Design and Evolution of Enhanced Peptide-Peptide Ligation for Modular Transglutaminase Assembly.

Robust and precise tools are needed to enhance the functionality and resilience of synthetic nanoarchitectures. Here, we have employed directed evolution and rational design to build a fast-acting molecular superglue from a bacterial adhesion protein. We have generated the SnoopLigase2 coupling system, a genetically encoded route for efficient transamidation between SnoopTag2 and DogTag2 peptides. Each peptide was selected for rapid reaction by phage display screening. The optimized set allows more than 99% completion and is compatible with diverse buffers, pH values, and temperatures, accelerating the reaction over 1000-fold. SnoopLigase2 directs a specific reaction in the mammalian secretory pathway, allowing covalent display on the plasma membrane. Transglutaminase 2 (TG2) has a network of interactions and substrates amidst the mammalian cell surface and extracellular matrix. We expressed a modified TG2 with resistance to oxidative inactivation and minimal self-reactivity. SnoopLigase2 enables TG2 functionalization with transforming growth factor alpha (TGFα) in routes that would be impossible through genetic fusion. The TG2:TGFα conjugate retained transamidase activity, stably anchored TGFα for signal activation in the extracellular environment, and reprogrammed cell behavior. This modular toolbox should create new opportunities for molecular assembly, both for novel biomaterials and complex cellular environments.

A first-in-human study of BLZ-100 (tozuleristide) demonstrates tolerability and safety in skin cancer patients.

BLZ-100 (tozuleristide) is an intraoperative fluorescent imaging agent that selectively detects malignant tissue and can be used in real time to guide tumor resection. The purpose of this study was to assess the safety, tolerability, and pharmacokinetics of BLZ-100 and to explore the pharmacodynamics of fluorescence imaging of skin tumors. In this first-in-human study, BLZ-100 was administered intravenously to 21 adult patients 2 days before excising known or suspected skin cancers. Doses were 1, 3, 6, 12, and 18 mg, with 3-6 patients/cohort. Fluorescence imaging was conducted before and up to 48 h after dosing. BLZ-100 was well tolerated. There were no serious adverse events, deaths, or discontinuations due to adverse events, and no maximum tolerated dose (MTD) was identified. Headache (n = 2) and nausea (n = 2) were the only BLZ-100 treatment-related adverse events reported for >1 patient. Median time to maximal serum concentration was <0.5 h. Exposure based on maximal serum concentrations increased in a greater than dose-proportional manner. For intermediate dose-levels (3-12 mg), 4 of 5 basal cell carcinomas and 4 of 4 melanomas were considered positive for BLZ-100 fluorescence. BLZ-100 was well tolerated at all dose levels tested and these results support further clinical testing of this imaging agent in surgical oncology settings. Clinicaltrials.gov: NCT02097875.

Factors Related to Nevus-Associated Cutaneous Melanoma: A Case-Case Study.

A proportion of cutaneous melanomas display neval remnants on histologic examination. Converging lines of epidemiologic and molecular evidence suggest that melanomas arising from nevus precursors differ from melanomas arising de novo. In a large, population-based study comprising 636 cutaneous melanomas subjected to dermatopathology review, we explored the molecular, host, and environmental factors associated with the presence of neval remnants. We found that nevus-associated melanomas were significantly associated with younger age at presentation, non-brown eye color, trunk site, thickness of less than 0.5 mm, and BRAFV600E mutation. Compared with patients with de novo melanomas, those with nevus-associated tumors were more likely to self-report many moles on their skin as a teenager (odds ratio = 1.94, 95% confidence interval = 1.01-3.72) but less likely to report many facial freckles (odds ratio = 0.49, 95% confidence interval = 0.25-0.96). They also had high total nevus counts (odds ratio = 2.18, 95% confidence interval = 1.26-3.78). On histologic examination, nevus-associated melanomas exhibited less dermal elastosis in adjacent skin compared with de novo melanomas (odds ratio = 0.55, 95% confidence interval = 0.30-1.01). These epidemiologic data accord with the emerging molecular paradigm that nevus-associated melanomas arise through a distinct sequence of causal events that differ from those leading to other cutaneous melanomas.

Adult Orbital and Adnexal Xanthogranulomatous Disease.

PURPOSE: Adult xanthogranulomatous disease of the orbit and ocular adnexa is a rare disease that can cause serious morbidity and mortality. Ophthalmologists are commonly the first clinicians to come in contact with affected patients and an understanding of the clinical features is essential. DESIGN: We present a retrospective case series of patients seen in the oculoplastic unit of a large tertiary referral hospital over a 20-year period. METHODS: The clinical files of 7 patients with adult xanthogranulomatous disease of the orbit and ocular adnexa were reviewed. Clinical, radiological, histopathological, and immunohistochemical findings were examined. RESULTS: Periocular clinical features included cutaneous xanthogranulomatous lesions, decreased visual acuity, proptosis, diplopia, skin ulceration, cicatricial ectropion, and mechanical ptosis. Systemic features included adult-onset asthma, disseminated xanthogranulomatous lesions with long bone involvement, and hematological disturbances such as monoclonal gammopathy and lymphoplasmacytic lymphoma. Lipid-laden macrophages and Touton multinucleated giant cells were histological hallmarks in all subtypes. Most lesions were strongly CD8 positive on immunohistochemistry. Radiologically, the lesions were diffuse and infiltrative in nature. Various treatments were employed with varying success including surgical excision, systemic and intralesional corticosteroids, other immunosuppressants, and systemic chemotherapy. CONCLUSIONS: Adult xanthogranulomatous disease of the orbit and ocular adnexa, although rare, may be sight or life threatening. Recognition by the ophthalmologist is critical as periocular features often constitute the initial presentation.

Histologic and Phenotypic Factors and MC1R Status Associated with BRAF(V600E), BRAF(V600K), and NRAS Mutations in a Community-Based Sample of 414 Cutaneous Melanomas.

Cutaneous melanomas arise through causal pathways involving interplay between exposure to UV radiation and host factors, resulting in characteristic patterns of driver mutations in BRAF, NRAS, and other genes. To gain clearer insights into the factors contributing to somatic mutation genotypes in melanoma, we collected clinical and epidemiologic data, performed skin examinations, and collected saliva and tumor samples from a community-based series of 414 patients aged 18 to 79, newly diagnosed with cutaneous melanoma. We assessed constitutional DNA for nine common polymorphisms in melanocortin-1 receptor gene (MC1R). Tumor DNA was assessed for somatic mutations in 25 different genes. We observed mutually exclusive mutations in BRAF(V600E) (26%), BRAF(V600K) (8%), BRAF(other) (5%), and NRAS (9%). Compared to patients with BRAF wild-type melanomas, those with BRAF(V600E) mutants were significantly younger, had more nevi but fewer actinic keratoses, were more likely to report a family history of melanoma, and had tumors that were more likely to harbor neval remnants. BRAF(V600K) mutations were also associated with high nevus counts. Both BRAF(V600K) and NRAS mutants were associated with older age but not with high sun exposure. We also found no association between MC1R status and any somatic mutations in this community sample of cutaneous melanomas, contrary to earlier reports.

Prospective study of patterns of surgical management in adults with primary cutaneous melanoma at high risk of spread, in Queensland, Australia.

BACKGROUND: Knowledge of variation in diagnosis and surgery in high-risk primary melanoma patients is limited. We assessed frequency and determinants of diagnostic procedures, wide local excision (WLE) and sentinel lymph node biopsy (SLNB). METHODS: People in Queensland newly diagnosed with melanoma, clinical stage 1b or 2, were recruited prospectively. Patient information was collected from questionnaires and pathology records. Differences in surgical procedures in relation to host and tumor characteristics were assessed. RESULTS: In 787 participants, primary melanoma was diagnosed by surgical excision (74%), shave (14%), punch (12%) or incisional (1%) biopsy. General practitioners (GPs) diagnosed 80%. Diagnostic procedure differed by remoteness of residence, health sector, treating doctor's specialty and melanoma site and thickness. 766 patients had WLE, 86% by surgeons. Of 134 residual melanomas, 13 (10%) were ≤ 1 mm at diagnosis but > 1 mm at WLE, mostly after shave biopsy. SLNB was performed in 261 (33%) patients. SLNB was more common in those under 50, in remoter locations or treated by GP initially, and less common with head and neck melanoma. CONCLUSION: Diagnostic and surgical procedures for primary melanoma vary substantially and partial biopsy can influence initial tumor microstaging. Patient, tumor and doctor characteristics influence SLNB practice.

Solar elastosis and cutaneous melanoma: a site-specific analysis.

Cutaneous melanomas are postulated to arise through at least two causal pathways, namely the "chronic sun exposure" and "nevus" pathways. While chronic sun exposure probably causes many head/neck melanomas, its role at other sites is unclear. In a population-based, case-case comparison study conducted in Brisbane, Australia, we determined the prevalence and epidemiologic correlates of chronic solar damage in skin adjacent to invasive, incident melanomas on the trunk (n = 418) or head/neck (n = 92) among patients aged 18-79 in 2007-2010. Participants self-reported information about environmental and phenotypic factors, and a dermatologist counted nevi and actinic keratoses. Dermatopathologists assessed solar elastosis adjacent to each melanoma using a four-point scale (nil, mild, moderate, marked), and noted the presence or absence of adjacent neval remnants. We measured associations between various factors and solar elastosis using polytomous logistic regression. Marked or moderate solar elastosis was observed in 10% and 27%, respectively, of trunk melanomas, and 60% and 17%, respectively, of head/neck melanomas. At both sites, marked elastosis was positively associated with age (p(trend) < 0.0001) and inversely associated with neval remnants (p(trend) < 0.001). For trunk melanomas, marked elastosis was associated with highest quartiles of total sun exposure [odds-ratio (OR) = 5.47, 95% confidence interval (CI) = 1.08-27.60] and facial freckling (OR = 2.98, 95% CI = 1.17-7.56), and inversely associated with deeply tanning skin (OR = 0.29, 95% CI = 0.08-1.11) and high nevus counts (OR = 0.08, 95% CI = 0.01-0.66). Mostly similar associations were observed with moderate solar elastosis. About one in three trunk melanomas in Queensland have evidence of moderate-to-marked sun damage, and they differ in risk associations from those without.

Site-specific determinants of cutaneous melanoma: a case-case comparison of patients with tumors arising on the head or trunk.

BACKGROUND: Cutaneous melanomas have been hypothesized to arise through different pathways according to phenotype, body site, and sun exposure. To further test this hypothesis, we explored associations between phenotype and melanoma at different sites using a case-case comparative approach. METHODS: Melanoma patients (n = 762) aged 18 to 79 years and diagnosed from 2007 to 2010 were ascertained from pathology laboratories in Brisbane, Australia. Patients reported phenotypic information and a dermatologist counted melanocytic nevi and solar keratoses. We compared data for patients with trunk melanoma (n = 541, the reference group), head/neck melanoma (n = 122), or lentigo maligna melanoma (LMM) of the head/neck (n = 69). ORs and 95% confidence intervals were calculated using classical or polytomous logistic regression models. RESULTS: Compared with trunk melanoma patients, those with head/neck melanoma were significantly less likely to have high nevus counts (≥135: OR = 0.27; Ptrend = 0.0004). Associations between category of nevus count and LMM head/neck were weaker and significantly different (≥135: OR = 1.09; Ptrend = 0.69; Phomogeneity = 0.02). Patients with head/neck melanoma were more likely than those with truncal melanoma to have high solar keratosis counts (≥7: OR = 1.78, Ptrend = 0.04). Again, associations with LMM head/neck were weaker, albeit not significantly different (≥7: OR = 1.61; Ptrend = 0.42; Phomogeneity = 0.86). CONCLUSION: Trunk melanomas are more strongly associated with nevus counts than head/neck melanomas, but are less strongly associated with number of solar keratoses, a marker of chronic sun exposure. IMPACT: These findings underscore the notion that melanomas on the trunk typically arise through a causal pathway associated with nevus propensity, whereas melanomas on the head/neck arise through a pathway associated with cumulative sun exposure.

Granulomas in common variable immunodeficiency: a diagnostic dilemma.

A 60-year-old man with common variable immunodeficiency presented with a 7-year history of violaceous plaques and papules on the thighs, arms and trunk. In the preceding 2 years he had developed new lesions on both hands. He had been previously diagnosed with sarcoidosis on the basis of skin and visceral histology, but subsequent opinion was that these were sarcoid-like granulomas rather than being representative of true sarcoidosis. Biopsy of the hand lesions showed necrotizing granulomas, and a single acid-fast bacillus (AFB) was identified on Wade-Fite stain. Subsequent repeat tissue biopsies for histology, culture and polymerase chain reaction testing failed to confirm the presence of mycobacterial organisms and it was felt that the organism was a contaminant introduced during tissue processing. The hand lesions responded well to intralesional injections of triamcinolone acetonide 10 mg/mL and oral tetracycline 500 mg b.d. was later introduced with a good clinical response. The diagnostic dilemma of finding granulomatous inflammation in a patient with common variable immunodeficiency, and the significance of a single AFB on histology are discussed. The treatment of sarcoid-like granulomas with tetracycline therapy is also commented on.