Epigenetic modifiers DNMT3A and BCOR are recurrently mutated in CYLD cutaneous syndrome.

Patients with CYLD cutaneous syndrome (CCS; syn. Brooke-Spiegler syndrome) carry germline mutations in the tumor suppressor CYLD and develop multiple skin tumors with diverse histophenotypes. Here, we comprehensively profile the genomic landscape of 42 benign and malignant tumors across 13 individuals from four multigenerational families and discover recurrent mutations in epigenetic modifiers DNMT3A and BCOR in 29% of benign tumors. Multi-level and microdissected sampling strikingly reveal that many clones with different DNMT3A mutations exist in these benign tumors, suggesting that intra-tumor heterogeneity is common. Integrated genomic, methylation and transcriptomic profiling in selected tumors suggest that isoform-specific DNMT3A2 mutations are associated with dysregulated methylation. Phylogenetic and mutational signature analyses confirm cylindroma pulmonary metastases from primary skin tumors. These findings contribute to existing paradigms of cutaneous tumorigenesis and metastasis.

Targeting Tropomyosin Receptor Kinase in Cutaneous CYLD Defective Tumors With Pegcantratinib: The TRAC Randomized Clinical Trial.

Importance: There are no medical interventions for the orphan disease CYLD cutaneous syndrome (CCS). Transcriptomic profiling of CCS skin tumors previously highlighted tropomyosin receptor kinases (TRKs) as candidate therapeutic targets. Objective: To investigate if topical targeting of TRK with an existing topical TRK inhibitor, pegcantratinib, 0.5% (wt/wt), is safe and efficacious in CCS. Design, Setting, and Participants: A phase 1b open-label safety study, followed by a phase 2a within-patient randomized (by tumor), double-blind, placebo-controlled trial (the Tropomyosin Receptor Antagonism in Cylindromatosis [TRAC] trial). The setting was a single-center trial based at a tertiary dermatogenetics referral center for CCS (Royal Victoria Infirmary, Newcastle, United Kingdom). Patients who had germline mutations in CYLD or who satisfied clinical diagnostic criteria for CCS were recruited between March 1, 2015, and July 1, 2016. Interventions: In phase 1b, patients with CCS applied pegcantratinib for 4 weeks to a single skin tumor. In phase 2a, allocation of tumors was to either receive active treatment on the right side and placebo on the left side (arm A) or active treatment on the left side and placebo on the right side (arm B). Patients were eligible if they had 10 small skin tumors, with 5 matched lesions on each body side; patients were randomized to receive active treatment (pegcantratinib) to one body side and placebo to the other side once daily for 12 weeks. Main Outcomes and Measures: The primary outcome measure was the number of tumors meeting the criteria for response in a prespecified critical number of pegcantratinib-treated tumors. Secondary clinical outcome measures included an assessment for safety of application, pain in early tumors, and compliance with the trial protocol. Results: In phase 1b, 8 female patients with a median age of 60 years (age range, 41-80 years) were recruited and completed the study. None of the participants experienced any adverse treatment site reactions. Three patients reported reduced pain in treated tumors. In phase 2a (15 patients [13 female; median age, 51 years], with 150 tumors), 2 tumors treated with pegcantratinib achieved the primary outcome measure of response compared with 6 tumors treated with placebo. The primary prespecified number of responses was not met. The incidence of adverse events was low. Conclusions and Relevance: In this study, pegcantratinib, 0.5% (wt/wt), applied once daily appeared to be well tolerated and to penetrate the tumor tissue; however, the low tumor drug concentrations demonstrated are likely to account for the lack of response. Dose-escalation studies to assess the maximal tolerated dose may be beneficial in future studies of CCS. Trial Registration: isrctn.org Identifier: ISRCTN75715723.

Overexpression of MYB drives proliferation of CYLD-defective cylindroma cells.

Cutaneous cylindroma is an adnexal tumour with apocrine differentiation. A predisposition to multiple cylindromas is seen in patients with Brooke-Spiegler syndrome, who carry germline mutations in the tumour suppressor gene CYLD. Previous studies of inherited cylindromas have highlighted the frequent presence of bi-allelic truncating CYLD mutations as a recurrent driver mutation. We have previously shown that sporadic cylindromas express either MYB-NFIB fusion transcripts or show evidence of MYB activation in the absence of such fusions. Here, we investigated inherited cylindromas from several families with germline CYLD mutations for the presence of MYB activation. Strikingly, none of the inherited CYLD-defective (n = 23) tumours expressed MYB-NFIB fusion transcripts. However, MYB expression was increased in the majority of tumours (69%) and global gene expression analysis revealed that well-established MYB target genes were up-regulated in CYLD-defective tumours. Moreover, knock-down of MYB expression caused a significant reduction in cylindroma cell proliferation, suggesting that MYB is also a key player and oncogenic driver in inherited cylindromas. Taken together, our findings suggest molecular heterogeneity in the pathogenesis of sporadic and inherited cutaneous cylindromas, with convergence on MYB activation. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The cylindromatosis gene product, CYLD, interacts with MIB2 to regulate notch signalling.

CYLD, an ubiquitin hydrolase, has an expanding repertoire of regulatory roles in cell signalling and is dysregulated in a number of cancers. To dissect CYLD function we used a proteomics approach to identify CYLD interacting proteins and identified MIB2, an ubiquitin ligase enzyme involved in Notch signalling, as a protein which interacts with CYLD. Coexpression of CYLD and MIB2 resulted in stabilisation of MIB2 protein levels and was associated with reduced levels of JAG2, a ligand implicated in Notch signalling. Conversely, gene silencing of CYLD using siRNA, resulted in increased JAG2 expression and upregulation of Notch signalling. We investigated Notch pathway activity in skin tumours from patients with germline mutations in CYLD and found that JAG2 protein levels and Notch target genes were upregulated. In particular, RUNX1 was overexpressed in CYLD defective tumour cells. Finally, primary cell cultures of CYLD defective tumours demonstrated reduced viability when exposed to γ-secretase inhibitors that pharmacologically target Notch signalling. Taken together these data indicate an oncogenic dependency on Notch signalling and suggest potential novel therapeutic approaches for patients with CYLD defective tumours.

Clinical signs of photodamage are associated with basal cell carcinoma multiplicity and site: a 16-year longitudinal study.

Although sun exposure is known to be associated with basal cell carcinoma (BCC), it is not known what determines multiple occurrences of BCCs among sporadically affected individuals or why BCCs develop on uncommonly sun-exposed body sites like the trunk. In a prospective community-based skin cancer study in Queensland, Australia, we studied all participants who experienced a histologically confirmed BCC from 1992 to 2007. Sun exposure history was monitored, and dermatologists documented phenotype at baseline and signs of photodamage over the study period. Anatomic sites of all incident BCCs were recorded. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using logistic regression. Of 401 participants who developed a new BCC during the 16 years of follow-up, 232 (58%) developed more than 1. Male sex (OR = 2.5, 95% CI 1.5-5.3) and age 60 or over (OR = 4.2, 95% CI 1.5-11.8) but not skin type were associated with highest BCC counts among those affected. Participants with high numbers of solar keratoses were most likely to experience the highest BCC counts overall (OR = 4.3, 95% CI 1.4-13.5). Moreover, occurrences of BCC on the trunk (OR = 3.3, 95% CI 1.4-7.6) and on the limbs (OR = 3.7, 95% CI 2.0-7.0) were strongly associated with high numbers of solar keratoses on these sites, respectively. Among those newly affected by BCC, chronic cutaneous sun damage predicts those who will be affected by more than 1 BCC, while chronic sun damage on the trunk and limbs predicts BCC occurrence on the trunk and limbs, respectively.

Incidence of basal cell carcinoma multiplicity and detailed anatomic distribution: longitudinal study of an Australian population.

A proportion of individuals are affected multiple times by basal cell carcinoma (BCC), but the rate and extent to which this occurs is unknown. We therefore prospectively estimated BCC incidence in a subtropical Australian population, focusing on the rate at which persons develop multiple primary BCCs and the precise anatomic sites of BCC occurrence. Between 1997 and 2006, 663 BCCs were confirmed in 301 of 1,337 participants in the population-based Nambour Skin Cancer Study. The incidence of persons affected multiple times by primary BCC was 705 per 100,000 person years compared to an incidence rate of people singly affected of 935 per 100,000 person years. Among the multiply and singly affected alike, site-specific BCC incidence rates were far highest on facial subsites, followed by upper limbs, trunk, and then lower limbs. We conclude that actual BCC tumor burden is much greater in the population than is apparent from normal incidence rates. Anatomic distribution of BCC is consistent with general levels of sun exposure across body sites.

Histologic and epidemiologic correlates of P-MAPK, Brn-2, pRb, p53, and p16 immunostaining in cutaneous melanomas.

Histologic, molecular, and epidemiologic data suggest that cutaneous melanomas arise through diverse causal pathways, one of which appears mediated by chronic sunlight exposure, and another associated with a nevus-prone phenotype. To further explore etiologic heterogeneity, we compared expression of key melanoma-related proteins according to histologic characteristics of the tumors and epidemiologic risk factors among a sample of 129 patients newly diagnosed with melanoma. Tumor tissue was stained with antibodies to phospho-mitogen-activated protein kinase (P-MAPK), Brn-2, retinoblastoma protein (pRb), p53, and p16. Using logistic regression analysis, we estimated the odds ratio (OR) and 95% confidence interval (95% CI) of protein expression associated with factors of interest. Except for pRb, candidate protein expression was detected in fewer than half the melanomas examined (P-MAPK, 39%; Brn-2, 30%; p53, 24%; p16, 41%). Strong pRb expression was associated with the presence of >20 solar keratoses (OR 6.5, 95% CI: 1.7-25.1) and melanomas with marked to moderate solar elastosis. p53 expression was positively associated with skin that readily burned (OR 3.8, 95% CI: 0.8-19.0) and was inversely associated with >60 nevi (OR 0.3, 95% CI: 0.04-1.5). Melanomas expressing P-MAPK were more likely to have contiguous neval remnants (OR 2.7, 95% CI: 1.1-6.5). P-MAPK and Brn-2 immunopositive melanomas were >/=4-fold more likely to be of the superficial spreading subtype. Brn-2 and p16 immunopositive melanomas had a greater Breslow thickness than melanomas that did not express these proteins. Brn-2, pRb, and p16 proteins were consistently coexpressed. These findings support the hypothesis that molecular profiles of melanoma reflect their histologic and epidemiologic characteristics, offering further evidence of more than one melanoma causal pathway. Exactly how the expression of each protein relates to the causal pathways needs to be further explored.