Johannes J. Manni

Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas

Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV‐DNA and the use of p16INK4A overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consumption on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16‐specific fluorescence in situ hybridization (FISH) and p16INK4A‐specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 integration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16INK4A accumulation. Only 5 out of 48 HPV‐negative tumors showed p16INK4A immunostaining (p < 0.0001). The presence of HPV furthermore correlates significantly with low tobacco (p = 0.002) and alcohol intake (p = 0.011), poor differentiation grade (p = 0.019), small tumor size (p = 0.024), presence of a local metastasis (p = 0.001) and a decreased (loco)regional recurrence rate (p = 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non‐smoking patients with HPV‐containing TSCC show a remarkably better disease‐specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16INK4A overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV‐positive tumors show a favorable prognosis as compared to those with HPV‐negative tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non‐smokers differ from those occurring in smokers, the former being related to HPV 16 infection.

A subset of head and neck squamous cell carcinomas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8.

Besides well‐known risk factors such as tobacco use and alcohol consumption, oncogenic human papillomavirus (HPV) infection also has recently been suggested to promote head and neck tumorigenesis. HPV is known to cause cancer by inactivation of cell cycle regulators p53 and pRb via expression of viral oncoproteins E6 and E7. This indicates that p53 mutations are not a prerequisite in HPV‐induced tumor development. However, discrepancy exists with respect to the frequency of head and neck squamous cell carcinomas (HNSCC) harboring DNA of oncogenic HPV and the fraction of these tumors showing p53 mutations. In our study, we examined the frequency of HNSCC demonstrating HPV 16/18 integration as identified by fluorescence in situ hybridization (FISH) and investigated their p53 (mutation) status by immunohistochemistry and single‐strand conformation polymorphism (SSCP) analysis of exons 5–8. Paraffin‐embedded, archival biopsy material from 27 premalignant mucosal lesions and 47 cases of HNSCC were analyzed. Ten of the 47 (21%) HNSCC unequivocally exhibited HPV 16 integration, including 8 of 12 (67%) tonsillar carcinomas. This is supported by the immunohistochemical detection of p16INK4A overexpression in all 10 HPV‐positive tumors. Although FISH is considered to be less sensitive than PCR‐based methods for HPV detection, our data clearly demonstrate clonal association of HPV with these tumors, as illustrated by the presence of integrated HPV 16 in both the primary tumor and their metastases in 2 patients. In contrast, HPV 16/18 DNA could not be detected in the premalignant lesions. In 30 of 47 (64%), HNSCC accumulation of p53 was observed, including 8 of the 10 HPV‐positive carcinomas. However, in none of the latter cases could mutations in exons 5–8 be identified, except for a polymorphism in codon 213 of exon 6 in one patient. Evaluation of clinical data revealed a significant inverse relation between tobacco use with or without alcohol consumption, and HPV positivity of the tumors.

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.

Glutathione S-transferase M1 and T1 and cytochrome P4501A1 polymorphisms in relation to the risk for benign and malignant head and neck lesions

Susceptibility to head and neck cancer in a particular individual may depend in part on the metabolic balance between Phase 1 enzymes, such as cytochromes P450 (CYPs), and Phase II enzymes, such as glutathione S‐transferases (GSTs). Genetic variability in CYP and GST isoenzymes may contribute to individual differences in susceptibility to chemical carcinogenesis. GSTM1 and GSTT1 null genotypes as well as polymorphic variants in the CYP1A1 gene that may help determine the risk for head and neck cancer have been described in previous reports.

A prospective longitudinal study on radiation-induced hearing loss

Recent clinical reports indicate that patients receiving radiotherapy that includes the auditory system in the treatment volume are likely to develop an irradiation-induced hearing loss. Although the early, presumed reversible, conductive hearing impairment due to secretory otitis media following radiotherapy is a sequela well known by radiation oncologists, permanent hearing loss, both conductive and sensorineural, is believed to be rare. A prospective study was performed enrolling patients receiving postoperative radiotherapy for unilateral parotid tumors. Audiometric results prior to irradiation were compared with those obtained 2 years later. Up to 50% of the patients (9 of 18) developed a clinically relevant hearing loss in the irradiated ear, both conductive and/or sensorineural. The contralateral ear remained unaltered. The hearing loss was permanent in 6 patients (33%) and affected their quality of life. The data suggest that changes occur in the inner ear as well as in the auditory nerve and auditory brain stem with conventional irradiation schemes with daily fractions of 2 to 2.5 Gy with a total dose of 50 Gy.

Reanimation of the paralyzed face by indirect hypoglossal-facial nerve anastomosis.

Abstract Background: The results of indirect hypoglossal facial nerve anastomosis with interposition of a free nerve graft, end-to-end to the periferal facial nerve stump, and end-to-side to the hypoglossal nerve are prospectively evaluated. This technique is supposed to overcome loss of hypoglossal function. Methods: Tongue function in 39 consecutive patients and facial reanimation in 29 patients who completed 24 months follow-up were assessed. Facial nerve function was judged using the House-Brackmann (HB) grading system. Results: Tongue movements were normal in all operated on patients. Initial facial movements occurred on average 7.5 months postoperatively. The results were graded HB II in 6 (20.9%), HB III in 13 (44.6%), HB IV in 7 (24.1%), HB V in 2 (6.8%) patients, and HB VI in 1 (3.4%) patient. The results were significantly better in young patients and when a short time interval between paralysis and surgery existed. Conclusions: Indirect hypoglossal-facial anastomosis is the preferred technique in most patients for whom the classical direct hypoglossofacial anastomosis is indicated.

Role of human papillomavirus in the development of head and neck squamous cell carcinomas

Objective To review the literature on the role of oncogenic human papillomaviruses (HPVs) in the carcinogenesis of the head and neck mucosa. Material and Methods Molecular and epidemiological studies concerning the high-risk HPV types and their role in carcinogenesis in the head and neck region were screened. Results Different studies revealed that: (i) 15–25% of head and neck squamous cell carcinomas (HNSCCs) are clonally associated with high risk HPV types (type 16); (ii) the oropharynx and particularly the tonsils are the most susceptible sites; (iii) patients with HPV-positive tumours present with more advanced stages of disease, are relatively younger, do not have extravagant tobacco and alcohol intake and seem to have a better survival; (iv) HPV-positive tumours are characterized by poor differentiation grade and a basaloid appearance; and (v) HPV-positive tumours exhibit integrated HPV DNA, wild-type p53, pRb downregulation and overexpression of p16INK4A. Conclusion Taken together, these data support the view that HPV-harbouring HNSCC can be considered a discrete tumour entity with, moreover, a favourable prognosis. Screening of patients, especially those with tonsillar cancers, for the presence of HPV may help to further optimize treatment protocols and to provide more accurate prognostic information.

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

P21Cip1/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis.

Human papillomavirus is involved in the carcinogenesis of tonsillar squamous cell carcinomas. Here, we investigated the expression and the prognostic value of key cell cycle proteins in the pRb and p53 pathways in both human papillomavirus type 16-positive and -negative tonsillar squamous cell carcinomas. Using immunohistochemistry, 77 tonsillar squamous cell carcinomas with known human papillomavirus type 16 status and clinical outcome were analyzed for expression of Ki67, p16INK4A, cyclin D1, pRb, p14ARF, MDM2, p53, p21Cip1/WAF1, and p27KIP1. Results were correlated with each other and with clinical and demographic patient data. A total of 35% of tonsillar carcinomas harbored integrated human papillomavirus type 16 DNA and p16INK4A overexpression, both being considered essential features for human papillomavirus association. These tumors also showed the overexpression of p14ARF (P<0.0001) and p21Cip1/WAF1 (P=0.001), and downregulation of pRb (P<0.0001) and cyclin D1 (P=0.027) compared with the human papillomavirus-negative cases. Univariate Cox regression analyses revealed a favorable survival rate for non-smokers (P=0.006), as well as for patients with T1-2 tumors (P<0.0001) or tumors showing low expression of cyclin D1 (P=0.028), presence of human papillomavirus and overexpression of p16INK4A (P=0.01), p14ARF (P=0.02) or p21Cip1/WAF1 (P=0.004). In multivariate regression analyses, smoking and tumor size, as well as expression of cyclin D1 and p21Cip1/WAF1, were found to be independent prognostic markers. We conclude that human papillomavirus positivity in tonsillar squamous cell carcinomas strongly correlates with p21Cip1/WAF1 and p14ARF overexpression and downregulation of pRb and cyclin D1. In particular p21Cip1/WAF1 overexpression is an excellent favorable prognosticator in tonsillar squamous cell carcinomas.

Meta-analysis and pooled analysis of GSTM1 and CYP1A1 polymorphisms and oral and pharyngeal cancers: A HuGE-GSEC review

The association of GSTM1 and CYP1A1 polymorphisms and oral and pharyngeal cancers was assessed through a meta-analysis of published case-control studies and a pooled analysis of both published and unpublished case-control studies from the Genetic Susceptibility to Environmental Carcinogens database (http://www.upci.upmc.edu/research/ccps/ccontrol/index.html). Thirty publications used in the meta-analysis included a total of 7783 subjects (3177 cases and 4606 controls); 21 datasets, 9397 subjects (3130 cases and 6267 controls) were included in the pooled analysis. The GSTM1 deletion was 2-fold more likely to occur in African American and African cases than controls (odds ratio: 1.7, 95% confidence interval: 0.9–3.3), although this was not observed among whites (odds ratio: 1.0, 95% confidence interval: 0.9–1.1). The meta-analysis and pooled analysis showed a significant association between oral and pharyngeal cancer and the CYP1A1 MspI homozygous variant (meta-ORm2/m2: 1.9, 95% confidence interval: 1.4–2.7; Pooled ORm2m2: 2.0, 95% confidence interval: 1.3–3.1; ORm1m2 or [infi]m2m2: 1.3, 95% confidence interval: 1.1–1.6). The association was present for the CYP1A1 (exon 7) polymorphism (ORVal/Val: 2.2, 95% confidence interval: 1.1–4.5) in ever smokers. A joint effect was observed for GSTM1 homozygous deletion and the CYP1A1 m1m2 variant on cancer risk. Our findings suggest that tobacco use and genetic factors play a significant role in oral and pharyngeal cancer.