Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA.

Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Hohlraum fields with monoenergetic proton radiography at OMEGA.

A more complete understanding of laser-driven hohlraum plasmas is critical for the continued development and improvement of ICF experiments. In these hohlraums, self-generated electric and magnetic fields can play an important role in modifying plasma properties such as heat transport; however, the strength and distribution of electromagnetic fields in such hohlraums remain largely uncertain. To explore this question, we conducted experiments at the OMEGA laser facility, using monoenergetic proton radiography to probe laser-driven vacuum hohlraums. We then utilized reconstructive methods to recover information about proton deflections. To interpret these reconstructions, a new technique for detangling the contributions of electric and magnetic fields to proton deflections was developed. This work was supported in part by the U.S. Department of Energy, the National Laser Users' Facility, and the Laboratory for Laser Energetics.

Oxidative stress inhibits distant metastasis by human melanoma cells.

Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. Here we studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NOD-SCID-Il2rg(-/-) (NSG) mice. We show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence on NADPH-generating enzymes in the folate pathway. Antioxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumours in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

Next-generation sequencing implicates oncogenic roles for p53 and JAK/STAT signaling in microcystic adnexal carcinomas.

Microcystic adnexal carcinoma is a locally aggressive sweat gland carcinoma characterized by its infiltrative growth and histopathologic overlap with benign adnexal tumors, often posing challenges to both diagnosis and management. Understanding the molecular underpinnings of microcystic adnexal carcinoma may allow for more accurate diagnosis and identify potential targetable oncogenic drivers. We characterized 18 microcystic adnexal carcinomas by targeted, multiplexed PCR-based DNA next-generation sequencing of the coding sequence of over 400 cancer-relevant genes. The majority of cases had relatively few (<8) prioritized somatic mutations, and lacked an ultraviolet (UV) signature. The most recurrent mutation was TP53 inactivation in four (22%) tumors. Frame-preserving insertions affecting the kinase domain of JAK1 were detected in three (17%) cases, and were nonoverlapping with TP53 mutations. Seven (39%) cases demonstrated copy number gain of at least one oncogene. By immunohistochemistry, p53 expression was significantly higher in microcystic adnexal carcinomas with TP53 mutations compared with those without such mutations and syringomas. Similarly, phospho-STAT3 expression was significantly higher in microcystic adnexal carcinomas harboring JAK1 kinase insertions compared with those with wild-type JAK1 and syringomas. In conclusion, microcystic adnexal carcinomas are molecularly heterogeneous tumors, with inactivated p53 or activated JAK/STAT signaling in a subset. Unlike most other nonmelanoma skin cancers involving sun-exposed areas, most microcystic adnexal carcinomas lack evidence of UV damage, and hence likely originate from a relatively photo-protected progenitor population in the dermis. These findings have implications for the biology, diagnosis, and treatment of microcystic adnexal carcinomas, including potential for therapeutic targeting of p53 or the JAK/STAT pathway in advanced tumors.

Primary Cutaneous Umbilical Melanoma: The Michigan Experience.

BACKGROUND: Primary cutaneous umbilical melanoma is rare. Thorough information regarding its characteristics and treatment, including use of sentinel lymph node biopsy (SLNB) staging, is difficult to obtain. The unique anatomy of the umbilicus adds to the complexity of diagnosing and treating melanoma at this site. OBJECTIVE: To improve understanding of diagnosis and treatment of primary cutaneous umbilical melanoma through presenting 7 new cases and reviewing 39 cases in the literature. MATERIALS AND METHODS: The University of Michigan melanoma database query and review of the literature regarding reported cases of primary umbilical melanoma. RESULTS: In 7 new and 39 previously reported cases of primary cutaneous umbilical melanoma, we describe signs and symptoms, histopathologic features, differential diagnosis, relevant anatomical considerations, and definitive treatment including SLNB when applicable. CONCLUSION: Our series, combined with a thorough literature review and compilation of findings, provides a better understanding and appreciation of melanoma in the unique anatomical site of the umbilicus, with a reminder to carefully examine the umbilicus during a full skin examination in patients at risk of melanoma. Primary umbilical melanoma presents and can be appropriately treated similarly to cutaneous melanoma in other sites, with attention to relevant anatomy.

Mohs Surgery versus Standard Local Excision for Basal Cell Carcinoma, Squamous Cell Carcinoma, and Melanoma Skin Cancer.

Basal cell carcinoma, squamous cell carcinoma, and melanoma represent the three most common skin cancers that occur on the face. The most common surgical treatments for facial skin cancers are Mohs' surgery and standard local excision. The effective utilization of either of these techniques is based on tumor and patient risk stratification incorporating known risk factors for occult invasion and local recurrence, combined with patient comorbidities, expectations, and desires. Best available evidence highlights multiple and consistent risk factors for each specific skin cancer type, and dictate local control rates reported in the literature. Recognizing gaps in the literature, we compare and review surgical treatment guidelines and data for standard local excision versus Mohs' surgery for cutaneous nonmelanoma and melanoma skin cancer. This article serves as a resource for optimal therapeutic decision making for surgical management of skin cancer on the face.

Guidelines of care for the management of primary cutaneous melanoma.

The incidence of primary cutaneous melanoma continues to increase each year. Melanoma accounts for the majority of skin cancer-related deaths, but treatment is usually curative following early detection of disease. In this American Academy of Dermatology clinical practice guideline, updated treatment recommendations are provided for patients with primary cutaneous melanoma (American Joint Committee on Cancer stages 0-IIC and pathologic stage III by virtue of a positive sentinel lymph node biopsy). Biopsy techniques for a lesion that is clinically suggestive of melanoma are reviewed, as are recommendations for the histopathologic interpretation of cutaneous melanoma. The use of laboratory, molecular, and imaging tests is examined in the initial work-up of patients with newly diagnosed melanoma and for follow-up of asymptomatic patients. With regard to treatment of primary cutaneous melanoma, recommendations for surgical margins and the concepts of staged excision (including Mohs micrographic surgery) and nonsurgical treatments for melanoma in situ, lentigo maligna type (including topical imiquimod and radiation therapy), are updated. The role of sentinel lymph node biopsy as a staging technique for cutaneous melanoma is described, with recommendations for its use in clinical practice. Finally, current data regarding pregnancy and melanoma, genetic testing for familial melanoma, and management of dermatologic toxicities related to novel targeted agents and immunotherapies for patients with advanced disease are summarized.

Subungual atypical lentiginous melanocytic proliferations in children and adolescents: A clinicopathologic study.

BACKGROUND: Most subungual melanocytic lesions in children are benign, but some are difficult to classify due to prominent lentiginous growth and high-grade cytologic atypia. OBJECTIVE: To characterize the clinicopathologic features of these rare lesions. METHODS: Subungual atypical lentiginous melanocytic proliferations from patients <20 years of age were collected for clinical and histopathologic review. Fluorescence in situ hybridization (FISH) was performed when possible. RESULTS: Eleven patients aged 2-19 years had expanding or darkening longitudinal pigmented streak(s) with or without Hutchinson sign. Microscopically, all revealed predominantly single-cell growth, pagetoid scatter, and poor circumscription. Eight (73%) cases showed focal or poor nesting, and 3 (27%) demonstrated confluence. Nuclear enlargement, hyperchromasia, and angulation were present in 8 (73%) cases, 7 (64%) cases, and 6 (55%) cases, respectively. One of 4 cases tested by FISH was positive. Three lesions recurred locally without other adverse outcome. LIMITATIONS: Small sample size and short clinical follow-up. Two cases were examined in partial biopsies only. CONCLUSION: Some subungual melanocytic lesions in children and adolescents are histologically indistinguishable from adult subungual melanoma in situ. While the biologic potential remains elusive, FISH might aid in risk stratification. Awareness of this rare group of lesions is crucial for facilitating future investigation into its biologic behavior.

The natural history of thin melanoma and the utility of sentinel lymph node biopsy.

BACKGROUND AND OBJECTIVES: Current literature may overestimate the risk of nodal metastasis from thin melanoma due to reporting of data only from lesions treated with SLNB. Our objective was to define the natural history of thin melanoma, assessing the likelihood of nodal disease, in order to guide selection for SLNB. METHODS: Retrospective review. The primary outcome was the rate of nodal disease. Clinicopathologic factors were evaluated to find associations with nodal disease. RESULTS: Five hundred and twelve lesions, follow up available for 488 (median: 48 months). Lesions treated with WLE/SLNB compared to WLE alone were more likely to have high-risk features. The rate of nodal disease was higher in the WLE/SLNB group (24 positive SLNB, five false-negative SLNB with nodal recurrence: 10.2%) compared to WLE alone (four nodal recurrences: 2.0%). Univariate analysis showed age ≤45, Breslow depth ≥0.85 mm, mitotic rate >1 mm2 , and ulceration were associated with nodal disease. Multivariate analysis confirmed the association of age ≤45 and ulceration. CONCLUSIONS: SLNB for melanoma 0.75-0.99 mm should be considered in patients age ≤45, Breslow depth ≥0.85 mm, mitotic rate >1 mm2 , and/or with ulceration. Thin melanoma <0.85 mm without high-risk features may be treated with WLE alone.

Genomic copy number analysis of a spectrum of blue nevi identifies recurrent aberrations of entire chromosomal arms in melanoma ex blue nevus.

Blue nevi may display significant atypia or undergo malignant transformation. Morphologic diagnosis of this spectrum of lesions is notoriously difficult, and molecular tools are increasingly used to improve diagnostic accuracy. We studied copy number aberrations in a cohort of cellular blue nevi, atypical cellular blue nevi, and melanomas ex blue nevi using Affymetrix's OncoScan platform. Cases with sufficient DNA were analyzed for GNAQ, GNA11, and HRAS mutations. Copy number aberrations were detected in 0 of 5 (0%) cellular blue nevi, 3 of 12 (25%) atypical cellular blue nevi, and 6 of 9 (67%) melanomas ex blue nevi. None of the atypical cellular blue nevi displayed more than one aberration, whereas complex aberrations involving four or more regions were seen exclusively in melanomas ex blue nevi. Gains and losses of entire chromosomal arms were identified in four of five melanomas ex blue nevi with copy number aberrations. In particular, gains of 1q, 4p, 6p, and 8q, and losses of 1p and 4q were each found in at least two melanomas. Whole chromosome aberrations were also common, and represented the sole finding in one atypical cellular blue nevus. When seen in melanomas, however, whole chromosome aberrations were invariably accompanied by partial aberrations of other chromosomes. Three melanomas ex blue nevi harbored aberrations, which were absent or negligible in their precursor components, suggesting progression in tumor biology. Gene mutations involving GNAQ and GNA11 were each detected in two of eight melanomas ex blue nevi. In conclusion, copy number aberrations are more common and often complex in melanomas ex blue nevi compared with cellular and atypical cellular blue nevi. Identification of recurrent gains and losses of entire chromosomal arms in melanomas ex blue nevi suggests that development of new probes targeting these regions may improve detection and risk stratification of these lesions.

Analysis of Prognostic Factors from 9387 Merkel Cell Carcinoma Cases Forms the Basis for the New 8th Edition AJCC Staging System.

BACKGROUND: The first consensus Merkel cell carcinoma (MCC) staging system was published in 2010. New information on the clinical course prompts review of MCC staging. METHODS: A total of 9387 MCC cases from the National Cancer Data Base Participant User File with follow-up and staging data (1998-2012) were analyzed. Prognostic differences based on clinical and pathological staging were evaluated. Survival estimates were compared by disease extent. RESULTS: Sixty-five percent of cases presented with local disease, whereas 26 and 8 % presented with nodal and distant disease. Disease extent at presentation was predictive of 5-year overall survival (OS) with estimates of 51, 35, and 14 % for local, nodal, and distant disease. Tumor burden at the regional nodal basin was predictive of 5-year OS with estimates of 40 and 27 % for clinically occult and clinically detected nodal disease. For local disease, we confirm improved prognosis when the regional nodal basin was negative by pathological compared with clinical staging. We identified 336 cases with clinically detected nodal disease and unknown primary tumor and showed improved prognosis over cases presenting with concurrent primary tumor (OS estimates of 42 vs. 27 %). CONCLUSIONS: Analysis of a national dataset of MCC cases validates the predictive value of disease extent at presentation. Separation of clinical and pathological stage groups and regrouping of unknown primary tumors are supported by the analysis. The revised staging system provides more accurate prognostication and has been formally accepted by the AJCC staging committee for inclusion in the 8th edition.

Elevated YAP and its downstream targets CCN1 and CCN2 in basal cell carcinoma: impact on keratinocyte proliferation and stromal cell activation.

Yes-associated protein (YAP) is a transcriptional co-activator of hippo signaling pathway, which plays an important role in organ size control and tumorigenesis. Here we report that YAP and its downstream transcriptional targets CCN1 and CCN2 are markedly elevated in keratinocytes in human skin basal cell carcinoma tumor islands. In human keratinocytes, knockdown of YAP significantly reduced expression of CCN1 and CCN2, and repressed proliferation and survival. This inhibition of proliferation and survival was rescued by restoration of CCN1 expression, but not by CCN2 expression. In basal cell carcinoma stroma, CCN2-regulated genes type I collagen, fibronectin, and α-smooth muscle actin were highly expressed. Furthermore, atomic force microscopy revealed increased tissue stiffness in basal cell carcinoma stroma compared to normal dermis. These data provide evidence that up-regulation of YAP in basal cell carcinoma impacts both aberrant keratinocyte proliferation, via CCN1, and tumor stroma cell activation and stroma remodeling, via CCN2. Targeting YAP and/or CCN1 and CCN2 may provide clinical benefit in basal cell carcinoma.

Sentinel Lymph Node Biopsy Use Among Melanoma Patients 75 Years of Age and Older.

INTRODUCTION: While SLN biopsy is recommended for melanoma ≥1 mm in depth, its use among the elderly population is more controversial. We reviewed our experience at the University of Michigan with melanoma patients ≥75 years of age. METHODS: A total of 952 melanoma patients ≥75 years of age from 1996 to 2011 were identified from our institutional review board-approved database. In addition to clinicopathologic features and outcome data, comorbidity data were collected to calculate the Charlson comorbidity index (CCI). Univariate and multivariate Cox regression analysis was performed to characterize predictors of outcome. Kaplan-Meier analysis was used to generate survival curves. RESULTS: Among 553 clinically node-negative patients with melanoma ≥1 mm in Breslow thickness, 213 had wide excision alone, whereas 340 had excision and SLN biopsy, with 83 (24 %) having a positive SLN. SLN biopsy was less likely with older age (p < 0.0001) and H&N location (p = 0.007), but not CCI. SLN involvement was associated with female gender [odds ratio (OR) 2.15, p = 0.009], Breslow thickness [OR 1.23/mm increase, p = 0.004], and satellitosis (OR 4.43, p = 0.004). Distant disease-specific survival was negatively associated with male gender (OR 1.5, p = 0.007), increasing age (OR 1.05/year, p < 0.001), increasing Breslow thickness (OR 1.07/year, p = 0.013), ulceration (OR 1.51, p = 0.004), a positive SLN (OR 2.61, p < 0.001), and not having a SLN biopsy (OR 1.72, p < 0.001). CCI did not predict worse disease-free or melanoma-specific survival. CONCLUSIONS: WLE and SLN biopsy was not only strongly prognostic, but compared with WLE alone was associated with improved outcome, even after factoring for age and comorbidities. If otherwise healthy, SLN biopsy should be strongly considered for this population.

Discordance in histopathologic evaluation of melanoma sentinel lymph node biopsy with clinical follow-up: results from a prospectively collected database.

BACKGROUND: Sentinel lymph node (SLN) status currently represents the single most important prognostic factor in clinically localized melanoma and is widely used in patients with melanoma at significant risk for nodal micrometastasis. Although several studies have looked at the rates and implications of inaccuracies in the histopathologic diagnosis of melanocytic lesions, accuracy in the histologic interpretation of the SLN in melanoma has not been addressed. The goal of this study was to determine the rates of discordance in the histopathologic evaluation of the SLN and the potential clinical impact on patients referred to a comprehensive melanoma center. METHODS: A prospectively collected database was queried for melanoma patients who had SLN biopsies performed at outside institutions before referral to the University of Michigan Multidisciplinary Melanoma Program between 2006 and 2009. These cases were reviewed and clinical follow-up obtained. RESULTS: After internal review of the SLN material, 13 (8 %) of 167 cases had major discrepancies in diagnosis that impacted patient management and prognosis. The disease of five patients was subsequently downstaged and the disease of eight patients was upstaged after internal review of the SLNs and reversal in diagnoses. CONCLUSIONS: There appears to be a small yet significant rate of discordance in diagnosis of the SLN for melanoma after expert histopathologic review. The implications of this discordance and revision of diagnosis is substantial. Expert histopathologic review of the SLN warrants consideration to provide the most accurate prognostic information and optimal patient care.

Age as a predictor of sentinel node metastasis among patients with localized melanoma: an inverse correlation of melanoma mortality and incidence of sentinel node metastasis among young and old patients.

PURPOSE: We have previously reported that older patients with clinical stage I and II primary cutaneous. Melanoma had lower survival rates compared to younger patients. We postulated that the incidence of nodal metastasis would therefore be higher among older melanoma patients. METHODS: The expanded American Joint Committee on Cancer melanoma staging database contains a cohort of 7,756 melanoma patients who presented without clinical evidence of regional lymph node or distant metastasis and who underwent a sentinel node biopsy procedure as a component of their staging assessment. RESULTS: Although older patients had primary melanoma features associated with more aggressive biology, we paradoxically observed a significant decrease in the incidence of sentinel node metastasis as patient age increased. Overall, the highest incidence of sentinel node metastasis was 25.8 % in patients under 20 years of age, compared to 15.5 % in patients 80 years and older (p < 0.001). In contrast, 5-year mortality rates for clinical stage II patients ranged from a low of 20 % for those 20-40 years of age up to 38 % for those over 70 years of age. Patient age was an independent predictor of sentinel node metastasis in a multifactorial analysis (p < 0.001). CONCLUSIONS: Patients with clinical stage I and II melanoma under 20 years of age had a higher incidence of sentinel lymph node metastasis but, paradoxically, a more favorable survival outcome compared to all other age groups. In contrast, patients >70 years had the most aggressive primary melanoma features and a higher mortality rate compared to all other age groups but a lower incidence of sentinel lymph node metastasis.