CYLD Mutations Research Articles

CYLD Alterations Are Associated With Metastasis and Poor Prognosis in Human Papilloma Virus-Positive Head and Neck Cancer.

Human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is an emerging epidemic and a subset of HPV-positive patients experience aggressive disease with metastases. The CYLD gene is frequently altered in HPV-positive HNSCC, but the role of these alterations in disease progression is poorly understood. We identified 11 HPV-positive HNSCC patients with CYLD alterations and assessed their clinical course. We also characterized a unique, HPV-positive, metastatic, HNSCC patient-derived xenograft (PDX). All 11 patients developed metastasis with reduced overall survival when compared with metastatic HPV-positive patients with wild-type CYLD. The metastatic PDX harbored a CYLD mutation (S371*) and exhibited reduced expression of connexin 43, a potentially antimetastatic protein. We also investigated the functional impact of the S371* mutation, as well as 2 CYLD mutations from our 11-patient cohort. Our findings indicate that alterations in CYLD in HPV-positive HNSCC are associated with metastasis and poor prognosis.

A computational approach for structural and functional analyses of disease-associated mutations in the human CYLD gene

Tumor suppressor cylindromatosis protein (CYLD) regulates NF-κB and JNK signaling pathways by cleaving K63-linked poly-ubiquitin chain from its substrate molecules and thus preventing the progression of tumorigenesis and metastasis of the cancer cells. Mutations in CYLD can cause aberrant structure and abnormal functionality leading to tumor formation. In this study, we utilized several computational tools such as PANTHER, PROVEAN, PredictSNP, PolyPhen-2, PhD-SNP, PON-P2, and SIFT to find out deleterious nsSNPs. We also highlighted the damaging impact of those deleterious nsSNPs on the structure and function of the CYLD utilizing ConSurf, I-Mutant, SDM, Phyre2, HOPE, Swiss-PdbViewer, and Mutation 3D. We shortlisted 18 high-risk nsSNPs from a total of 446 nsSNPs recorded in the NCBI database. Based on the conservation profile, stability status, and structural impact analysis, we finalized 13 nsSNPs. Molecular docking analysis and molecular dynamic simulation concluded the study with the findings of two significant nsSNPs (R830K, H827R) which have a remarkable impact on binding affinity, RMSD, RMSF, radius of gyration, and hydrogen bond formation during CYLD-ubiquitin interaction. The principal component analysis compared native and two mutants R830K and H827R of CYLD that signify structural and energy profile fluctuations during molecular dynamic (MD) simulation. Finally, the protein–protein interaction network showed CYLD interacts with 20 proteins involved in several biological pathways that mutations can impair. Considering all these in silico analyses, our study recommended conducting large-scale association studies of nsSNPs of CYLD with cancer as well as designing precise medications against diseases associated with these polymorphisms.

Inactivation of the Tumor Suppressor CYLD Sensitizes Mice to Breast Cancer Development.

Breast cancer is a leading cause of cancer-related deaths among women. Down-regulation of the tumor suppressor gene Cyld in breast cancer has been linked to a poor prognosis. This study investigated the role of Cyld in breast cancer using conditional mutant mouse models carrying a Cyld mutation, which inactivates the deubiquitinating activity of its protein product CYLD in mammary epithelial cells. We examined the potential of CYLD inactivation to induce mammary tumors spontaneously or modify the susceptibility of mice to mammary tumorigenesis by DMBA treatment or ErbB2 over-expression. CYLD inactivation significantly increased susceptibility to breast cancer induced by either DMBA treatment or ErbB2 over-expression. Moreover, while CYLD inactivation alone did not lead to spontaneous mammary tumorigenesis, it did contribute to the formation of multifocal hyperplastic lesions in virgin mice of predominantly FVB/NJ background. Our study demonstrates the tumor enhancing potential of CYLD inactivation in mammary tumorigenesis in vivo and establishes novel relevant mouse models that can be exploited for developing prognostic and therapeutic protocols.

A Misdiagnosed Familiar Brooke-Spiegler Syndrome: Case Report and Review of the Literature.

Aim of the report: Brooke-Spiegler syndrome (BSS) is a rare autosomal dominant disease characterized by the growth of cylindromas, spiradenomas, trichoepitheliomas, or their combination. These neoplasms usually begin in the second decade and progressively increase in number and size over the years. Diagnosis necessitates consideration of family history, clinical examination, histological findings, and genetic analysis. The aim of this paper is to explore the clinical overlap between Brooke-Spiegler syndrome (BSS) and neurofibromatosis type 1 (NF1). We aim to highlight the challenges associated with their differential diagnosis and emphasize the lack of standardized diagnostic criteria and treatment approaches. Case presentation: Hereby, we introduce the case of a 28-year-old male referred for suspicion of neurofibromatosis type 1 (NF1) who initially declined the recommended surgical excision for a scalp mass. After four years, he returned with larger masses of the scalp, and underwent excision of multiple masses, revealing cylindromas, spiradenomas, and spiradenocylindromas. Family history reported similar tumors in his father, who was also diagnosed with NF1 for the presence of multiple subcutaneous lesions on the scalp. Clinical overlap led to a genetic consultation, but testing for CYLD mutations yielded no significant variations. Despite this, the strong family history and consistent findings led to a revised diagnosis of Brooke-Spiegler syndrome, correcting the initial misdiagnosis of NF1 syndrome. Conclusions: Thanks to the evolving landscape of BSS research over the past two decades, its molecular underpinnings, clinical presentation, and histopathological features are now clearer. However, a thorough family history assessment is mandatory when BSS is suspected. It is our belief that a multidisciplinary approach and cooperation between specialists are essential when dealing with BSS. By sharing this case, we hope to underscore the importance of considering BSS as a differential diagnosis, especially in cases with atypical presentations or overlapping features with other syndromes like NF1.

Abstract HER2-05: HER2-05 Comprehensive genomic characterization of HER2-low breast cancer

Abstract Background: About half of all breast cancers exhibit low HER2 expression. Despite lack of ERBB2 amplification, HER2-low tumors respond to trastuzumab deruxtecan (T-DXd), leading to the NCCN recommendation of T-DXd both for patients with HER2+ and HER2-low metastatic breast cancer (MBC). It remains however unclear if HER2-low represents a distinct molecular entity, as compared to HER2-0 MBC. Here, we compare the genomic landscape of HER2-low versus HER2-0 breast cancers in a large, single institution cohort. Methods: We identified consecutive patients with MBC seen at Dana-Farber Cancer Institute between 07/2013 and 12/2020. Patients were included if they had HER2-negative MBC per ASCO/CAP Guidelines and had undergone next generation sequencing (NGS) testing with a targeted, tumor-only platform (OncoPanel). Based on the HER2 status of the specimen tested by NGS, patients were divided into 2 groups: (i) HER2-low if immunohistochemistry (IHC) 1+ or 2+ non-amplified, or (ii) HER2-0 if IHC 0. Mutations of interest detected on NGS were classified as oncogenic using the OncoKB tool and additional annotation. Genomic profiles of HER2-low and HER2-0 tumors were compared using Chi-Square and Kruskal-Wallis tests. To determine genomic event enrichment between the two HER2 groups, logistic regression models were used, accounting for background rate and estrogen receptor (ER) expression. ERBB2 copy counts were calculated for tumors with recorded histology-estimated purities and copy-number segmentation using a simple model of allelic gain/loss. Results: Among 1847 patients with HER2-negative MBC, 1043 underwent NGS testing on a HER2-low (n=489, 47%) or HER2-0 sample (n=554, 53%). Most samples were metastatic (71%, n=743) while 29% (n=300) were from primary tumors. 73% had ductal histology, 13% were lobular and 14% had mixed or other histology. ER expression was enriched among HER2-low vs. HER2-0 tumors (76% vs. 60%; p< 0.001). Focusing on the most commonly occurring genetic mutations, no major differences were observed in HER2-low vs. HER2-0 tumors, after correcting for ER status (Table 1). Among all mutational events, any mutation in MPL, CYLD, and MAP3K and oncogenic mutations in TP53 and NF1 were more common in HER2-0, while any mutation in MTOR, RAD21, DNMT3A, and PDGFRA were enriched in HER2-low patients, when controlling for ER status and background mutational rate (p< 0.05). However, no mutation reached significance after accounting for multiple hypothesis testing. Similarly, no deep deletion or high amplification CNV events reached significance for either group. Analysis of tumor mutational burden in HER2-low vs. HER-0 tumors revealed no significant differences (median: 7.26 muts/Mb vs. 7.60 muts/Mb, p=1.00), including when accounting for ER status. Finally, among tumors with sufficient tumor purity for ERBB2 copy count analysis (n=374 and 419 for HER2-low and HER2-0, respectively), HER2-low tumors had a significantly higher number of ERBB2 alleles as compared to HER2-0 (< 2 copies, 15.0% vs. 30.9%, 2 copies 67.4% vs. 60.5%, and >2 copies, 17.6% vs. 8.6%; p< 0.001 by Kruskal-Wallis). Conclusions: To our knowledge, this is the largest comprehensive genomic analysis of HER2-low MBC to date. In our cohort of patients with HER2-negative MBC, the genomic landscape of HER2-low and HER2-0 tumors did not differ significantly, apart from a higher number of ERBB2 alleles. These data further support the notion that HER2-low, as currently defined, is not a distinct molecular subtype of breast cancer. Citation Format: Paolo Tarantino, Hersh V. Gupta, Melissa E. Hughes, Janet L. Files, Sarah Strauss, Gregory Kirkner, Anne-Marie Feeney, Yvonne Y. Li, Ana C. Garrido-Castro, Romualdo Barroso-Sousa, Brittany Bychkovsky, Laura MacConaill, Neal Lindeman, Bruce Johnson, Matthew Meyerson, Sheheryar Kabraji, Rinath Jeselsohn, Xintao Qiu, Rong Li, Henry W. Long, Eric Winer, Deborah A. Dillon, Giuseppe Curigliano, Andrew Cherniack, Sara Tolaney, Nancy U. Lin. HER2-05 Comprehensive genomic characterization of HER2-low breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr HER2-05.

Differential diagnosis of Brooke–Spiegler syndrome in a young woman with multiple trichoepitheliomas

BrookeSpiegler syndrome (Brooke-Spiegler syndrome; OMIM #605041) is a rare, autosomal dominant inherited monogenic disease caused by mutations in gene CYLD with its different penetrance.
 It is clinically manifested by the development of multiple neoplasms of skin appendages such as spiradenoma, cylindroma, spiradenocylindroma and trichoepithelioma. Several phenotypic variants with mutations in gene CYLD have been described in the scientific literature. They are classic BrookeSpiegler syndrome; multiple familial trichoepithelioma syndrome (multiple trichoepitheliomas without cylindromas, spiradenomas, etc.); familial cylindromatosis (multiple cylindromas on the scalp i.e., "turban tumor"); syndrome of multiple spiradenomas or spiradenocylindromas without other neoplasms of the skin appendages. For four diseases associated with CYLD mutations, the prevalence is 1/1 000 000.
 The scientific literature describes more than 200 cases of BrookeSpiegler syndrome, which is more common in women. Phenotypic manifestations of the CYLD mutation are variable, so there is no reliable statistics on the frequency of occurrence of clinical variants of BrookeSpiegler syndrome. The variety of neoplasms of the skin appendages, the commonality of their histogenesis, the similarity of clinical and histological patterns significantly complicate the diagnosis. Verification of the diagnosis of BrookeSpiegler syndrome is based on histological examination and sequencing of the CYLD gene.

A novel frameshift CYLD mutation in a family with multiple familial trichoepithelioma

Introduction: Multiple familial trichoepithelioma is a rare autosomal dominant genodermatosis caused by a mutation in the CYLD gene. This disease is characterized by the development of multiple skin-colored papules and nodules mainly on the central face. We herein describe a family with multiple familial trichoepithelioma with a novel frameshift mutation in CYLD. Case presentation: The proband was an 8-year-old girl who presented with a 2-year history of numerous progressive, asymptomatic, firm skin-colored papules on her nasal dorsum, nasolabial folds, and eyelids. Histopathological examination was compatible with the typical findings of trichoepithelioma. Her father displayed similar but inconspicuous smaller lesions on the face. Mutation analysis of the proband identified a heterozygous frameshift mutation c.1181_1182insCTTTGAC (p.D394fs) in exon 10 of CYLD. This mutation was also detected in the proband’s father, but not in either her mother or 100 unrelated control individuals Discussion: There is no obvious genotype–phenotype correlation in patients with multiple familial trichoepithelioma. In our study, the girl had a severe clinical appearance characterized by facial disfiguration, whereas her father had inconspicuous smaller lesions despite having the same mutation. We also identified a novel frameshift mutation c.1181_1182insCTTTGAC (p.D394fs) in exon 10 of the CYLD gene. The presence of this mutation leads to premature translation termination and thus to the synthesis of a truncated protein. Conclusion: We found a novel frameshift mutation of the CYLD gene in a Chinese family, thus expanding the database of CYLD gene mutations in multiple familial trichoepithelioma.

NF-κB over-activation portends improved outcomes in HPV-associated head and neck cancer.

Evolving understanding of head and neck squamous cell carcinoma (HNSCC) is leading to more specific diagnostic disease classifications. Among HNSCC caused by the human papilloma virus (HPV), tumors harboring defects in TRAF3 or CYLD are associated with improved clinical outcomes and maintenance of episomal HPV. TRAF3 and CYLD are negative regulators of NF-κB and inactivating mutations of either leads to NF-κB overactivity. Here, we developed and validated a gene expression classifier separating HPV+ HNSCCs based on NF-κB activity. As expected, the novel classifier is strongly enriched in NF-κB targets leading us to name it the NF-κB Activity Classifier (NAC). High NF-κB activity correlated with improved survival in two independent cohorts. Using NAC, tumors with high NF-κB activity but lacking defects in TRAF3 or CYLD were identified; thus, while TRAF3 or CYLD gene defects identify the majority of tumors with NF-κB activation, unknown mechanisms leading to NF-kB activity also exist. The NAC correctly classified the functional consequences of two novel CYLD missense mutations. Using a reporter assay, we tested these CYLD mutations revealing that their activity to inhibit NF-kB was equivalent to the wild-type protein. Future applications of the NF-κB Activity Classifier may be to identify HPV+ HNSCC patients with better or worse survival with implications for treatment strategies.

Impact of CYLD on chromatin structure and histone methylation in malignant melanoma.

The tumor suppressive role of CYLD lysine 63 deubiquitinase (CYLD) is known in melanoma. To the best of our knowledge, however, the precise mechanism underlying the tumor suppressive function of CYLD has yet to be clarified. In the present study, a novel melanoma mouse model was generated, which revealed accelerated tumor growth in Cyld-knockout (Cyld−/−) compared with Cyld-wild-type (Cyld+/+) mice. To determine the underlying molecular mechanism, mutation analysis of primary tumor-derived cell lines from Cyld+/+ and Cyld−/− mice was performed using RNA sequencing data. Variant calling revealed no common mutations in Cyld−/− compared with Cyld+/+ cells. Thus, the epigenetic processes influencing development and progression of melanoma were investigated. Initial analysis of expression pattern of known hypermethylated genes in melanoma (suppressor of cytokine signalling, methylthioadenosine phosphorylase, cadherin 1) in the presence or absence of 5′-Aza-deoxyctidine treatment revealed that CYLD does not play a key role in DNA methylation. Chromatin accessibility and histone H3 modification assay uncovered a role of CYLD in the formation of chromatin structure. Subsequent inhibitor experiments confirmed the effect of CYLD on H3K9me2 level associated with heterochromatin. Furthermore, enhanced H3K9 dimethylation in Cyld−/− melanoma cells was associated with upregulation of euchromatic histone lysine methyltransferase 2 (EHMT2). Moreover, the specific inhibitor of EHMT2, CM272, resulted in decreased proliferation and relaxation of compact chromatin in Cyld-deficient melanoma cells. These results reveal a novel role of CYLD in histone methylation and chromatin packaging.

Prevalence of CYLD mutations in Vietnamese patients with polycythemia vera.

Polycythemia vera (PV) is characterized by increased proliferation and accumulation of erythroid and mature myeloid cells and megakaryocyte in the bone marrow and peripheral blood. The JAK2V617F mutation is present in most PV patients. Deubiquitinase (DUB) genes, including TNFAIP3 (A20), CYLD and Cezanne, function as negative regulators of inflammatory reaction through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ęB) signaling. To determine single nucleotide polymorphisms (SNPs) profiling and gene expression of the DUB genes as well as the immunophenotype of PV cells. Seventy-seven patients with PV and 55 healthy individuals with well-characterized clinical profiles were enrolled. Gene expression profile was determined using quantitative real-time polymerase chain reaction (qRT-PCR), the immunophenotype with flow cytometry, secretion of cytokines using enzyme-linked immunosorbent assay (ELISA), and gene polymorphisms using direct DNA sequencing. Inactivation of A20, CYLD and Cezanne, and increases in interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-á) levels, as well as the enhanced number of CD25+CD4 T, Th1 and regulatory T cells were observed in PV patients. The genetic analysis of the CYLD gene identified 11 SNPs, in which a novel W736G nsSNP in exon 15 and a SNP c.2483+6 T>G in intron 15 were observed in PV cases with the frequencies of 18.2% and 5.2%, respectively. The W736G non-synonymous SNP (nsSNP) was found to be most likely to exert deleterious effect and the intronic SNP c.2483+6 T>G was identified as aberrant splicing. Sequencing of Cezanne gene identified 7 SNPs in intron 10 and PV carriers of the SNPs had at least 2 SNPs in this gene. Importantly, PV carriers of the W736G nsSNP had multiple SNPs in CYLD, but not in A20 or Cezanne gene. Two identified SNPs, including the W736G nsSNP and the SNP c.2483+6 T>G, in CYLD gene might be associated with a risk of PV disease, in which the deleterious effect of the W736G nsSNP in CYLD gene could contribute to the pathogenesis of PV.

Deficiency of the CYLD Impairs Fear Memory of Mice and Disrupts Neuronal Activity and Synaptic Transmission in the Basolateral Amygdala.

Fear learning and memory are crucial for animal survival. Abnormal fear memory is a hallmark of many neuropsychiatric disorders. Appropriate neuronal activation and excitability in the basolateral amygdala (BLA) are necessary for the formation of fear memory. The gene cylindromatosis (Cyld), which encodes a lysine-63 deubiquitinase, is expressed in several brain regions including the amygdala. The functions of the cylindromatosis protein (CYLD) in the regulation of the neuronal activity, neural circuits and fear memory, remain largely unknown, however. Here, we report that Cyld knockout impairs amygdala-dependent tone-cued fear memory. The number of c-Fos+ neurons responding to the tone-cued fear test was reduced in the BLA of Cyld–/– mice, suggesting that the absence of CYLD causes aberrant neuronal activation. We found that this aberrant neuronal activation in the BLA of Cyld–/– mice may relate to the decreased excitability of principal neurons. Another possibility of aberrant neuronal activation could be the impaired excitatory synaptic transmission in the BLA of Cyld–/– mice. Specifically, both the frequency of spontaneous excitatory postsynaptic currents and the amplitude of miniature excitatory postsynaptic currents in BLA principal neurons were decreased. In addition, Cyld mutation caused an increase in both the frequency of miniature inhibitory postsynaptic currents in principal neurons and the number of parvalbumin+ interneurons, consistent with excessive local circuit inhibition in the BLA of Cyld–/– mice. Taken together, these results suggest that CYLD deficiency disrupts the neuronal activity and synaptic transmission in the BLA of mice which may contribute to the impaired fear memory observed in Cyld–/– mice.

Abstract 2252: Genome profiling of thymic carcinoma identifies putative driver mutations in the NF-κB signaling pathway

Abstract Background: Thymic epithelial tumors (TETs), including thymoma and thymic carcinoma (TC), are one of the rarest cancers. Unlike thymoma, TC is clinically agressive and responds poorly to first-line chemotherapy and second-line systemic therapy. Currently, sunitinib is the only receptor tyrosine kinase inhibitor approved for TC treatment. However, most TC patients do not have the corresponding c-Kit mutations. Because the molecular drivers of TC are almost unknown, characterization of the mutational landscape of TC will broaden our understanding of TC etiology and facilitate the development of effective targeted therapies. Methods: Comprehensive genomic profiling, including SNV, indels, copy number alterations and re-arrangement analysis, was performed with a 830-gene next-generation sequencing panel on tumor tissues from a cohort of 32 TET patients (16 TCs and 16 thymomas). Results: Recurrent mutated genes in this TET cohort were TP53, CYLD, KMT2C, BRCA1, CHD2, PBRM1, ARID2, ASXL1, BRD7, CDKN2A, EGFR, KIT, MYH9, NFKB1A, RB1 and TET2. TP53 is the most frequently mutated gene, seen in 31% (5/16) of TCs and 6% (1/16) of thymomas. We found two actionable mutations in TCs (KIT D579del and IDH1 R132C) and one in thymomas (KIT V560D). Consistent with previous reports, we observed recurrent mutations in multiple genes encoding epigenetic regulator proteins (ASXL1, TET2 KMT2C, CHD2, PBRM1, ARID2 and BRD7), all in TCs but not thymomas. Most importantly, we found four CYLD mutations (two stop gain, one frameshift and one missense) and two NFKBIA mutations (one frameshift and one in splicing-site) in 6 TC samples. Both CYLD and NFKBIA genes encode negative regulators of NF-κB signaling pathway. CYLD is a Ub-specific protease (USP)-type deubiquitinase that preferentially disassembles Lys63-Ub, a post-translational modification required for the activation of NF-κB, while NFKBIA causes trapping REL dimers induced inhibitory activity of dimeric NF-κB/REL protein complexes. Furthermore, genomic alterations in NFKBIA and CYLD were mutually exclusive. Our results indicated that loss-of-function mutations in CYLD and NFKBIA may function as the molecular drivers of TC through constitutive activation of NF-κB signaling pathway. Conclusion: Our findings provide an enhanced road map for the etiology of TC. Further investigation is warranted into the roles of NF-κB pathway and epigenetic regulators dysfunction in TC pathogenesis and their potential for clinical trials. Citation Format: Naixin Liang, Xiaomo Li, Tonghui Ma, Chao Gao, Zhili Cao, Lei Cao, Zhongxing Bing, Zhenhuan Tian, Chao Guo, Shanqing Li, Yadong Wang, Jianchao Xue, Ji Li, Xiaoguang Li, Peng Liu, Shuyang Zhang. Genome profiling of thymic carcinoma identifies putative driver mutations in the NF-κB signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2252.

CYLD-mutant cylindroma-like basaloid carcinoma of the anus: a genetically and morphologically distinct class of HPV-related anal carcinoma

Rare reports of anal carcinoma (AC) describe histologic resemblance to cutaneous cylindroma, but mutations in the tumor suppressor CYLD, the gene responsible for familial and sporadic cylindromas, have not been systematically investigated in AC. Here, we investigate CYLD-mutant AC, focusing on molecular correlates of distinct histopathology. Comprehensive genomic profiling (hybrid-capture-based DNA sequencing) was performed on 574 ACs, of which 75 unique cases (13%) harbored a CYLD mutation. Clinical data, pathology reports, and histopathology were reviewed for each CYLD-mutant case. The spectrum of CYLD mutations included truncating (n = 50; 67%), homozygous deletion (n = 10; 13%), missense (n = 16; 21%), and splice-site (n = 3; 4%) events. Compared with CYLD-wildtype AC (n = 499), CYLD-mutant ACs were significantly enriched for females (88% vs. 67%, p = 0.0001), slightly younger (median age 59 vs. 61 years, p = 0.047), and included near-universal detection of high-risk HPV sequences (97% vs. 88%, p = 0.014), predominantly HPV16 (96%). The CYLD-mutant cohort also showed significantly lower tumor mutational burden (TMB; median 2.6 vs. 5.2 mut/Mb, p < 0.00001) and less frequent alterations in PIK3CA (13% vs. 31%, p = 0.0015). On histopathologic examination, 73% of CYLD-mutant AC (55/75 cases) showed a striking cylindroma-like histomorphology, composed of aggregates of basaloid cells surrounded by thickened basement membranes and containing characteristic hyaline globules, while only 8% of CYLD-wildtype tumors (n = 34/409) contained cylindroma-like hyaline globules (p < 0.0001). CYLD-mutant carcinomas with cylindroma-like histomorphology (n = 55) showed significantly lower TMB compared with CYLD-mutant cases showing basaloid histology without the distinctive hyaline globules (n = 14) (median 1.7 vs. 4.4 mut/Mb, p = 0.0058). Only five CYLD-mutant cases (7%) showed nonbasaloid conventional squamous cell carcinoma histology (median TMB = 5.2 mut/Mb), and a single CYLD-mutant case showed transitional cell carcinoma-like histology. Within our cohort of ACs, CYLD mutations characterize a surprisingly large subset (13%), with distinct clinical and genomic features and, predominantly, a striking cylindroma-like histopathology, representing a genotype-phenotype correlation which may assist in classification of AC.

CYLD mutation characterizes a subset of HPV-positive head and neck squamous cell carcinomas with distinctive genomics and frequent cylindroma-like histologic features

Mutations in the tumor suppressor CYLD, known to be causative of cylindromas, were recently described in a subset of high-risk (hr) HPV-positive head and neck squamous cell carcinomas (HNSCC). Pathologic and genetic characterization of these CYLD-mutant carcinomas, however, remains limited. Here, we investigated whether CYLD mutations characterize a histopathologically and genomically distinct subset of hrHPV-positive HNSCC. Comprehensive genomic profiling via hybrid capture-based DNA sequencing was performed on 703 consecutive head and neck carcinomas with hrHPV sequences, identifying 148 unique cases (21%) harboring CYLD mutations. Clinical data, pathology reports, and histopathology were reviewed. CYLD mutations included homozygous deletions (n = 61/148; 41%), truncations (n = 52; 35%), missense (n = 26; 18%) and splice-site (n = 9; 6%) mutations, and in-frame deletion (n = 1; 1%). Among hrHPV-positive HNSCC, the CYLD-mutant cohort showed substantially lower tumor mutational burden than CYLD-wildtype cases (n = 555) (median 2.6 vs. 4.4 mut/Mb, p < 0.00001) and less frequent alterations in PIK3CA (11% vs. 34%, p < 0.0001), KMT2D (1% vs. 16%, p < 0.0001), and FBXW7 (3% vs. 11%, p = 0.0018). Male predominance (94% vs. 87%), median age (58 vs. 60 years), and detection of HPV16 (95% vs. 89%) were similar. On available histopathology, 70% of CYLD-mutant HNSCC (98/141 cases) contained hyalinized material, consistent with basement membrane inclusions, within crowded aggregates of tumor cells. Only 7% of CYLD-wildtype cases demonstrated this distinctive pattern (p < 0.0001). Histopathologic patterns of CYLD-mutant HNSCC lacking basement membrane inclusions included nonkeratinizing (n = 22, 16%), predominantly nonkeratinizing (nonkeratinizing SCC with focal maturation; n = 10, 7%), and keratinizing (n = 11, 8%) patterns. The latter two groups showed significantly higher frequency of PTEN alterations compared with other CYLD-mutant cases (38% [8/21] vs. 7% [8/120], p = 0.0004). Within our cohort of hrHPV-positive HNSCCs, CYLD mutations were frequent (21%) and demonstrated distinctive clinical, histopathologic, and genomic features that may inform future study of prognosis and treatment.

Clonal Mutations Activate the NF-κB Pathway to Promote Recurrence of Nasopharyngeal Carcinoma.

The genetic events occurring in recurrent nasopharyngeal carcinoma (rNPC) are poorly understood. Here, we performed whole-genome and whole-exome sequencing in 55 patients with rNPC and 44 primarily diagnosed NPC (pNPC), with 7 patients having paired rNPC and pNPC samples. Previously published pNPC exome data were integrated for analysis. rNPC and pNPC tissues had similar mutational burdens, however, the number of clonal mutations was increased in rNPC samples. TP53 and three NF-κB pathway components (TRAF3, CYLD, and NFKBIA) were significantly mutated in both pNPC and rNPC. Notably, mutations in TRAF3, CYLD, and NFKBIA were all clonal in rNPC, however, 55.6% to 57.9% of them were clonal in pNPC. In general, the number of clonal mutations in NF-κB pathway-associated genes was significantly higher in rNPC than in pNPC. The NF-κB mutational clonality was selected and/or enriched during NPC recurrence. The amount of NF-κB translocated to the nucleus in samples with clonal NF-κB mutants was significantly higher than that in samples with subclonal NF-κB mutants. Moreover, the nuclear abundance of NF-κB protein was significantly greater in pNPC samples with locoregional relapse than in those without relapse. Furthermore, high nuclear NF-κB levels were an independent negative prognostic marker for locoregional relapse-free survival in pNPC. Finally, inhibition of NF-κB enhanced both radiosensitivity and chemosensitivity in vitro and in vivo. In conclusion, NF-κB pathway activation by clonal mutations plays an important role in promoting the recurrence of NPC. Moreover, nuclear accumulation of NF-κB is a prominent biomarker for predicting locoregional relapse-free survival. SIGNIFICANCE: This study uncovers genetic events that promote the progression and recurrence of nasopharyngeal carcinoma and has potential prognostic and therapeutic implications.See related commentary by Sehgal and Barbie, p. 5915.

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.

305 Putative phenotype modifying genetic factors associated with phenotypic diversity in Brooke-Spiegler syndrome

Brooke-Spiegler syndrome (BSS; OMIM 605041) is a rare monogenic skin disease characterized by the development of skin appendage tumors caused by mutations in the cylindromatosis gene (CYLD). Concerning the so far reported phenotypes and the underlying CYLD mutations, it is difficult to establish genotype-phenotype correlations in BSS. We have recently investigated a Hungarian family (with Bukovinian origin) and an Anglo-Saxon BSS pedigree, in whom the affected family members - despite of carrying the same disease-causing mutation (c.2806C>T, p.Arg936X) of the CYLD gene - show striking differences in their phenotypes. The aim of our study was to identify phenotype modifier genetic factors, which could help the understanding of genotype-phenotype correlations in BSS. Comparing the WES data of the Hungarian and Anglo-Saxon BSS patients, here we have identified three putative phenotype modifying genetic variants: the rs1053023 SNP of the signal transducer and activator of transcription 3 (STAT3) gene, the rs1131877 SNP of the tumor necrosis factor receptor-associated factor 3 (TRAF3) gene and the rs202122812 SNP of the neighbor of BRCA1 gene 1 (NBR1) gene. Our study contributes to the accumulating evidence describing the clinical importance of genetic phenotype modifying factors, which have high potential in the elucidation of genotype-phenotype correlations and disease prognosis.

Abstract 1722: Spectrum of solid malignancies associated with mutations in CYLD gene

Abstract Introduction: The CYLD gene product is a de-ubiqutinating enzyme involved in regulation of NF-kappaB signaling. Inactivating germline mutations in CYLD are the cause of Brooke-Spiegler syndrome characterized by multiple cutaneous tumors (cylindromas, trichoepitheliomas and spiradenomas) and tumors of salivary glands. Little is known about the role of CYLD in the development of solid organ malignancies. Methods: We retrospectively analyzed CYLD gene mutations detected in a large database of solid tumors genetically profiled (NGS of 592 genes) at a reference laboratory (Caris Life Science, Phoenix, AZ). Results: CYLD pathogenic mutations were identified in 235 tumor samples (0.4% of the tumors analyzed). The most common primary sites included lung (60), endometrium (15) and anus (10). Histologically, the majority of the tumors were classified as squamous (71) and adenocarcinoma (59). A number of mutations were identified, most commonly affecting codons p.N722fs (27), p.S371* (16) and p.P771S/p.P771L (12). 38 cases had only CYLD mutations detected, while the majority of associated mutations occurred in TP53 (45%). Numerous co-occurring pathogenic mutations were present in oncogenes (e.g. PIK3CA in 41, KRAS in 33, BRAF 17) and/or tumor suppressor genes (e.g. APC in 40, PTEN in 36, BRCA2 in 25, RB1 in 23). Conclusion: Our study provides the largest database analysis of CYLD gene mutations in solid tumors and shows the distribution in numerous primary tumor sites previously unrecognized in association with CYLD. Additional studies may reveal unexpected germ-line associations and will be pursued in the future. Citation Format: Michelle Ellis, Jeff Swensen, Zoran Gatalica. Spectrum of solid malignancies associated with mutations in CYLD gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1722.

Abstract 2525: Comprehensive analysis of thymic epithelial tumors using an integrated bioinformatics platform reveals novel biological characteristics

Abstract Introduction:Thymic epithelial tumors (TETs) are rare malignancies. Among TETs, thymoma is the most predominant, characterized by a unique association with myasthenia gravis, followed by thymic carcinoma which is less common but more clinically aggressive. The TCGA recently published a comprehensive analysis of 117 TETs using molecular profiling by exome-seq, RNA-seq, RPPA, and methylation. Using an integrated informatics platform we re-analyzed the TCGA TET data to identify novel biological characteristics of these rare tumors. Methods: Molecular and clinical data from the TCGA TET project was downloaded from the cbioportal database and imported into the tag.bio informatics platform. Tag.bio is a commercial cloud-based analysis platform that combines data, algorithms and question-based analysis protocols. It is used to for the rapid and reproducible statistical comparison of cohorts.Statistical analysis for differential expression, methylation, RPPA and mutational enrichment was performed using the Mann-Whitney test with multiple correction testing. Pathway and upstream regulator analysis were performed using Ingenuity Pathway Analysis. Results: Differential expression analysis of myasthenia gravis (MG) patients (N=32) compared to those with no history of MG (N=85), revealed the most significant gene as PPARGC1A (overexpressed, p&amp;lt;1e-8). This gene has not been previously associated with MG. PPARGC1A is highly expressed in skeletal muscle and mediates mitochondrial biogenesis. Supporting this observation, upstream regulator analysis was performed which identifies regulators that can explain the observed gene expression changes in a dataset. The results demonstrated that PPARGC1A as the most activated transcriptional regulator in MG+ tumors and may represent a novel autoantigen for MG. TETs are relatively indolent tumors, and only a small subset metastasizes. Differential expression in a subset of Stage IV tumors (N=6) identified overexpressed FGFR3. Of note, a previous study identified two patients with activating mutations in FGFR3. A genomic hallmark of Thymic Carcinoma (TC) is Chr16q loss. Several tumor suppressors reside on 16q, including:CYLD, CBFB, CDH1, CDH11, CTCF, ZFHX3, but the exact causative candidate is unknown. Analysis of the mutational data identified 2 of 10 patients with concurrent 16q loss and deleterious mutations in CYLD, congruent with previous reports of recurrent mutations in CYLD in TC. CYLD has been previously demonstrated to regulate STAT3. Differential expression followed by upstream regulator analysis of TC vs Thymoma demonstrated activated STAT3 in TC. Conclusion:Re-analysis of the TCGA TET dataset using a novel integrated bioinformatics platform identified novel biology of TETs including: PPARGC1A association with MG; FGFR3 expression in TET metastasis; and CYLD/STAT3 axis in TC. Citation Format: Milan Radovich, Jeffrey P. Solzak, Wade Webster, Jesse Paquette. Comprehensive analysis of thymic epithelial tumors using an integrated bioinformatics platform reveals novel biological characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2525.

Abstract 3442: Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD, in nasopharyngeal carcinoma

Abstract Background: NF-κB is an important signaling pathway for regulating multiple signaling cascades involved in cancer development such as inflammation, immune response, cell survival, and cell proliferation. Constitutive activation of the NF-κB pathway plays a key role in the development of NPC. Our previous whole-exome sequencing (WES) study in NPC biopsies has identified five mutations in CYLD, including R758Q. As R758X is a mutational hotspot in CYLD, it has been reported in multiple familial trichoepithelioma type 1, Brooke-Spiegler syndrome and familial cylindromatosis. Thus, we attempted to evaluate the functional effect of CYLD WT and R758Q in regulating NF-κB activity in NPC, which is crucial in NPC progression and development. Methods: C17, C666-1 and NPC43 EBV-positive cell lines and HK1 were co-infected by CYLD wild-type (WT) and mutant constructs with a NF-κB specific dual luciferase promoter plasmid. CRISPR-CAS9 system was used to knock out CYLD. The functional role of CYLD was determined by MTT, CFA and wound healing assays. NF-κB downstream targets expression was evaluated by the method of RT-QPCR. Co-immunoprecipitation (Co-IP) experiments were performed with the Sigma Anti-FLAG M2 magnetic beads and the results were confirmed by Western blot analysis. Female athymic BALB/cAnN (nude) mice were utilized to investigate tumorigenicity. NPC cell lines were treated with cisplatin to check cell sensitivity. Results: The reporter assay showed that CYLD can downregulate NF-κB binding activities in comparison to the vector-alone (VA), but R758Q failed to inhibit the NF-κB binding activities. Over-expression of CYLD can suppress NF-κB downstream target genes expression. Knocking out of CYLD results in higher NF-κB downstream genes expression compared with VA, which confirmed that CYLD can suppress NF-κB activity. R758Q increases these genes expression compared with WT. CYLD can suppress cell growth rate, while R758Q promoted cell proliferation and migration compared to WT. Furthermore, WT CYLD can significantly suppress tumor growth rate, whereas R758Q has the same growth rate with VA, consistent with R758Q loss of WT function, causing tumor suppression. Moreover, CYLD can enhance cell sensitivity to cisplatin treatment compared to the VA. Conclusions: These results suggest that CYLD can suppress NF-κB activity compared to VA, while R758Q lost WT function in NPC. CYLD can inhibit tumorigenesis compared to VA, whereas R758Q induces tumor growth compared to WT. The NPC cell line with CYLD WT is more sensitive to cisplatin treatment. Acknowledgement: Research Grants Council Area of Excellence grant AoE-M06/08 to MLL. Citation Format: Mingdan Deng, Wei Dai, Maria Lung. Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD, in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3442.