title: Next-generation sequencing of the basal cell carcinoma miRNome and a description of novel microRNA candidates under neoadjuvant vismodegib therapy: an integrative molecular and surgical case study.
abstract: BACKGROUND
  MicroRNAs (miRNAs) have been identified as key players in posttranscriptional gene regulation and have a significant impact on basal cell carcinoma (BCC) development. The Sonic hedgehog pathway inhibitor vismodegib has been approved for oral therapy of metastatic or advanced BCC. Here, a high-throughput miRNA sequencing analysis was carried out to identify differentially expressed miRNAs and possible novel miRNA candidates in vismodegib-treated BCC tissue. Additionally, we described our surgical experience with neoadjuvant oral vismodegib therapy.
  
  
  METHODS
  A punch biopsy (4 mm) from a patient with an extensive cranial BCC under oral vismodegib therapy and a corresponding nonlesional epithelial skin biopsy were harvested. Total RNA was isolated, after which a sequencing cDNA library was prepared, and cluster generation was carried out, which was followed by an ultra-high-throughput miRNA sequencing analysis to indicate the read number of miRNA expression based on miRBase 21. In addition to the identification of differentially expressed miRNAs from RNA sequencing data, additional novel miRNA candidates were determined with a tool for identifying new miRNA sequences (miRDeep2).
  
  
  RESULTS
  We identified 33 up-regulated miRNAs (fold change ≥2) and 39 potentially new miRNA candidates (miRDeep scores 0-43.6). A manual sequence analysis of the miRNA candidates on the genomic locus of chromosome 1 with provisional IDs of chr1_1913 and chr1_421 was further carried out and rated as promising (chr1_1913) and borderline (chr1_421). Histopathology revealed skip lesions in clinically healthy appearing skin at the tumor margins, which were the cause of seven re-excisions by micrographic controlled surgery to achieve tumor-free margins.
  
  
  CONCLUSIONS
  miRNA sequencing revealed novel miRNA candidates that need to be further confirmed in functional Dicer knockout studies. Clinically, on the basis of our surgical experience described here, neoadjuvant vismodegib therapy in BCC appears to impede histopathologic evaluations with effects on surgical therapy. Thus, larger studies are necessary, but are not preferable at this time if other options are available.
authors.affiliation: Dermatologic Surgery Unit, Department of Dermatology, Venereology and Allergology, Ruhr-University Bochum, Bochum Department of Plastic Surgery, St Josef Hospital, Catholic Clinics of the Ruhr Peninsula, Essen, Germany michael.sand@ruhr-uni-bochum.de.
authors.forename: M
authors.initials: M
authors.lastname: Sand
authors.affiliation: Dermatologic Surgery Unit, Department of Dermatology, Venereology and Allergology, Ruhr-University Bochum, Bochum.
authors.forename: F G
authors.initials: FG
authors.lastname: Bechara
authors.affiliation: Dermatologic Surgery Unit, Department of Dermatology, Venereology and Allergology, Ruhr-University Bochum, Bochum.
authors.forename: T
authors.initials: T
authors.lastname: Gambichler
authors.affiliation: University of Michigan, Kellogg Eye Center, Ann Arbor, USA.
authors.forename: D
authors.initials: D
authors.lastname: Sand
authors.affiliation: Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
authors.forename: M R
authors.initials: MR
authors.lastname: Friedländer
authors.affiliation: Department of Plastic Surgery, St Josef Hospital, Catholic Clinics of the Ruhr Peninsula, Essen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Bromba
authors.affiliation: Institut für Pathologie, Essen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Schnabel
authors.affiliation: Dermatologic Surgery Unit, Department of Dermatology, Venereology and Allergology, Ruhr-University Bochum, Bochum.
authors.forename: S
authors.initials: S
authors.lastname: Hessam
mesh_terms: D000368:Aged; D000813:Anilides / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001483:Base Sequence; D001706:Biopsy; D002280:Basal Cell Carcinoma / Q000188:drug therapy / Q000235:genetics* / Q000601:surgery; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D011725:Pyridines / Q000627:therapeutic use; D017423:Sequence Analysis, RNA; D012867:Skin / Q000473:pathology*; D012878:Skin Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000601:surgery
chemical_list: D000813:Anilides; D000970:Antineoplastic Agents; D053823:Hedgehog Proteins; C538724:HhAntag691; D035683:MicroRNAs; D011725:Pyridines; C509239:SHH protein, human
keywords: basal cell carcinoma; miRNAs; next-generation sequencing; noncoding RNAs