BACKGROUND Exome and targeted sequencing studies have identified potential driver mutations for a variety of tumor types. Cutaneous squamous cell carcinoma (cSCC) is one of the most highly mutated cancers but is typically associated with low rates of metastasis and high survival rates. Nevertheless, metastatic cSCC is a significant health threat; up to 8800 individuals die yearly from this disease. METHODS As it is difficult to predict which cSCCs are more likely to metastasize, and because there are no targeted therapies specifically designated for metastatic cSCC, we performed exome and/or targeted sequencing of 18 metastatic and 10 primary cSCCs to identify mutations that were more frequent in metastatic tumors and might be targeted for therapeutic benefit. We compared our results to published sequencing results of an additional 223 primary tumors and 68 metastatic cSCCs. RESULTS We identified genes showing higher mutation frequencies in metastatic cSCC relative to primary tumors including the chromatin remodeling gene KMT2D and the classic skin tumor suppressor TP53 which was mutated in 54% of primary tumors relative to 85% of metastatic tumors (p <0.0001). CONCLUSIONS These studies uncover potential pathways important in metastatic cSCC that broaden our understanding of the biology contributing to aggressive tumor behavior and may lead to new therapeutic strategies.

Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous due to detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. 23 of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.

Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1, genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in BRIP1. This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population (P < 0.0001, x 2 test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60-3.30; P < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants. Significance: Functional characterization of rare variants of uncertain significance in BRIP1 revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in BRIP1 confer risk for both breast and ovarian cancer.

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Mus spretus mice are highly resistant to several types of cancer compared to Mus musculus mice. To determine whether differences in microRNA (miRNA) expression account for some of the differences in observed skin cancer susceptibility between the strains, we performed miRNA expression profiling of skin RNA for over 300 miRNAs. Five miRNAs, miR-1, miR-124a-3, miR-133a, miR-134, miR-206, were differentially expressed by array and/or qPCR. miR-1 was previously shown to have tumor suppressing abilities in multiple tumor types. We found miR-1 expression to be lower in mouse cutaneous squamous cell carcinomas (cSCCs) compared to normal skin. Based on the literature and our expression data, we performed detailed studies on predicted miR-1 targets and evaluated the effect of miR-1 expression on two murine cSCC cell lines, A5 and B9. Following transfection of miR-1, we found decreased mRNA expression of three validated miR-1 targets, Met, Twf1 and Ets1 and one novel target Bag4. Decreased expression of Ets1 was confirmed by Western analysis and by 3’ reporter luciferase assays containing wildtype and mutated Ets1 3’UTR. We evaluated the effect of miR-1 on multiple tumor phenotypes including apoptosis, proliferation, cell cycle and migration. In A5 cells, expression of miR-1 led to decreased proliferation compared to a control miR. miR-1 expression also led to increased apoptosis at later time points (72 and 96 h) and to a decrease in cells in S-phase. In summary, we identified five miRNAs with differential expression between cancer resistant and cancer susceptible mice and found that miR-1, a candidate tumor suppressor, has targets with defined roles in tumorigenesis.

Background Variants at the Oculocutaneous albinism 2 (OCA2)/HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase 2 (HERC2) locus have been associated with pigmentation phenotypes as well as risk of developing multiple types of skin cancer. Objectives The goal of this study was to evaluate OCA2/HERC2 locus variants for impact on time to develop cutaneous squamous cell carcinoma (cSCC) in organ transplant recipients (OTRs) who are at elevated risk of developing cSCC. Methods Participants were solid organ transplant recipients ascertained from two centers (n=125 and 261) with an average of 13.1 years follow-up post-transplant. DNA was available for genotyping for all participants in addition to medical records and questionnaire data. The Ohio State University (OSU) study design was a case-control with prospective follow-up, and the University of California San Francisco (UCSF) study design was a national cross-sectional survey with retrospective chart review. Results OCA2 variants rs12913832 and rs916977 were significantly associated with time to first cSCC post-transplant. OTRs homozygous for the brown eye alleles of rs916977 (GG) and rs12913832 (AA) had significant delays of time to first cSCC post-transplant compared to individuals homozygous for the blue eye alleles [HR=0.34, p<0.001and HR=0.54, p=0.012, respectively]. Both variants were highly associated with eye color in combined studies (p<0.001). Conclusions This study is the first to show an association between OCA2/HERC2 variants and time to first cSCC post-transplant which may impact dermatologic screening recommendations for high-risk populations.

Desmoid fibromatosis is a locally aggressive clonal fibroblastic proliferation with high recurrence rates and no metastatic potential. Implicated molecular aberrations occur within the Wnt/β-catenin pathway (APC and β-catenin gene mutations). Transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) are profibrotic growth factors, downstream from nuclear translocation of β-catenin, that lead to increased fibrogenesis. CTGF (a downstream effector of TGF-β) is a matricellular protein that modulates the activity of growth factors, adhesion molecules, integrins, and extracellular matrix thus playing a central role in tissue remodeling and fibrosis. Recently there has been growing interest in use of extracellular matrix inhibitors for treatment of various fibrogenic diseases. Desmoid fibromatosis samples (n=15) were evaluated for expression of β-catenin, TGF-β, and CTGF using immunohistochemistry on formalin paraffin-embedded material. A control group comprising scar tissue and adjacent normal skin (n=10) were simultaneously immunostained with above mentioned markers. Real-time polymerase chain reaction was performed on frozen specimens of desmoid fibromatosis (n=6) and normal skin (n=2). All 15 desmoid tumors were positive for β-catenin (surrogate marker of Wnt/β-catenin pathway dysregulation) which was negative in control normal skin and scar samples. TGF-β and CTGF were negative in 9 of 10 normal skin controls. TGF-β and CTGF were positive in all cases of scar tissue. All 15 cases of desmoid tumors were positive for TGF-β and CTGF. The real-time polymerase chain reaction showed higher expression levels of TGF-β and CTGF in desmoid fibromatosis compared with normal skin. The high constitutive expression of β-catenin downstream effectors; TGF-β, CTGF has the potential for enabling targeted therapy.

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