UV-signature Mutations Research Articles

Whole Genome Sequencing Elucidates Etiological Differences in MCPyV-negative Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine neoplasm of the skin. Immunosuppression, ultraviolet radiation and the integration of Merkel cell polyomavirus (MCPyV) have all been shown to be involved in the pathogenesis of this malignancy. We performed whole genome sequencing on two MCPyV-negative cases of MCC that demonstrated very different clinical presentations and outcomes, and mutational profiles. The first case exhibited a highly aggressive clinical course, absence of UV-signature mutations and a low tumor mutational burden. A rearrangement in the tumor suppressor gene SUFU was identified, a likely driver and potential target of the Hedgehog signaling pathway. Meanwhile, the second case exhibited a less aggressive behavior, harbored UV-signature mutations, and a high mutational burden including mutations in TP53 and RB1.

CK20-negative Merkel Cell Carcinoma with Concomitant Squamous Cell Carcinoma In-Situ and Metastasis to the Parotid Gland

Abstract Introduction/Objective This report presents a patient with an aggressive CK20-negative neuroendocrine tumor of the dermis with metastasis to the parotid gland and a concomitant squamous cell carcinoma in-situ (SCCIS) and highlights diagnostic challenges of CK20-negative tumors. Methods/Case Report A centennial female with a history of non-melanoma skin cancer and leukemia presented to the dermatology clinic with a one-year history of an enlarging skin lesion on her right cheek and a subcutaneous mass on the right central lateral neck. On examination, the lesion appeared as a rough and raised red papule with a central crust. On CT, a large heterogeneously enhancing right parotid mass with surrounding satellite nodules, adjacent necrotic adenopathy, and an ulcerating subcutaneous mass in the adjacent right face. An incidental 3 mm right upper lobe nodule was identified. The pathology report noted two distinct malignant neoplasms within the specimen. Microscopy revealed ulceration with acanthosis and full thickness keratinocyte atypia, suggesting Bowen’s disease. On microscopic examination of the separate dermal tumor, there were sheets of uniform small cells with a high nuclear to cytoplasmic ratio and scant cytoplasm. IHC staining was positive for the neuroendocrine markers AE1/AE3, CAM5.2, synaptophysin, and CD56. CK20, CK7, and TTF-1 were negative. As some MCC may be negative for CK20, CK20 was performed twice. Differential diagnoses included SCCIS with CK20-negative MCC versus a metastatic neuroendocrine carcinoma of parotid gland to the skin. The patient transitioned to hospice care soon after the diagnosis. Results (if a Case Study enter NA) NA. Conclusion Our patient presents with an interesting case of a CK20-negative MCC with metastasis to the parotid gland. Since CK20 was stained negative twice, it complicated the diagnosis of MCC vs. small cell carcinoma. We suspected a more likely diagnosis of MCC metastasis to the parotid gland due to multiple reported cases (Day et al. 2016). Our patient also had an identifiable Bowen’s disease borderline to the neuroendocrine carcinoma. It is well known of the coexistence of MCC and squamous cell carcinoma in a cutaneous lesion (Suaiti et al. 2019). Thus, a metastatic MCC to the parotid gland is more likely in this case. As MCC is an aggressive cancer that metastasizes quickly, accurate diagnosis is crucial. With recent literature reporting the association of CK20-negativity with VN-MCC, and the presence of UV signature mutations in CK20- negative MCC, genomic screening would be a useful diagnostic tool.

Pervasive Induction of Regulatory Mutation Microclones in Sun-exposed Skin.

Carcinogen-induced mutations are thought near-random, with rare cancer-driver mutations underlying clonal expansion. Using high-fidelity Duplex Sequencing to reach a mutation frequency sensitivity of 4×10 -9 per nt, we report that sun exposure creates pervasive mutations at sites with ∼100-fold UV-sensitivity in RNA-processing gene promoters - cyclobutane pyrimidine dimer (CPD) hyperhotspots - and these mutations have a mini-driver clonal expansion phenotype. Numerically, human skin harbored 10-fold more genuine mutations than previously reported, with neonatal skin containing 90,000 per cell; UV signature mutations increased 8,000-fold in sun-exposed skin, averaging 3×10 -5 per nt. Clonal expansion by neutral drift or passenger formation was nil. Tumor suppressor gene hotspots reached variant allele frequency 0.1-10% via 30-3,000 fold clonal expansion, in occasional biopsies. CPD hyperhotspots reached those frequencies in every biopsy, with modest clonal expansion. In vitro, tumor hotspot mutations arose occasionally over weeks of chronic low-dose exposure, whereas CPD hyperhotspot mutations arose in days at 1000-fold higher frequencies, growing exponentially. UV targeted mini-drivers in every skin cell.

Squamoid Eccrine Ductal Carcinoma Displays Ultraviolet Mutations and Intermediate Gene Expression Relative to Squamous Cell Carcinoma, Microcystic Adnexal Carcinoma, and Porocarcinoma

Squamoid eccrine ductal carcinoma is a rare infiltrative tumor with morphologic features intermediate between squamous cell carcinoma (SCC) and sweat gland carcinomas such as microcystic adnexal carcinoma. Although currently classified as a sweat gland carcinoma, it has been debated whether squamoid eccrine ductal carcinoma is better classified as a variant of SCC. Furthermore, therapeutic options for patients with advanced disease are lacking. Here, we describe clinicopathologic features of a cohort of 15 squamoid eccrine ductal carcinomas from 14 unique patients, with next-generation sequencing DNA profiling for 12 cases. UV signature mutations were the dominant signature in the majority of cases. TP53 mutations were the most highly recurrent specific gene alteration, followed by mutations in NOTCH genes. Recurrent mutations in driver oncogenes were not identified. By unsupervised comparison of global transcriptome profiles in squamoid eccrine ductal carcinoma (n = 7) to SCC (n = 10), porocarcinoma (n = 4), and microcystic adnexal carcinoma (n = 4), squamoid eccrine ductal carcinomas displayed an intermediate phenotype between SCC and sweat gland tumors. Squamoid eccrine ductal carcinoma displayed significantly higher expression of 364 genes (including certain eccrine markers) and significantly lower expression of 525 genes compared with other groups. Our findings support the classification of squamoid eccrine ductal carcinoma as a carcinoma with intermediate features between SCC and sweat gland carcinoma.

Undifferentiated and Dedifferentiated Metastatic Melanomas Masquerading as Soft Tissue Sarcomas: Mutational Signature Analysis and Immunotherapy Response

The distinction between undifferentiated melanoma (UM) or dedifferentiated melanoma (DM) from undifferentiated or unclassifiable sarcoma can be difficult and requires the careful correlation of clinical, pathologic, and genomic findings. In this study, we examined the utility of mutational signatures to identify patients with UM/DM with particular attention as to whether this distinction matters for treatment because the survival of patients with metastatic melanoma has dramatically improved with immunologic therapy, whereas durable responses are less frequent in sarcomas. We identified 19 cases of UM/DM that were initially reported as unclassified or undifferentiated malignant neoplasm or sarcoma and submitted for targeted next-generation sequencing analysis. These cases were confirmed as UM/DM by harboring melanoma driver mutations, UV signature, and high tumor mutation burden. One case of DM showed melanoma in situ. Meanwhile, 18 cases represented metastatic UM/DM. Eleven patients had a prior history of melanoma. Thirteen of 19 (68%) of the tumors were immunohistochemically completely negative for 4 melanocytic markers (S100, SOX10, HMB45, and MELAN-A). All cases harbored a dominant UV signature. Frequent driver mutations involved BRAF (26%), NRAS (32%), and NF1 (42%). In contrast, the control cohort of undifferentiated pleomorphic sarcomas (UPS) of deep soft tissue exhibited a dominant aging signature in 46.6% (7/15) without evidence of UV signature. The median tumor mutation burden for DM/UM vs UPS was 31.5 vs 7.0 mutations/Mb (P < .001). A favorable response to immune checkpoint inhibitor therapy was observed in 66.6% (12/18) of patients with UM/DM. Eight patients exhibited a complete response and were alive with no evidence of disease at the last follow-up (median 45.5 months). Our findings support the usefulness of the UV signature in discriminating DM/UM vs UPS. Furthermore, we present evidence suggesting that patients with DM/UM and UV signatures can benefit from immune checkpoint inhibitor therapy.

The Role of Acetyl Zingerone and Its Derivatives in Inhibiting UV-Induced, Incident, and Delayed Cyclobutane Pyrimidine Dimers.

Cyclobutane pyrimidine dimers (CPDs) are ultraviolet radiation (UV)-induced carcinogenic DNA photoproducts that lead to UV signature mutations in melanoma. Previously, we discovered that, in addition to their incident formation (iCPDs), UV exposure induces melanin chemiexcitation (MeCh), where UV generates peroxynitrite (ONOO-), which oxidizes melanin into melanin-carbonyls (MCs) in their excited triplet state. Chronic MeCh and energy transfer by MCs to DNA generates CPDs for several hours after UV exposure ends (dark CPD, dCPDs). We hypothesized that MeCh and the resulting dCPDs can be inhibited using MeCh inhibitors, and MC and ONOO- scavengers. Here, we investigated the efficacy of Acetyl Zingerone (AZ), a plant-based phenolic alkanone, and its chemical analogs in inhibiting iCPDs and dCPDs in skin fibroblasts, keratinocytes, and isogenic pigmented and albino melanocytes. While AZ and its methoxy analog, 3-(4-Methoxy-benzyl)-Pentane-2,4-dione (MBPD) completely inhibited the dCPDs, MBPD also inhibited ~50% of iCPDs. This suggests the inhibition of ~80% of total CPDs at any time point post UV exposure by MBPD, which is markedly significant. MBPD downregulated melanin synthesis, which is indispensable for dCPD generation, but this did not occur with AZ. Meanwhile, AZ and MBPD both upregulated the expression of nucleotide excision repair (NER) pathways genes including Xpa, Xpc, and Mitf. AZ and its analogs were non-toxic to the skin cells and did not act as photosensitizers. We propose that AZ and MBPD represent "next-generation skin care additives" that are safe and effective for use not only in sunscreens but also in other specialized clinical applications owing to their extremely high efficacy in blocking both iCPDs and dCPDs.

Genomic landscape of multiple Bowen's disease using whole-exome sequencing.

The genomic landscape of Bowen's disease (BD), with multiple manifestations, has not yet been determined. This study aimed to investigate the genomic alterations in multiple BD. We performed whole-exome sequencing of BD lesions (n=9) and matched germlines collected from three patients with multiple (≥3) BD to detect somatic and germline mutations. We found a median of 64 somatic mutations in each sample (range 20-267). UV-signature mutations accounted for 64.9% (median, range 26.0%-82.1%) of point mutations. Putative driver mutations were found in five BDs (RB1 p.R445*, ARID2 p.R274*, TP53 p.Y163D/p.Y205D/p.R342*, KMT2C p.R4549C) but not in the other four lesions. Somatic mutations were not shared between multiple BD lesions collected from the same patient, indicating a different clonal origin. We also found no known pathogenic germline mutations in cancer-related genes. The mutational signature analysis revealed that UV signatures (SBS7a/7b) and age-related signatures (SBS1/5) were the main active signatures. Copy number alterations (CNAs) were found in two BDs: one with extensive CNA regions (21.7% of the genome), including driver genes (PIK3CA/SOX2/TP63 and MYC gain, and CDKN2A loss), and the other with 1q gain. Our study revealed that multiple BD lesions harbor distinct genomic landscapes, suggesting that they have different risks of malignant progression.

The Role of Circular RNAs in Keratinocyte Carcinomas.

Simple SummaryKeratinocyte carcinomas include BCC and cSCC and represent the most frequent cancer among fair-skinned people. Tumor development is associated with mutations and dysregulation of many genes involved in biological processes such as cell proliferation, differentiation and apoptosis. The expression of these genes is controlled in many ways, including transcriptional and post-transcriptional control by circular RNAs. In recent studies, a number of circular RNAs have been identified that are dysregulated in BCC and cSCC. Biological functions relevant to tumor development were shown for some of these circRNAs, which may represent biomarkers for disease progression and targets for novel treatment approaches.Keratinocyte carcinomas (KC) include basal cell carcinomas (BCC) and cutaneous squamous cell carcinomas (cSCC) and represents the most common cancer in Europe and North America. Both entities are characterized by a very high mutational burden, mainly UV signature mutations. Predominately mutated genes in BCC belong to the sonic hedgehog pathway, whereas, in cSCC, TP53, CDKN2A, NOTCH1/2 and others are most frequently mutated. In addition, the dysregulation of factors associated with epithelial to mesenchymal transition (EMT) was shown in invasive cSCC. The expression of factors associated with tumorigenesis can be controlled in several ways and include non-coding RNA molecules, such as micro RNAs (miRNA) long noncoding RNAs (lncRNA) and circular RNAs (circRNA). To update findings on circRNA in KC, we reviewed 13 papers published since 2016, identified in a PubMed search. In both BCC and cSCC, numerous circRNAs were identified that were differently expressed compared to healthy skin. Some of them were shown to target miRNAs that are also dysregulated in KC. Moreover, some studies confirmed the biological functions of individual circRNAs involved in cancer development. Thus, circRNAs may be used as biomarkers of disease and disease progression and represent potential targets of new therapeutic approaches for KC.

073 A non-invasive genomic test for early assessment of UV damage in human skin

UV-exposure leads to mutations in skin cells, which may clonally expand and ultimately turn into cancerous lesions ('field cancerization'). Early detection of low-frequency cancer-causing UV-signature mutations in still normal-appearing skin remains a challenge. We have developed a non-invasive genomic test capable of quantifiably detecting UV-signature mutations with high sensitivity to change this paradigm. This genomic test combines DermTech's non-invasive skin sample collection technology 'adhesive skin collection kits' with an ultrasensitive and multiplexed MALDI-TOF mass spectrometry-based mutation detection platform, 'the MassARRAY' from Agena Bioscience. The test includes over two dozen DNA driver mutations in genes such as TP53, CDKN2A, NOTCH1, and NOTCH2, known to be associated with the development of non-melanoma skin cancers. In a proof-of-concept study, contralaterally collected skin samples from sun-exposed body sites in subjects of varying race, age, and gender were tested. UV-signature mutations were detected in sun-exposed skin, with the numbers of mutations increasing with the subjects' age. In our reference control neonatal keratinocytes, no UV signature mutations were detected. This genomic test demonstrates reliable detection of UV-signature mutations in sun-exposed skin samples collected using DermTech's non-invasive skin sample collection technology and offers the opportunity to non-invasively assess UV-damage of skin that lacks visual elements of UV damage. Additional studies will help understand the correlation of UV-damage detected in normal-appearing skin to future non-melanoma skin cancer risk. We envision this proactive genomic approach to measure UV damage to change how photoaging is assessed and treated.

064 Genome analysis reveals UV signature mutations in sun-exposed skin tumors in tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a tumor syndrome caused by pathogenic variants in TSC1 or TSC2. Individuals with TSC develop skin tumors with second-hit somatic mutations in TSC1 or TSC2 in fibroblast-like tumor cells. These somatic mutations in facial angiofibromas, but not fibrous tumors in non-sun-exposed areas, are commonly UV signature mutations, prompting recommendations to limit UV exposure to reduce the severity of TSC skin tumors. In order to investigate whether UV light induces additional genetic alterations that promote TSC tumor pathogenesis, we performed whole genome sequencing of DNA from patient peripheral blood and fibroblast-like cells grown from TSC skin tumors. Unique dual-indexed PCR-free libraries were sequenced as pools on an Illumina NovaSeq 6000 generating 2x150 read pairs with a Picard mean coverage of ∼38X for blood samples and ∼63X for tumor samples. TSC1 and TSC2 germline mutations were identified in 39 out of 48 patients (5 TSC1, 34 TSC2 including 12 mosaic). TSC1 and TSC2 somatic mutations were found in 27 out of 45 tumor samples (2 TSC1, 25 TSC2), including copy neutral LOH, point mutations, large deletions, and intra- and interchromosomal gene fusions. The mutation rate per Mb was higher in sun-exposed (median 5.7/Mb, IQR 4.1-11.8) than non-sun-exposed locations (median 1.7/Mb, IQR 1.5-2.0). C>T mutations and a UV signature were more frequent throughout the genome in sun-exposed than other sites, but there were no recurrent mutations in known driver genes. In summary, TSC skin tumors have relatively low somatic mutation rates that are increased in sun-exposed sites; however, somatic alteration of TSC1 or TSC2, whether random or UV-induced, combined with a TSC1/TSC2 germline mutation, appears to be sufficient to drive tumorigenesis.

Light and Skin.

Sunlight comprises radiation of different wavelengths, of which UVA and UVB are most important with respect to human skin diseases. Next to erythema, edema, and sunburns, UV radiation causes skin cancer. UV radiation of any wavelength is now considered as a class I carcinogen to humans. The mutagenic effects of UV radiation depend on DNA damage following direct absorption by nuclear DNA, resulting in cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts that, if not repaired by nucleotide excision repair pathway, result in characteristic UV signature mutations (C→T or CC→TT transition). In addition, increased formation of reactive oxygen species by UV exposure may cause formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine leading to T→G transversion. In addition, UV radiation has been shown to induce a number of immune modulations that largely result in local and potentially also in systemic immunosuppression, which may not only impair control of dysplastic and neoplastic skin lesions but also affect immuno-pathological and infectious skin diseases. Recent find-ings have shown that ambient doses of high-energy visible light, beyond the UV range, may also cause damage to human skin.

Invasive squamous cell carcinomas and precursor lesions on UV-exposed epithelia demonstrate concordant genomic complexity in driver genes

Although squamous cell carcinomas (SCC) are the most frequent human solid tumor at many anatomic sites, the driving molecular alterations underlying their progression from precursor lesions are poorly understood, especially in the context of photodamage. Therefore, we used high-depth, targeted next-generation sequencing (NGS) of RNA and DNA from routine tissue samples to characterize the progression of both well- (cutaneous) and poorly (ocular) studied SCCs. We assessed 56 formalin-fixed paraffin-embedded (FFPE) cutaneous lesions (n = 8 actinic keratosis, n = 30 carcinoma in situ [CIS], n = 18 invasive) and 43 FFPE ocular surface lesions (n = 2 conjunctival/corneal intraepithelial neoplasia, n = 20 CIS, n = 21 invasive), from institutions in the US and Brazil. An additional seven cases of advanced cutaneous SCC were profiled by hybrid capture-based NGS of >1500 genes. The cutaneous and ocular squamous neoplasms displayed a predominance of UV-signature mutations. Precursor lesions had highly similar somatic genomic landscapes to SCCs, including chromosomal gains of 3q involving SOX2, and highly recurrent mutations and/or loss of heterozygosity events affecting tumor suppressors TP53 and CDKN2A. Additionally, we identify a novel molecular subclass of CIS with RB1 mutations. Among TP53 wild-type tumors, human papillomavirus transcript was detected in one matched pair of cutaneous CIS and SCC. Amplicon-based whole-transcriptome sequencing of select 20 cutaneous lesions demonstrated significant upregulation of pro-invasion genes in cutaneous SCCs relative to precursors, including MMP1, MMP3, MMP9, LAMC2, LGALS1, and TNFRSF12A. Together, ocular and cutaneous squamous neoplasms demonstrate similar alterations, supporting a common model for neoplasia in UV-exposed epithelia. Treatment modalities useful for cutaneous SCC may also be effective in ocular SCC given the genetic similarity between these tumor types. Importantly, in both systems, precursor lesions possess the full complement of major genetic changes seen in SCC, supporting non-genetic drivers of invasiveness.

Whole-Exome and Transcriptome Analysis of UV-Exposed Epidermis and Carcinoma In Situ Reveals Early Drivers of Carcinogenesis

Squamous cell carcinoma in situ (SCCIS) is a prevalent precancerous lesion that can progress to cutaneous squamous cell carcinoma. Although SCCIS is common, its pathogenesis remains poorly understood. To better understand SCCIS development, we performed laser captured microdissection of human SCCIS and the adjacent epidermis to isolate genomic DNA and RNA for next-generation sequencing. Whole-exome sequencing identified UV-signature mutations in multiple genes, including NOTCH1-3 in the epidermis and SCCIS and oncogenic TP53 mutations in SCCIS. Gene families, including SLFN genes, contained UV/oxidative-signature disruptive epidermal mutations that manifested positive selection in SCCIS. The frequency and distribution of NOTCH and TP53 mutations indicate that NOTCH mutations may precede TP53 mutations. RNA sequencing identified 1,166 differentially expressed genes; the top five enriched gene ontology biological processes included (i) immune response, (ii) epidermal development, (iii) protein phosphorylation, (iv) regulation of catalytic activity, and (v) cytoskeletal regulation. The NEURL1 ubiquitin ligase, which targets Notch ligands for degradation, was upregulated in SCCIS. NEURL1 protein was found to be elevated in SCCIS suggesting that increased levels could represent a mechanism for downregulating Notch during UV-induced carcinogenesis. The data from DNA and RNA sequencing of epidermis and SCCIS provide insights regarding SCCIS formation.

Abstract PR01: Genomic UV-hypersensitive sites as sentinels for personal UV exposure

Abstract The largest risk factor for skin cancers such as melanoma is past sun exposure, so an objective measurement of traces of an individual’s sun history would allow a general practitioner to identify people who should be monitored for early cancer detection. If the genome contains outlier DNA sequences hypersensitive to environmental agents such as ultraviolet light (UV), these would be genomic dosimeters for monitoring personal carcinogen exposure and would facilitate noninvasive measurements on small skin samples. Such DNA sites might also allow UV to drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, tagged rare UV-induced cyclobutane pyrimidine dimers (CPDs) and provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequent than UV-induced CPDs. UV exposure revealed hyperhotspots acquiring CPDs up to 200-fold more frequently than the genomic average; these were 20-fold more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within two motifs. One motif occurred at ETS1 transcription factor binding sites, known to be UV targets, and at sites of mTOR/TOP-tract translation regulation; the second occurred at a sequence that developed delayed CPDs after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ~20 cell pathways targeted, rendering CPD hyperhotspots epigenetic marks. This abstract is also being presented as Poster A28. Citation Format: Sanjay Premi, Lynn Han, Sameet Mehta, James Knight, Dejian Zhao, Antonella Bacchiocchi, Ruth Halaban, Meg A. Palmatier, Karl Kornacker, Douglas E. Brash. Genomic UV-hypersensitive sites as sentinels for personal UV exposure [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr PR01.

Evidence That STK19 Is Not an NRAS-dependent Melanoma Driver

SummarySTK19 was proposed to be a cancer driver, and recent work by Yin et al. (2019) in Cell suggested that the frequently recurring STK19 D89N substitution represents a gain-of-function change, allowing increased phosphorylation of NRAS to enhance melanocyte transformation. Here we show that the STK19 gene has been incorrectly annotated, and that the expressed protein is 110 amino acids shorter than indicated by current databases. The “cancer driving” STK19 D89N substitution is thus outside the coding region. We also fail to detect evidence of the mutation affecting STK19 expression; instead, it is a UV signature mutation, found in the promoter of other genes as well. Furthermore, STK19 is exclusively nuclear and chromatin-associated, while no evidence for it being a kinase was found. The data in this Matters Arising article raise fundamental questions about the recently proposed role for STK19 in melanoma progression via a function as an NRAS kinase, suggested by Yin et al. (2019) in Cell. See also the response by Yin et al. (2020), published in this issue.

Multiple interaction nodes define the postreplication repair response to UV-induced DNA damage that is defective in melanomas and correlated with UV signature mutation load.

Ultraviolet radiation‐induced DNA mutations are a primary environmental driver of melanoma. The reason for this very high level of unrepaired DNA lesions leading to these mutations is still poorly understood. The primary DNA repair mechanism for UV‐induced lesions, that is, the nucleotide excision repair pathway, appears intact in most melanomas. We have previously reported a postreplication repair mechanism that is commonly defective in melanoma cell lines. Here we have used a genome‐wide approach to identify the components of this postreplication repair mechanism. We have used differential transcript polysome loading to identify transcripts that are associated with UV response, and then functionally assessed these to identify novel components of this repair and cell cycle checkpoint network. We have identified multiple interaction nodes, including global genomic nucleotide excision repair and homologous recombination repair, and previously unexpected MASTL pathway, as components of the response. Finally, we have used bioinformatics to assess the contribution of dysregulated expression of these pathways to the UV signature mutation load of a large melanoma cohort. We show that dysregulation of the pathway, especially the DNA damage repair components, are significant contributors to UV mutation load, and that dysregulation of the MASTL pathway appears to be a significant contributor to high UV signature mutation load.

519 Paranuclear cytokeratin aggregates help Merkel cell carcinoma evade extrinsic apoptosis

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. Most MCC tumors are virus positive and express Merkel cell polyomavirus oncogenes, whereas virus negative MCC are associated with abundant UV signature mutations. The majority of MCC tumors express CK20. Staining for CK20 and other intermediate filaments in a paranuclear dot pattern is a histological feature used to diagnose MCC. The functional significances of these paranuclear protein aggregates is unknown. We immunostained native MCC tumors with a pan-CK antibody and found areas of paranuclear dot staining in 94% of tumors. Immunofluorescent microscopy (IF) of MCC cell lines showed that paranuclear dot proteins often extend into a lattice-like structure surrounding the nucleus. Electron microscopy revealed that the intermediate filaments in the paranuclear dot did not resided within any cellular organelles but formed a cytoplasmic inclusion surrounding the centrosome. Using IF we confirmed that in 100% of interphase cells the paranuclear dot colocalized with the centrosome. In contrast, mitotic MCC cells showed partial disaggregation of the paranuclear dot and stochastic segregation of dot proteins into daughter cells, suggesting the paranuclear dot is a dynamic structure that reforms with each cell division. Disruption of microtubules with nocodazole resulted in dot disaggregation, demonstrating that microtubules are essential for dot maintenance. In contrast, inhibition of ubiquitin ligation enhanced cytokeratin aggregation. Interestingly, IF staining showed colocalization of the paranuclear dot with Fas Associate Death Domain (FADD). Consistent with functional sequestration of FADD in the paranuclear dot, TNFα and Fas ligand failed to induce apoptosis in dot-positive MCC cell lines. Taken together, the paranuclear dot may allow MCC tumors to evade extrinsic apoptosis by sequestering FADD and disrupting death receptor signaling.

UV Signature Mutations Reclassify Salivary High-grade Neuroendocrine Carcinomas as Occult Metastatic Cutaneous Merkel Cell Carcinomas.

Salivary high-grade neuroendocrine carcinomas (NECs) are rare, occur predominantly in the parotid gland, and are difficult to differentiate from metastatic cutaneous Merkel cell carcinomas (MCCs), which have overlapping morphologic, immunophenotypic, and molecular profiles. Oncogenic Merkel cell polyomavirus (MCPyV), found in 70% to 80% of MCCs, has also been reported in a few salivary NECs, but this is controversial. A promising biomarker to distinguish the 2 tumor types are UV signature mutations. UV signature mutations indicate a sun damage-induced mechanism of pathogenesis and recently have been found to be highly prevalent in MCPyV-negative MCCs but would be inconsistent with salivary origin. Here, we examine UV signature mutations as a molecular marker to distinguish primary salivary high-grade NEC from MCC. Whole exome DNA sequencing was performed on matched tumor-normal tissue from 4 MCPyV-negative high-grade salivary NECs with no other primary source identified, as well as 3 melanomas and 3 lung NECs as positive and negative controls, respectively. UV signature mutations were found in all salivary NECs, when defined as ≥60% of total mutations being C-to-T transitions at dipyrimidine sites, and when compared with known human cancer-related mutational signatures. The presence of UV signature mutations in salivary high-grade NECs strongly favors these to be occult metastatic MCCs. True salivary primary NECs are likely exceedingly rare. When a high-grade NEC is encountered in the salivary gland, the presence of UV signature mutations or MCPyV may be useful to exclude occult unknown primary MCC.

130 Novel method for non-invasive assessment of molecular markers of UV exposure in normal skin

The use of markers of UV-induced DNA damage as a dosimeter of exposure and predictor of skin cancer risk has been proposed by multiple groups. While UV-induced mutations and adducts are well described and present in normal appearing UV-exposed epidermis, sampling normal non-lesional skin requires non-invasive methods to extract epidermal DNA for analysis. Here we demonstrate the feasibility of such an approach. Sampling was performed using a felt-tip pen soaked in a mixture of surfactants (Brij-30/DPS). In mice, we show that the epidermis can be selectively removed without scarring, with complete healing within 2 weeks. We exposed Hairless mice to low-dose solar simulated light (Newport) over a period of 3 months for a total exposure of 175 kJ/m2, a dose which normally produces tumors within 3 months. Over 2 micrograms / cm2 of DNA could be purified and exome sequenced. We noted a progressive increase in a UV-signature mutational burden over the following 2 months following the cessation of UV exposure, consistent with accumulation of mutant clones. Next we sought to assess whether this was practical to perform in human patients. Skin samples were collected from neck or shoulder (sun-exposed areas with dermatoheliosis) and lateral chest or back (minimally sun-exposed areas) with no prior preparation. Approximately 300 ng of total DNA could be isolated from an approximately 1 cm2 area of sampled skin. These samples were subjected to targeted exome sequencing using a commercially available panel (QIAseq Cancer Panel) to a depth of approximately 3500X and referenced to saliva. We identified considerable heterogeneity in multiple samples obtained from the same patient, suggesting widespread mosaicism, but also an enrichment for UV-signature mutations in key tumor suppressors including TP53 and NOTCH1/2. Our next steps are to use hypermutated areas of the genome to further refine this dosimetry method and assess its dynamic range across a larger spectrum of patients.

Mechanistic considerations on the wavelength-dependent variations of UVR genotoxicity and mutagenesis in skin: the discrimination of UVA-signature from UV-signature mutation

Ultraviolet radiation (UVR) predominantly induces UV-signature mutations, C → T and CC → TT base substitutions at dipyrimidine sites, in the cellular and skin genome. I observed in our in vivo mutation studies of mouse skin that these UVR-specific mutations show a wavelength-dependent variation in their sequence-context preference. The C → T mutation occurs most frequently in the 5'-TCG-3' sequence regardless of the UVR wavelength, but is recovered more preferentially there as the wavelength increases, resulting in prominent occurrences exclusively in the TCG sequence in the UVA wavelength range, which I will designate as a "UVA signature" in this review. The preference of the UVB-induced C → T mutation for the sequence contexts shows a mixed pattern of UVC- and UVA-induced mutations, and a similar pattern is also observed for natural sunlight, in which UVB is the most genotoxic component. In addition, the CC → TT mutation hardly occurs at UVA1 wavelengths, although it is detected rarely but constantly in the UVC and UVB ranges. This wavelength-dependent variation in the sequence-context preference of the UVR-specific mutations could be explained by two different photochemical mechanisms of cyclobutane pyrimidine dimer (CPD) formation. The UV-signature mutations observed in the UVC and UVB ranges are known to be caused mainly by CPDs produced through the conventional singlet/triplet excitation of pyrimidine bases after the direct absorption of the UVC/UVB photon energy in those bases. On the other hand, a novel photochemical mechanism through the direct absorption of the UVR energy to double-stranded DNA, which is called "collective excitation", has been proposed for the UVA-induced CPD formation. The UVA photons directly absorbed by DNA produce CPDs with a sequence context preference different from that observed for CPDs caused by the UVC/UVB-mediated singlet/triplet excitation, causing CPD formation preferentially at thymine-containing dipyrimidine sites and probably also preferably at methyl CpG-associated dipyrimidine sites, which include the TCG sequence. In this review, I present a mechanistic consideration on the wavelength-dependent variation of the sequence context preference of the UVR-specific mutations and rationalize the proposition of the UVA-signature mutation, in addition to the UV-signature mutation.