Intraoperative Molecular Margin Analysis in Head and Neck Cancer | Genetics and Genomics | JAMA Otolaryngology–Head & Neck Surgery | JAMA Network
[Skip to Navigation]
Access to paid content on this site is currently suspended due to excessive activity being detected from your IP address Please contact the publisher to request reinstatement.
Gandour-Edwards  RFDonald  PJWiese  DA Accuracy of intraoperative frozen section diagnosis in head and neck surgery: experience at a university medical center.  Head Neck.1993;15:33-38.PubMedGoogle Scholar
Looser  KGShah  JPStrong  EW The significance of "positive" margins in surgically resected epidermoid carcinomas.  Head Neck Surg.1978;1:107-111.PubMedGoogle Scholar
Batsakis  JG Surgical excision margins: a pathologist's perspective.  Adv Anat Pathol.1999;6:140-148.PubMedGoogle Scholar
Fearon  ERVogelstein  B A genetic model for colorectal tumorigenesis.  Cell.1990;61:759-767.PubMedGoogle Scholar
Brennan  JAMao  LHruban  RH  et al Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck.  N Engl J Med.1995;332:429-435.PubMedGoogle Scholar
Boyle  JOHakim  JKoch  W  et al The incidence of p53 mutations increases with progression of head and neck cancer.  Cancer Res.1993;53:4477-4480.PubMedGoogle Scholar
Maestro  RDolcetti  RGasparotto  D  et al High frequency of p53 gene alterations associated with protein overexpression in human squamous cell carcinoma of the larynx.  Oncogene.1992;7:1159-1166.PubMedGoogle Scholar
Nathan  CAAmirghahri  NRice  CAbreo  FWShi  RStucker  FJ Molecular analysis of surgical margins in head and neck squamous cell carcinoma patients.  Laryngoscope.2002;112:2129-2140.PubMedGoogle Scholar
Nathan  CAFranklin  SAbreo  FWNassar  RDe Benedetti  AGlass  J Analysis of surgical margins with the molecular marker eIF4E: a prognostic factor in patients with head and neck cancer.  J Clin Oncol.1999;17:2909-2914.PubMedGoogle Scholar
Baylin  SBHerman  JGGraff  JRVertino  PMIssa  JP Alterations in DNA methylation: a fundamental aspect of neoplasia.  Adv Cancer Res.1998;72:141-196.PubMedGoogle Scholar
Esteller  M CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future.  Oncogene.2002;21:5427-5440.PubMedGoogle Scholar
Okami  KWu  LRiggins  G  et al Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumors.  Cancer Res.1998;58:509-511.PubMedGoogle Scholar
Gleich  LLSalamone  FN Molecular genetics of head and neck cancer.  Cancer Control.2002;9:369-378.PubMedGoogle Scholar
Merlo  AHerman  JGMao  L  et al 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers.  Nat Med.1995;1:686-692.PubMedGoogle Scholar
Reed  ALCalifano  JCairns  P  et al High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma.  Cancer Res.1996;56:3630-3633.PubMedGoogle Scholar
Papadimitrakopoulou  VIzzo  JLippman  SM  et al Frequent inactivation of p16INK4a in oral premalignant lesions.  Oncogene.1997;14:1799-1803.PubMedGoogle Scholar
Esteller  MHamilton  SRBurger  PCBaylin  SBHerman  JG Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia.  Cancer Res.1999;59:793-797.PubMedGoogle Scholar
Pegg  AEByers  TL Repair of DNA containing O6-alkylguanine.  FASEB J.1992;6:2302-2310.PubMedGoogle Scholar
Gerson  SLAllay  EVitantonio  KDumenco  LL Determinants of O6-alkylguanine-DNA alkyltransferase activity in human colon cancer.  Clin Cancer Res.1995;1:519-525.PubMedGoogle Scholar
Citron  MGraver  MSchoenhaus  M  et al Detection of messenger RNA from O6-methylguanine-DNA methyltransferase gene MGMT in human normal and tumor tissues.  J Natl Cancer Inst.1992;84:337-340.PubMedGoogle Scholar
Costello  JFFutscher  BWTano  KGraunke  DMPieper  RO Graded methylation in the promoter and body of the O6-methylguanine DNA methyltransferase (MGMT) gene correlates with MGMT expression in human glioma cells.  J Biol Chem.1994;269:17228-17237.PubMedGoogle Scholar
Qian  XCBrent  TP Methylation hot spots in the 5' flanking region denote silencing of the O6-methylguanine-DNA methyltransferase gene.  Cancer Res.1997;57:3672-3677.PubMedGoogle Scholar
Watts  GSPieper  ROCostello  JFPeng  YMDalton  WSFutscher  BW Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene.  Mol Cell Biol.1997;17:5612-5619.PubMedGoogle Scholar
Rosas  SLKoch  Wda Costa Carvalho  MG  et al Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients.  Cancer Res.2001;61:939-942.PubMedGoogle Scholar
Sanchez-Cespedes  MEsteller  MWu  L  et al Gene promoter hypermethylation in tumors and serum of head and neck cancer patients.  Cancer Res.2000;60:892-895.PubMedGoogle Scholar
Capone  RBPai  SIKoch  WM  et al Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma.  Clin Cancer Res.2000;6:4171-4175.PubMedGoogle Scholar
Heid  CAStevens  JLivak  KJWilliams  PM Real time quantitative PCR.  Genome Res.1996;6:986-994.PubMedGoogle Scholar
Holland  PMAbramson  RDWatson  RGelfand  DH Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.  Proc Natl Acad Sci U S A.1991;88:7276-7280.PubMedGoogle Scholar
Davidson  TMNahum  AMAstarita  RW Microscopic controlled excisions for epidermoid carcinoma of the head and neck.  Otolaryngol Head Neck Surg.1981;89:244-251.PubMedGoogle Scholar
Gath  HJBrakenhoff  RH Minimal residual disease in head and neck cancer.  Cancer Metastasis Rev.1999;18:109-126.PubMedGoogle Scholar
Partridge  M Oral cancer, 2: clinical presentation and use of new knowledge about the biology of cancer to establish why tumours may recur.  Dent Update.2000;27:288-294.PubMedGoogle Scholar
Not Available Cepheid technology: solving the problem of DNA analysis  Cepheid Web site. Available at: Accessed October 15, 2003.
Yamashita  KUpadhyay  SOsada  M  et al Pharmacologic unmasking of epigenetically silenced tumor suppressor genes in esophageal squamous cell carcinoma.  Cancer Cell.2002;2:485-495.PubMedGoogle Scholar
Original Article
January 2004

Intraoperative Molecular Margin Analysis in Head and Neck Cancer

Author Affiliations

From the Departments of Otolaryngology–Head and Neck Surgery (Drs Goldenberg, Harden, Masayesva, Ha, Koch, Sidransky, and Califano and Ms Benoit) and Pathology(Dr Westra), The Johns Hopkins University School of Medicine, Baltimore, Md. The authors have no relevant financial interest in this article.

Arch Otolaryngol Head Neck Surg. 2004;130(1):39-44. doi:10.1001/archotol.130.1.39

Background  Tumor-specific molecular alterations in surgical margins have been shown to predict risk of local recurrence. However, assays used for these analyses are time-consuming and therefore cannot be used in the intraoperative setting.

Objective  To detect and quantify tumor-specific methylated promoter sequences in surgical margins in a time frame suitable for intraoperative use.

Design  A novel quantitative methylation-specific polymerase chain reaction (QMSP) protocol.

Methods  A total of 13 patients with head and neck squamous cell carcinoma (HNSCC) were initially characterized for molecular alterations in their tumor at the time of biopsy. Six primary tumors were found to harbor promoter hypermethylation for p16 and O6–methylguanine-DNA-methyltransferase (MGMT) genes. Rapid QMSP was then used to identify promoter hypermethylation of these genes in the surgical margins. Results were compared with standard intraoperative histologic frozen section analysis and with conventional QMSP.

Results  Using our rapid QMSP assay, we found that 3 patients had methylation-positive margins. Tumor margins from 2 patients were methylated for p16 alone, and margins from 1 patient were methylated for p16 and MGMT simultaneously. Molecular margin analysis was completed in less than 5 hours, a time frame appropriate for selected major HNSCC resections that require combined primary tumor resection, cervical lymphadenectomy, and complex reconstruction. This technique was comparable in sensitivity to conventional QMSP.

Conclusion  Rapid molecular margin analysis using QMSP is feasible and may be performed intraoperatively in selected patients with HNSCC that requires extensive resection.