title: Identification of inguinofemoral lymph node metastases by methylation markers in vulvar cancer.
abstract: OBJECTIVE
  Lymph node status in early-stage vulvar cancer can be accurately assessed by the sentinel-node (SN) procedure. Molecular techniques, such as DNA-methylation assay, might improve SN assessment. In this study, we selected methylation markers for vulvar cancer and determined if these methylation markers were suitable for lymph node assessment.
  
  
  METHODS
  We performed methylation specific PCR on DNA isolated from primary tumors, metastatic lymph nodes, and negative lymph nodes from twenty vulvar cancer patients using the following genes: P16INK4a, MGMT, TWIST1, CADM1, TERT, and TFPI2. For P16INK4a and MGMT immunohistochemistry was performed on primary tumors and metastatic lymph nodes in order to explore intratumor heterogeneity in gene expression patterns.
  
  
  RESULTS
  TERT was methylated in all vulvar cancers, P16INK4a in 13/20, TFPI2 in 12/20, CADM1 in 11/20, MGMT in 9/20, and TWIST1 in 7/20. A panel of three methylation markers (P16INK4a, TERT and TFPI2) reached a sensitivity of 67% and specificity of 100% for detection of metastatic lymph nodes. Immunohistochemistry showed intratumor heterogeneity for expression of P16INK4a and MGMT in respectively 55% and 45% of primary tumors.
  
  
  CONCLUSIONS
  Our study shows methylation for one or more methylation markers in all vulvar cancers. Despite a specificity of 100% our panel of three methylation markers had only moderate sensitivity for metastatic lymph node detection, thereby limiting its applicability for lymph node assessment. Intratumor heterogeneity for expression of P16INK4a and MGMT may reflect intratumor heterogeneity for methylation patterns and thereby in general explain the moderate sensitivity of our marker panel for detection of metastases.
authors.affiliation: Department of Obstetrics and Gynecology, University Medical Center Groningen, University of Groningen, PO Box 30 001, 9700RB Groningen, The Netherlands.
authors.forename: M H M
authors.initials: MH
authors.lastname: Oonk
authors.forename: J J H
authors.initials: JJ
authors.lastname: Eijsink
authors.forename: H H
authors.initials: HH
authors.lastname: Volders
authors.forename: H
authors.initials: H
authors.lastname: Hollema
authors.forename: G B A
authors.initials: GB
authors.lastname: Wisman
authors.forename: E
authors.initials: E
authors.lastname: Schuuring
authors.forename: A G J
authors.initials: AG
authors.lastname: van der Zee
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000096:biosynthesis / Q000235:genetics; D045643:DNA Repair Enzymes / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D019942:Genes, p16; D005819:Genetic Markers; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis / Q000235:genetics; D007150:Immunohistochemistry; D007264:Inguinal Canal; D008198:Lymph Nodes / Q000378:metabolism / Q000473:pathology*; D008207:Lymphatic Metastasis; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics; D051879:Twist-Related Protein 1 / Q000096:biosynthesis / Q000235:genetics; D014846:Vulvar Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*
chemical_list: C403514:CADM1 protein, human; C000614131:CDKN2A protein, human; D000075982:Cell Adhesion Molecule-1; D015815:Cell Adhesion Molecules; D019941:Cyclin-Dependent Kinase Inhibitor p16; D005819:Genetic Markers; D006023:Glycoproteins; D007136:Immunoglobulins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D025521:Tumor Suppressor Proteins; D051879:Twist-Related Protein 1; C021240:tissue-factor-pathway inhibitor 2; D015254:DNA Modification Methylases; C515491:MGMT protein, human; C509186:TERT protein, human; D019098:Telomerase; D045643:DNA Repair Enzymes