 expression and regression of the tumour, TTP or OS was found. In light of other recent findings, these data altogether do support a role as biomarker for MHC II expression by tumour cells; however, its exact immunological pathomechanism(s) remain to be established