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

Archives of Pathology & Laboratory Medicine, Oct 2008 by Lee, Walter T, Tubbs, Raymond R, Teker, Aysenur M, Scharpf, Joseph, Strome, Marshall, Wood, Benjamin, Lorenz, Robert R, Hunt, Jennifer

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 history 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 anatomic 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 carcinoma. Our data show that high-risk HPV is an uncommon 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)

Tobacco and alcohol use are well known and established risk factors for head and neck squamous cell carcinoma (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 papillomavirus (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. Furthermore, the most common site of infection was the oropharynx. 1,4 There appear to be clinical implications from knowing the HPV status in HNSCC. Numerous reports have demonstrated a survival outcome advantage in patients 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 variation may be due to the variety and high sensitivity of the methods used to identify HPV. One of the most common methods is polymerase chain reaction (PCR).10 However, this method is known to be sensitive to contamination and may be prone to false negatives.11 If accurate investigation 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 alcohol 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 samples. Furthermore, it allows for direct visualization of HPV in HNSCC cells.

MATERIALS AND METHODS

All patients with HNSCC who did not have a history of alcohol or tobacco use were screened from October 2004 through October 2005 after Cleveland Clinic Institutional Review Board approval. Patients were surveyed by the physician on initial presentation and diagnosis. Nonsmokers constituted patients who replied that they had never smoked currently or in the past. Nondrinkers constituted patients who claimed no regular or routine use of alcoholic beverages currently or in the past.

Histologic sections were reviewed, and the diagnosis was confirmed. Blocks were selected to contain both tumor and normal adjacent epithelium as an internal control. Fresh formalin-fixed, paraffin-embedded tissue sections were cut from the tissue block and mounted on coated slides. These slides were used to perform chromogenic in situ hybridization (ISH), according to standard protocols. The probe sets used for the assay included are well validated to identify low- or high-risk HPV (Ventana Medical Systems). The high-risk family 16 probe cocktail has an affinity to high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, and 66. The low-risk family 6 probe cocktail has an affinity to HPV genotypes 6 and 11. The slides were examined for the presence of integrated HPV using light microscopy. Positive signal is indicated by dark blue dotlike staining within the cytoplasm of tumor cells. Copy number can be estimated by comparison to the controls, which are xenograft controls that have known copy numbers. Internal negative controls were examined for each tissue section.