Arch Pathol Lab Med—Vol 132, October 2008 ISH to Detect HPV in HNSCC Patients—Lee et al 1653 Use of In Situ Hybridization to Detect Human Papillomavirus in Head and Neck Squamous Cell Carcinoma Patients Without a History of Alcohol or Tobacco Use WalterT. Lee, MD; Raymond R.Tubbs, MD; Aysenur M. Teker, MD; Joseph Scharpf, MD; Marshall Strome, MD; Benjamin Wood, MD; Robert R. Lorenz, MD; Jennifer Hunt, MD ● Context.—Head and neck squamous cell carcinoma is commonly associated with tobacco and alcohol use. There are, however, a group of patients without a significant his- tory of tobacco or alcohol use, and the etiology of these tumors is incompletely understood. Objective.—To examine tumors in this subpopulation for association with human papillomavirus (HPV) using newly available in situ hybridization probes. Design.—Between October 2004 and October 2005, 22 patients who did not use alcohol or tobacco were included. Formalin-fixed, paraffin-embedded tissue sections were used to perform in situ hybridization using newly available probe sets (Ventana Medical Systems, Tucson, Ariz). The slides were examined for the presence of integrated HPV using light microscopy. Positive and negative xenograft controls were run with the assay. Results.—The mean age of the patients was 64 years. There were 14 men and 8 women. The most common an- atomic sites included tongue (n  8), tonsil (n  7), and larynx (n  7). All cases and controls were successfully stained. Only 2 cases were positive for high-risk HPV, and both demonstrated an integrated pattern. Both cases were tumors of the tonsil. No cases were positive for low-risk HPV. Conclusions.—These results demonstrate that the new probe sets for HPV can be used very efficiently in clinical pathology material of head and neck squamous cell car- cinoma. Our data show that high-risk HPV is an uncom- mon finding in head and neck squamous cell carcinoma from patients who do not have a history of tobacco or alcohol use; low-risk HPV was not seen in any case. (Arch Pathol Lab Med. 2008;132:1653–1656) T obacco and alcohol use are well known and established risk factors for head and neck squamous cell carci- noma (HNSCC). However, there is a subset of HNSCC patients who do not report a history of tobacco or alcohol use. These patients comprise a unique population, and other etiologies for their HNSCC have been investigated. One such area of research is the role of human papillo- mavirus (HPV). It has been reported that HPV may be associated with HNSCC in patients without a history of tobacco or alcohol consumption. 1–3 These studies found a significantly higher incidence of HPV compared with other cohorts. Further- more, the most common site of infection was the oro- pharynx. 1,4 There appear to be clinical implications from Accepted for publication March 11, 2008. From the Head and Neck Institute (Drs Lee, Teker, Scharpf, Strome, Wood, and Lorenz) and the Division of Pathology and Laboratory Med- icine (Drs Tubbs and Hunt), Cleveland Clinic, Cleveland, Ohio. Dr Lee is now located at Duke University Medical Center, Durham, NC. Dr Tubbs has received research support from Ventana Medical Sys- tems as well as honoraria for speaking on its behalf. The other authors have no relevant financial interest in the products or companies de- scribed in this article. Reprints: Walter T. Lee, MD, Division of Otolaryngology-Head and Neck Surgery, Duke University Medical Center, DUMC 3805, Durham, NC 27710 (e-mail: walter.lee@duke.edu). knowing the HPV status in HNSCC. Numerous reports have demonstrated a survival outcome advantage in pa- tients with HPV-positive tumors. 5–7 Furthermore, with the recent approval of the HPV vaccine for cervical cancer, research is ongoing as to the utility of such a vaccine against HNSCC in this patient population. 4,8 Human papillomavirus serotypes 16 and 18 have been associated with cancer development. 7–9 The incidence of these patients with HPV varies widely. Across published reports, HPV positivity varies from 20% to 90%. 7 This var- iation may be due to the variety and high sensitivity of the methods used to identify HPV. One of the most com- mon methods is polymerase chain reaction (PCR). 10 How- ever, this method is known to be sensitive to contamina- tion and may be prone to false negatives. 11 If accurate in- vestigation of HPV-associated tumors is to be done, then it will be necessary to provide an accurate and sensitive method of determining HPV involvement. We present a series of patients without a history of al- cohol or tobacco use who underwent detection of HPV using novel and newly available, well-validated, low- and high-risk probe sets (Ventana Medical Systems, Tucson, Ariz). Both low- and high-risk serotypes were screened in pathologic specimens. This technique allows for accurate and rapid detection of HPV infectivity in pathologic sam- ples. Furthermore, it allows for direct visualization of HPV in HNSCC cells.