Jens Peter Klussmann

Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers.

There are few well‐established patient risk factors associated with human papillomavirus (HPV) infection in cancers of the oral cavity and oropharynx. The purpose of this study was to determine if there were significant different risk factors and tumor characteristics between HPV‐positive and HPV‐negative cancer cases. HPV was evaluated in cancer tissue and exfoliated oral cells of 193 oral cavity/oropharynx cancer patients using PCR and direct DNA sequencing. A patient questionnaire collected information about risk factors, sexual practices and medical history. The prevalence of HPV high‐risk (HR) types was 20% in cancer cases. Three types were identified: HPV‐16 (87%), HPV‐18 (3%) and HPV‐33 (11%). Risk factors for HPV‐HR included younger age (≤ 55 years vs. > 55 years; adjusted OR = 3.4; 95% CI = 1.6–7.3) and younger‐age cases who had more lifetime sex partners (adjusted OR = 3.8; 95% CI = 1.4–10.1), practiced oral‐genital sex (adjusted OR = 4.3; 95% CI = 1.8–10.4) or oral‐anal sex (adjusted OR = 19.5; 95% CI = 3.4–113). Compared to HPV‐negative cancers, HPV‐HR cancers were more likely to have a positive HPV‐HR exfoliated oral cytology test (adjusted OR = 7.8; 95% CI = 3.4–18.4), later stage (adjusted OR = 3.0), nodal involvement (adjusted OR = 4.1) and advanced grade (adjusted OR = 3.0). This study shows new evidence that the prevalence of oncogenic mucosal HPV is higher in younger‐age oral cavity/oropharynx cancer cases whose sexual practices are typically associated with sexual transmission of the virus. HPV detection also appears to be an indicator of advanced disease characteristics that may require different clinical treatment for this subset of patients. An exfoliated oral cytology test for HPV was a significant predictor of HR types in the cancers, suggesting that an oral rinse may provide an early biomarker of infected tumors.

Human papillomavirus infection as a prognostic factor in carcinomas of the oral cavity and oropharynx

Although studies have established human papillomaviruses (HPVs) as a risk factor for oral and oropharyngeal cancer, it is not clear whether viral infection affects survival in head and neck malignancies. This investigation examined the relationship between HPV and survival in carcinomas of the oral cavity and oropharynx. Formalin‐fixed, paraffin‐embedded tumor specimens from 139 newly diagnosed cases were tested for HPVs by PCR and DNA sequencing. Patient and tumor characteristics were obtained from questionnaires, pathology reports and cancer registries. Odds ratios (ORs) and relative risks (RRs) were based on logistic and Cox regression models, respectively. HPVs were detected in 21% of the tumors; 83% were HPV‐16. Greater risk of HPV infection was associated with males (OR = 2.9, 95% CI = 1.0–8.6), a history of oral‐genital sex (OR = 4.2, 95% CI = 1.5–11.7), and oropharyngeal tumors (OR = 10.4, 95% CI = 3.5–31.2). As tobacco usage increased, the odds of HPV detection decreased (OR = 0.97/pack‐year, 95% CI = 0.96–0.99). HPV infected patients had better overall survival (RR = 0.3, 95% CI = 0.1–0.8) than those with HPV‐negative tumors. There was an interaction between gender and HPV for overall (p = 0.05) and disease‐specific (p = 0.03) survival that suggested that HPV infected males had better prognosis than HPV‐negative males, but this was not the case among females. HPV status was identified as an independent prognostic factor in oral and oropharyngeal cancers. This result appeared to be gender‐specific, suggesting the need for further study of the interaction between HPV and gender on survival.

Combined analysis of HPV-DNA, p16 and EGFR expression to predict prognosis in oropharyngeal cancer

Molecular prognostic indicators for oropharyngeal squamous cell carcinoma (OSCC), including HPV‐DNA detection, epidermal growth factor receptor (EGFR) and p16 expression, have been suggested in the literature, but none of these are currently used in clinical practice. To compare these predictors, 106 newly diagnosed OSCC for the presence of HPV‐DNA and expression of p16 and EGFR were analyzed. The 5‐year disease‐free survival (DFS) and overall survival (OS) were calculated in relation to these markers and a multivariate Cox analysis was performed. Twenty‐eight percent of the cases contained oncogenic HPV‐DNA and 30% were positive for p16. The p16 expression was highly correlated with the presence of HPV‐DNA (p < 0.001). Univariate analysis of the 5‐year DFS revealed a significantly better outcome for patients with p16‐positive tumors (84% vs. 49%, p = 0.009). EGFR‐negative tumors showed a tendency toward a better prognosis in DFS (74% vs. 47%, p = 0.084) and OS (70% vs. 45%, p = 0.100). Remarkable and highly significant was the combination of p16 and EGFR expression status, leading to 5‐year DFS of 93% for p16+/EGFR− tumors vs. 39% for p16−/EGFR+ tumors (p = 0.003) and to a 5‐year OS of 79% vs. 38%, respectively (p = 0.010). In multivariate analysis p16 remained a highly significant prognostic marker for DFS (p = 0.030) showing a 7.5‐fold increased risk for relapse in patients with p16‐negative tumors. Our data indicate that p16 expression is the most reliable prognostic marker for OSCC and further might be a surrogate marker for HPV‐positive OSCC. HPV+/p16+ tumors tended to have decreased EGFR expression, but using both immunohistological markers has significant prognostic implications.

Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus.

Recent analyses of head and neck squamous cell carcinomas revealed frequent infections by oncogenic human papillomavirus (HPV) type 16 in tonsillar carcinomas. Concerning involvement of risk factors, clinical course of the disease, and prognosis there are strong indications arguing that the HPV-positive tonsillar carcinomas may represent a separate tumor entity. Looking for a surrogate marker, which in further epidemiological studies could replace the laborious and expensive HPV detection and typing we analyzed p16 protein expression in 34 tonsillar carcinoma for correlation to HPV status and load of viral DNA. p16 has been shown to be of diagnostic value for clinical evaluation of cervical dysplasia. We found 53% of the tested tonsillar carcinomas to be HPV-positive. Fifty-six percent of all tumors tested were immunohistochemically positive for the p16 protein. In 16 of 18 of the HPV-positive carcinomas diffuse p16 expression was observed. In contrast, only one of the HPV-negative carcinomas showed focal p16 staining (P < 0.001). As determined by laser-assisted microdissection and quantitative real-time polymerase chain reaction, p16 expression correlated with the presence of HPV-DNA in the individual tumor specimens. Clinical outcome analysis revealed significant correlation of p16 expression with increased disease-free survival (P = 0.02). These data indicate that p16 is a technically simple immunohistological marker, applicable for routine pathological histology, and its prognostic value for survival is fully equivalent to HPV-DNA detection.

Genetic Signatures of HPV-related and Unrelated Oropharyngeal Carcinoma and Their Prognostic Implications

Purpose: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. Experimental Design: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16INK4A immunostaining. The results were correlated with HPV status and clinical data from patients. Results: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P = 0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P = 0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P = 0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P = 0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P = 0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P = 0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P = 0.008; disease-free survival, P = 0.01) and none of these patients had a tumor recurrence. Conclusions: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.

Histopathology of Pleomorphic Adenoma in the Parotid Gland: A Prospective Unselected Series of 100 Cases

Objectives/Hypothesis Histopathological characteristics of pleomorphic adenomas, especially of capsular alterations such as thin capsule areas, capsule‐free regions, capsule penetration, satellite nodules, and pseudopodia in the different subtypes, are described.

Human papillomavirus-positive tonsillar carcinomas: a different tumor entity?

Abstract. Human papillomavirus (HPV) infections are thought to be one of the causal factors in the development of head and neck squamous cell carcinomas (HNSCC), particularly in tumors arising from the Waldeyers tonsillar ring. We screened 98 carefully stratified HNSCC and different control tissues for the presence of HPV DNA by nested polymerase chain reaction (PCR) specific for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs and by HPV16-specific single step PCR. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. On average HNSCC showed rather low HPV DNA prevalences; 18% of the oral cavity cancers, 8% of nasopharyngeal cancers, 25% of hypopharyngeal cancers and 7% of laryngeal cancers were HPV DNA positive. In contrast, HPV sequences could be detected in 45% of the oropharyngeal cancers, particularly tonsillar carcinomas (58%). Tonsillar carcinomas were significantly more likely to be HPV positive than tumors from any other site (P<0.001). All tonsillar cancers contained oncogenic HPV types, predominantly HPV16 (13 of 14; 93%). Unaffected tonsils were available from two of these patients, but both tested negative for HPV DNA. Furthermore, no HPV DNA could be found in tonsillar biopsy specimens from control groups. Localization and load of HPV DNA was determined in HPV16-positive tonsillar carcinomas, their metastases and in unaffected mucosa using laser-assisted microdissection and subsequent real time fluorescence PCR. We demonstrated that the HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Quantification of HPV16 DNA in samples of seven patients yielded viral loads from 6 to 153 HPV DNA copies per β-globin gene copy and the load values in both locations were roughly comparable. These loads are comparable with data shown for other HPV-associated lesions. Statistical evaluation of data related to clinicopathological parameters showed a significant correlation of the HPV positivity of tonsillar carcinomas with tumor grading (P=0.008) and alcohol consumption (P=0.029). Taken together our findings show a preferential association of HPV DNA with tonsillar carcinomas. Furthermore our results argue for HPV-positive tonsillar carcinomas representing a separate tumor entity, which is less dependent on conventional HNSCC risk factors.

T cells specific for HPV16 E7 epitopes in patients with squamous cell carcinoma of the oropharynx

Squamous cell carcinomas of the oropharynx (SCCO) are often infected with oncogenic human papilloma virus (HPV) subtype 16. To determine the frequency of T cells specific for human leukocyte antigen (HLA)‐A2.1 restricted HPV16 E7 protein‐derived epitopes, tetramer analysis was performed using peripheral blood lymphocytes of 20 HLA‐A2.1+ patients and 20 HLA‐A2.1+ healthy individuals. Tetramers specific for 3 HPV16 peptides (E711–20, E782–90 and E786–93), an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were used in multicolor flow analysis. The HPV‐specific T‐cell frequencies were correlated with the HPV16 E7 and p16 status in tumor sections. In vitro stimulation (IVS) with autologous dendritic cells (DC) pulsed with HPV16 E7 epitopes was performed to demonstrate proliferation and antitumor activity of the HPV‐responsive T cells. Frequencies of CD8+ T cells specific for HPV16 E7 peptides were not significantly different in patients with SCCO relative to normal donors. However, patients with tumors expressing HPV16 E7 (60%) and p16 (50%) had an increased frequency (p < 0.05) of T cells specific for the E711–20 epitope compared to those with tumors negative for both markers. HPV16 E711–20 and HPV16 E786–93 specific T cells were expandable upon IVS with cognate peptide‐pulsed DC and were reactive against peptide‐pulsed targets or, in case of the E711–20 epitope‐specific T cells, against HPV16 E7 expressing CaSki cell line. Thus, in patients with HPV16+ SCCO, precursor T cells specific for E711–20 epitope are present (1/3,947) in the circulation, are responsive to stimulation with the cognate viral peptide and recognize in vitro HPV16 E7+ tumor cells. Further studies have to elucidate why those T cells are unable to eliminate the tumor in vivo and this might also allow for finding potential strategies that will increase the chances of developing a future HPV‐based vaccine in patients with SCCO.

p16INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Klingenberg B, Hafkamp H C, Haesevoets A, Manni J J, Slootweg P J, Weissenborn S J, Klussmann J P & Speel E‐J M
(2010) Histopathology 56, 957–967
p16INK4Aoverexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

COMPLICATIONS OF SUSPENSION LARYNGOSCOPY

Although suspension laryngoscopy (SL) is routinely used in operative laryngology, no prospectively gathered data on the complications of this procedure have so far been available. We prospectively analyzed 339 consecutive procedures for ***intervention-related complications. The survey included preoperative dental status and assessment of postoperative dental, mucosal, and nerve injuries. Minor mucosal lesions were found in 75% of all patients. All healed spontaneously within a few days. Dental injuries occurred in 6.5% of all patients. These were more frequent in therapeutic laryngoscopy than in diagnostic procedures (6.8% versus 6.0%). Highly significant correlations were found between dental injury rate and preoperative dental disease (p < .04) and grade of periodontitis (p < .001). Temporary nerve lesions were observed in 13 patients (9 of the lingual nerve and 4 of the hypoglossal nerve). Although minor complications frequently occur during SL, it is a relatively safe procedure with a low risk of significant morbidity.