title: Rapid molecular detection of metastatic head and neck squamous cell carcinoma as an intraoperative adjunct to sentinel lymph node biopsy.
abstract: OBJECTIVE
  Clinical staging of early head and neck squamous cell carcinoma (SCCHN) is often inaccurate, leading to elective neck dissection to detect the 30% of patients with micrometastatic disease. Sentinel node biopsy accurately stages the regional lymphatics, but intraoperative pathology is only moderately sensitive, and final pathology takes several days to complete. To facilitate immediate neck dissection where necessary, we have identified several promising marker genes of SCCHN metastasis and developed a rapid, accurate, and automated quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) assay for intraoperative use.
  
  
  METHODS
  Prospective tissue collection, retrospective pathologic correlation with qRT-PCR.
  
  
  METHODS
  From a 40-gene marker screen, we quantified expression of 11 potential tumor genes using a test set of primary tumors (n = 32) and metastatic (n = 19) and benign (n = 10) lymph nodes. Eight patients' paired primary tumor and metastatic nodes were included. A validation set of 442 grossly tumor-negative nodes was evaluated for expression of the most promising markers, comparing metastasis detection by qRT-PCR with pathologic analysis (hematoxylin and eosin and immunohistochemistry). A novel multiplexed, automated, single-tube qRT-PCR assay was used to analyze more than 100 lymph nodes using a two-marker, 35-minute assay to determine its negative predictive value.
  
  
  RESULTS
  Based on expression of 11 tumor-associated genes from the marker screen, the two most promising markers of SCCHN metastasis in the test set, pemphigus vulgaris antigen (PVA) and tumor-associated calcium signal transducer 1 (TACSTD1), also known as epithelial cell adhesion molecule (EpCAM), were selected. Development of a multiplexed qRT-PCR assay for the detection of metastasis compared favorably with pathologic analysis in the additional 442-node set. A rapid, multiplexed assay using PVA and TACSTD1 demonstrated excellent reproducibility, linearity, and accuracy (≈96% negative predictive value) for identifying positive (n = 40) and negative (n = 62) nodes in a validation subset.
  
  
  CONCLUSIONS
  Detection of metastatic SCCHN using multiplexed qRT-PCR can be rapid, accurate, and automated and may enable sentinel node biopsy to be used for intraoperative decision-making. PCR amplification of tumor marker genes is an effective method of intraoperative molecular staging of SCCHN and could more appropriately guide application of neck dissection in pN+ SCCHN patients, sparing 60% to 70% of pN0 patients from unnecessary neck dissection. This technique may also be used for identifying residual neck disease posttreatment, using outpatient fine-needle aspiration biopsy specimens.
authors.affiliation: Department of Otolaryngology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. ferrrl@upmc.edu
authors.forename: Robert L
authors.initials: RL
authors.lastname: Ferris
authors.forename: Patrick
authors.initials: P
authors.lastname: Stefanika
authors.forename: Liqiang
authors.initials: L
authors.lastname: Xi
authors.forename: William
authors.initials: W
authors.lastname: Gooding
authors.forename: Raja R
authors.initials: RR
authors.lastname: Seethala
authors.forename: Tony E
authors.initials: TE
authors.lastname: Godfrey
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis* / Q000556:secondary / Q000601:surgery; D005260:Female; D006258:Head and Neck Neoplasms / Q000175:diagnosis* / Q000556:secondary / Q000601:surgery; D006801:Humans; D007432:Intraoperative Period; D008207:Lymphatic Metastasis; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009367:Neoplasm Staging / Q000379:methods*; D011446:Prospective Studies; D012334:RNA, Neoplasm / Q000032:analysis*; D015203:Reproducibility of Results; D021701:Sentinel Lymph Node Biopsy; D000077195:Squamous Cell Carcinoma of Head and Neck; D013997:Time Factors
chemical_list: D014408:Biomarkers, Tumor; D012334:RNA, Neoplasm