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Cylindroma is a rare skin tumour that is inherited in several skin-tumour syndromes caused by germline mutations in the tumour suppressor gene, CYLD. In this Review, we provide insight into the clinical features of patients who develop multiple cylindromas and other related tumours. The CYLD protein is also dysfunctional in various sporadic cancers and we discuss how such dysfunction relates to the role of CYLD in regulating key cellular pathways. Clinical management of patients with germline CYLD mutations is challenging and we discuss genetic counselling and surgical interventions. Finally, we discuss how the study of these rare syndromes might provide insights into understanding more common diseases.

CYLD lysine 63 deubiquitinase (CYLD) is a ubiquitin hydrolase with important roles in immunity and cancer. Complete CYLD ablation, truncation and expression of alternate isoforms, including short CYLD, drive distinct phenotypes and offer insights into CYLD function in inflammation, cell death, cell cycle progression and cell transformation. Research in diverse model systems has shown that these are mediated via CYLD regulation of cellular pathways including the NF-κB, Wnt and TGF-β pathways. Recent biochemical advances and models have offered new insights into the regulation and function of CYLD. In addition, recent discoveries of gain-of-function germline pathogenic CYLD variants in patients with a neurodegenerative phenotype contrast with the more widely known loss-of-function mutations seen in patients with CYLD cutaneous syndrome and with sporadic cancers. Here, we provide a current review of mechanistic insights into CYLD function gained from CYLD animal models, as well as an update on the role of CYLD in human disease.

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. CYLD cutaneous syndrome (also known as Brooke-Spiegler syndrome) is characterised by germline mutations in the tumor suppressor CYLD . Here, the authors highlight recurrent mutations in DNMT3A and BCOR , indicating a role for epigenetic dysregulation in this rare genetic skin disease.

Checkpoint inhibitors (CPIs) are monoclonal antibodies which, by disrupting interactions of immune checkpoint molecules with their ligands, block regulatory immune signals otherwise exploited by cancers. Despite revolutionary clinical benefits, CPI use is associated with an array of immune-related adverse events (irAEs) that mirror spontaneous autoreactivity. Severe irAEs necessitate pausing or stopping of CPI therapy and use of corticosteroids and/or other immunomodulatory interventions. Despite increasingly widespread CPI use, irAE pathobiology remains poorly understood; its elucidation may point to targeted mitigation strategies and uncover predictive biomarkers for irAE onset in patients, whilst casting new light on mechanisms of spontaneous immune-mediated disease. This review focuses on common CPI-induced irAEs of the gut, skin and synovial joints, and how these compare to immune-mediated diseases such as ulcerative colitis, vitiligo and inflammatory arthritis. We review current understanding of the immunological changes reported following CPI therapy at the level of peripheral blood and tissue. Many studies highlight dysregulation of cytokines in irAE-affected tissue, particularly IFNγ and TNF. IrAE-affected tissues are also predominantly infiltrated by T-cells, with low B-cell infiltration. Whilst there is variability between studies, patients treated with anti-programmed cell death-1 (PD-1)/PDL-1 therapies seem to exhibit CD8+ T-cell dominance, with CD4+ T-cells dominating in those treated with anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) monotherapy. Interestingly, CD8+CXCR3+ T-cells have been reported to be elevated in gastrointestinal, dermatological and musculoskeletal -irAE affected tissues. These findings may highlight potential opportunities for therapeutic development or re-deployment of existing therapies to prevent and/or improve the outcome of irAEs.

CYLD cutaneous syndrome (CCS) is an inclusive label for the inherited skin adnexal tumour syndromes Brooke-Spiegler Syndrome (BSS-OMIM 605041), familial cylindromatosis (FC--OMIM 132700) and multiple familial trichoepitheliomas (MFTOMIM 601606). All three syndromes arise due to germline pathogenic variants in CYLD, a tumour suppressor gene (OMIM 605018). CCS is transmitted in an autosomal dominant pattern, and has variable expressivity, both of the three syndromic phenotypes, and of the severity of tumour burden. Age-related penetrance figures are not precisely reported. The first tumours typically appear during puberty and progressively accumulate through adulthood. Penetrance is typically high, with equal numbers of males and females affected. Genetic testing is important for confirmation of the clinical diagnosis, genetic counselling and family planning, including preimplantation diagnosis. Additionally, identified CCS patients may be eligible for future clinical trials of non-surgical pre-emptive interventions that aim to prevent tumour growth. In this update, we review the clinical presentations of germline and mosaic CCS. An overview of the germline pathogenic variant spectrum of patients with CCS reveals more than 100 single nucleotide variants and small insertions and deletions in coding exons, most frequently resulting in predicted truncation. In addition, a minority of patients have large deletions involving the CYLD gene, intronic pathogenic variants that affect splicing, or inversions. We discuss germline and somatic testing approaches. Somatic testing of tumour tissue, relevant in mosaic CCS, can reveal recurrently detected pathogenic variants when two or more tumours are tested. This can influence genetic testing of children, who may inherit this as a germline variant, and inform genetic counselling and prenatal diagnosis. Finally, we discuss testing technologies that are currently used, their benefits and limitations, and future directions for genetic testing in CCS. Keywords: CYLD gene testing

Background Checkpoint inhibitors (CPIs) are widely used in cancer treatment, with transformative impacts on survival. They nonetheless carry a significant risk of toxicity in the form of immune-related adverse events (IrAEs), which may be sustained and life-altering. IrAEs may require high-dose and/or prolonged steroid use and represent a significant healthcare burden. They mimic immune-mediated inflammatory diseases (IMIDs) but understanding of their pathogenesis is limited. The MEDALLION project aims to determine targetable mechanisms of immune dysregulation in IrAE development, employing an immune monitoring approach to determine changes in circulating and tissue resident cells of CPI recipients who do/do not develop them and assessing the contribution of the microbiome in parallel. Methods MEDALLION is a non-randomised longitudinal cohort study aiming to recruit 66 cancer patient recipients of anti-PD1/PD-L1, anti-CTLA-4 or combination therapy. Eligible participants include those with malignant melanoma in the adjuvant or metastatic setting, mesothelioma and non-small cell lung carcinoma (NSCLC) treated in the metastatic setting. Comprehensive clinical evaluation is carried out alongside blood, skin swab and stool sampling at the time of CPI initiation (baseline) and during subsequent routine hospital visits on 6 occasions over a 10-month follow-up period. It is conservatively anticipated that one third of enrolled patients will experience a “significant IrAE” (SirAE), defined according to pre-determined criteria specific to the affected tissue/organ system. Those developing such toxicity may optionally undergo a biopsy of affected tissue where appropriate, otherwise being managed according to standard of care. Peripheral blood mononuclear cells will be analysed using multi-parameter flow cytometry to investigate immune subsets, their activation status and cytokine profiles. Stool samples and skin swabs will undergo DNA extraction for 16 S ribosomal RNA (rRNA) sequencing and internal transcribed spacer (ITS) gene sequencing to determine bacterial and fungal microbiome diversity, respectively, including species associated with toxicity. Stored tissue biopsies will be available for in situ and single-cell transcriptomic evaluation. Analysis will focus on the identification of biological predictors and precursors of SirAEs. Discussion The pathogenesis of IrAEs will be assessed through the MEDALLION cohort, with the potential to develop tools for their prediction and/or strategies for targeted prevention or treatment. Trial Registration The study was registered on 18/09/2023 in the ISRCTN registry (43,419,676).

The phenotype-based prevalence of Birt-Hogg-Dubé syndrome (BHD) is commonly estimated at 1 in 200,000–500,000. However, we demonstrate that BHD-causing FLCN variants are 75 to 180 times more prevalent in the multi-ethnic large genomic registry population. We highlight the urgent need for updated prevalence and penetrance estimates for BHD and other tumor suppressor gene syndromes, particularly among underrepresented non-European populations.

Transcriptomic profiling of skin disease using next generation sequencing allows for detailed information on aspects of RNA biology including gene expression, non-coding regulatory elements and gene splicing. The application of RNA sequencing to human skin disease and cancer is often hampered by degraded RNA. Here we describe a protocol that allows for consistently intact RNA to be extracted from snap frozen skin biopsy samples, which has been validated in a clinical trial setting. Human skin tumour punch biopsies (n=28) ranging from 4-6mm in diameter were obtained from 14 patients with an inherited skin tumour syndrome (CYLD cutaneous syndrome) and frozen in liquid nitrogen prior to being stored at -80°C. These samples were then subject to cyrostat sectioning, allowing for histological assessment, and were homogenised using a bead-based lysis platform. RNA extraction was performed using a silica column-based system. RNA concentration was measured using fluorescent quantitation and RNA integrity assessed using microfluidic gel electrophoresis. We also processed normal skin biopsies using the same protocol (n=10). The mean RNA integrity score of the tumour and normal samples was 9.5, and the quantity of RNA obtained from the small amounts of tissue used exceeded requirements for RNA-seq library generation. We propose that the method of RNA extraction suggested here allows for transcriptomic profiling from small pieces of human tissue without the need for PCR amplification during library preparation. This protocol could be utilised in healthy and diseased skin to improve mechanistic understanding in a range of human skin diseases.

Birt-Hogg-Dubé syndrome (BHD syndrome) is an autosomal dominant multisystem disorder with variable expression due to pathogenic constitutional variants in the FLCN gene. Patients with BHD syndrome are predisposed to benign cutaneous fibrofolliculomas/trichodischomas, pulmonary cysts with an associated risk of spontaneous pneumothorax, and renal cell carcinoma. A requirement for updated International consensus recommendations for the diagnosis and management of BHD syndrome was identified. Based on a comprehensive literature review and expert consensus within the fields of respiratory medicine, urology, radiology, dermatology, clinical oncology and clinical genetics, updated recommendations for diagnosis, surveillance and management in BHD syndrome were developed. With the widespread availability of FLCN genetic testing, clinical scenarios in which a diagnosis should be considered and criteria for genetic testing were defined. Following a clinical and/or molecular diagnosis of BHD syndrome, a multidisciplinary approach to disease management is required. Regular renal cancer surveillance is recommended in adulthood and life-long, but the evidence base for additional tumour surveillance is limited and further research warranted. Recommendations for the treatment of cutaneous, pulmonary and renal manifestations are provided. Awareness of BHD syndrome needs to be raised and better knowledge of the clinical settings in which the diagnosis should be considered should enable earlier diagnosis. Further details, including areas for future research topics are available at: https://www.genturis.eu/l=eng/Guidelines-and-pathways/Clinical-practice-guidelines.html .