Hagen Weidauer

Tobacco and alcohol and the risk of head and neck cancer

SummaryWe carried out two case-control studies on the relative risk of head and neck cancer in association with tobacco and alcohol consumption. The first study carried out at the ENT Department of the University hospitals of Heidelberg and Giessen (FRG) comprised 200 male patients with squamous cell cancer of the head and neck and 800 control subjects matched for sex, age, and residential area (1:4 matching design). Of the tumour patients, 4.5% had never smoked, in contrast to 29.5% of the control group. The average tobacco and alcohol consumption of the patients was approximately twice as high as in the control subjects. The highest alcohol and tobacco consumption was observed in patients suffering from oropharyngeal cancer. Tobacco and alcohol increased the risk of head and neck cancer in a dose-dependent fashion and acted as independent risk factors. In heavy smokers (> 60 pack-years) a relative risk of 23.4 (alcohol adjusted) was calculated. Combined alcohol and tobacco consumption showed a synergistic effect. The risk ratio increased more in a multiplicative than in an additive manner. Oral and laryngeal cancer were associated with the highest tobacco-associated risk values. The highest ethanol-associated risk values were associated with oropharyngeal and laryngeal cancer. The second study was carried out at the ENT Department of the University of Heidelberg on 164 males with squamous cell carcinoma of the larynx and 656 control subjects matched for sex, age and residential area (1:4 matching design). Of the cases, 4.2% had never smoked, compared with 28.5% of the control subjects. The risk of laryngeal cancer by tobacco consumption was dose dependent, reaching a maximum value of 9.1 (adjusted for alcohol) for a consumption of more than 50 tobacco-years (TY). The relative risk of laryngeal cancer associated with alcohol intake was also dose dependent, reaching a value of 9.0 (adjusted for tobacco) for a mean daily consumption of more than 75 g alcohol. An analysis of subsite specific risks showed that heavy smokers (> 50 TY) carried a nearly ten times higher risk of supraglottic cancer than of glottic cancer. The risk of supraglottic cancer from alcohol consumption was also higher than that of glottic cancer.

TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer

Recent studies have suggested that different mutation types within the core domain of the tumour suppressor protein p53, i.e. DNA contact mutations and structural mutations, confer different biological properties. We have analysed in 86 head and neck squamous cell carcinomas (HNSCC), whether these p53 mutation types have a differential clinical impact. Thirty-seven missense mutations were identified. Thirteen of these (36%) were DNA contact mutations, occurring in the L3 loop, in the H2 loop sheet helix motif, in the S10 β strand and in Zinc binding residues. Microsatellite marker analysis revealed a selective association between these mutations and the loss of wild-type alleles (100% LOH vs 50% LOH in tumours with structural mutations; P=0.0034, Fishers exact, 2-tailed). In comparison to structural mutations or to the absence of mutations in the core domain, DNA contact mutations were associated with higher tumour stages (84.6% vs 62%), a higher incidence of lymph node metastasis (91.7% vs 56%; P=0.014, Fishers exact, 2-tailed), a shortened recurrence-free survival (8.1 months vs 23.7 months, P=0.047, log rank test) and overall survival (11 months vs 29.2 months; P=0.003, log rank test). The latter was also the case when only stage IV tumours were analysed (P=0.0055, log rank test). These data indicate that in HNSCC, TP53 DNA contact mutations confer a strong selection pressure to eliminate wild-type alleles, and that they result in an accelerated tumour progression and reduced therapeutic responsiveness.

Infection of the oral mucosa with defined types of human papillomaviruses

Biopsies from 9 different oral papillomatous proliferations were analysed for human papilloma viral (HPV) sequences of types 1 to 19 and 21 to 26 by Southern blot analysis with 32p-labelled cellular DNA. HPV sequences were detected in 7 out of 9 biopsies obtained from individual patients. Of three cases with the clinical diagnosis focal hyperplasia Heck, two contained HPV-6 related sequences and one HPV 13. In addition, one tongue base papilloma contained HPV 11. A papilloma of the palate revealed HPV 11 sequences. HPV 6 could be demonstrated twice in the ramaining 4 oral papillomatous proliferations. Two biopsies remained negative. The data shows that HPV DNA can be regularly demonstrated in oral papillomas.

Antitumor Vaccination in Patients with Head and Neck Squamous Cell Carcinomas with Autologous Virus-Modified Tumor Cells

Prognosis of patients with advanced head and neck squamous cell carcinomas (HNSCC) is still poor. Therefore, we analyzed whether antitumor vaccination with a virus-modified autologous tumor cell vaccine is feasible and safe in HNSCC patients. Furthermore, we determined the influence on disease-free survival and overall survival and the vaccination-induced antitumor reactivity. In a nonrandomized pilot study, 20 patients were vaccinated postoperatively. Vaccine was prepared from the tumor cell cultures of patients by infection of the cells with Newcastle Disease Virus, followed by γ-irradiation, and vaccine was applied up to five times. Antitumor immune reactivity was determined in the skin by delayed type hypersensitivity skin reaction and in the blood by enzyme-linked immunospot assay. Establishment of tumor cell cultures was successful in about 80% of the cases. After vaccination, we observed no severe side effects. Percentages of survival of vaccinated patients with stage III and stage IV tumors (n = 18) were 61% at 5 years. Immune monitoring revealed significant increases of antitumor delayed type hypersensitivity reactivity especially in disease-free patients, and in a significant proportion of vaccinated patients the presence of tumor-reactive T-cells in the peripheral blood even 5 to 7 years after the last vaccination. Postoperative vaccination with virus-modified autologous tumor cells seems to be feasible and safe and may improve the prognosis of HNSCC patients with advanced tumors. This could be supported by antitumor immune responses that we observed especially in long-term surviving patients.

Head and neck tumor sites differ in prevalence and spectrum of p53 alterations but these have limited prognostic value.

The tumor site is a strong clinical factor in head and neck squamous cell carcinoma (HNSCC). To clarify the biologic and clinical role of p53 alterations in HNSCC, we have examined the prevalence and the nature of p53 alterations in a large cohort of tumors from the different sites. For immunohistochemical analysis of p53 protein expression, we introduced tyramide signal amplification immunohistochemistry (TSA‐IHC) on a tissue microarray. This allowed the discrimination between normal low‐level expression and reduced or lost expression. Two hundred fifty‐three tumors were subjected to mutational analysis by genomic DNA sequencing, employing also the p53 GeneChip from Affymetrix. The prevalence of all p53 alterations, i.e., mutations, overexpression and loss of expression, was significantly higher in hypopharyngeal tumors than in the other sites (p = 0.001). Laryngeal tumors showed the lowest rate of p53 alterations, but revealed a distinct mutation spectrum: most mutations affected exon 5 (p = 0.013) and the S2′ domain (p = 0.002), and most hot‐spot 248 mutations occurred in the larynx (p < 0.001). Sequencing by p53GeneChip technology was shown to be only insignificantly more sensitive than dideoxy sequencing. In agreement with p53 mutations occurring prior to invasiveness, their prevalence did not increase with tumor stage, and all mutation classes lacked prognostic significance. The large patient cohort of this study showed that p53 is differentially affected in the different tumor sites of the head and neck, but its mode of inactivation does not play a major role in tumor progression.

Expression of the histone H3 gene in benign, semi-malignant and malignant lesions of the head and neck: a reliable proliferation marker

To search for a reliable proliferation marker in epithelial head and neck lesions, we have analysed the expression of the histone H3 gene by in situ hybridisation and compared this with the immunoreactivity of the widely used monoclonal antibody Ki-67. In many lesions, the Ki-67 staining failed to delineate proliferation. In contrast, the H3 hybridisation signals were in accordance with the histopathology of the biopsies: in hyperplastic epithelia, significant H3 mRNA levels were only seen in areas with inflammation. Dysplastic cells showed distinctly elevated H3 expression. Benign and semi-malignant tumours, i.e. basal cell carcinomas, showed moderate H3 signals at the periphery. In squamous cell carcinomas, H3 expression was always high at the expanding zone of the tumour and was most extensive in undifferentiated carcinomas. Thus, the expression of the histone H3 gene closely reflected the dynamics of neoplastic growth within and around head and neck tumours.

Prognostic relevance of serum levels of the angiogenic peptide bFGF in advanced carcinoma of the head and neck treated by primary radiochemotherapy.

The objective of this trial was to analyze the prognostic relevance of the angiogenic peptides basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and matrix metalloproteinase‐2 (MMP‐2) in the serum of patients with advanced carcinoma of the head and neck treated by primary radiochemotherapy.

Chronic Alcohol Consumption-The Key Risk Factor for Pharyngeal Cancer

A case-control study on oropharynx and hypopharynx cancer was carried out in the region of Heidelberg (in the southwest region of Germany). This report presents an analysis of the risk associated with alcohol and tobacco consumption based on 105 cases and 420 controls matched for age, sex, and area of residency.

Prognostic impact of reoxygenation in advanced cancer of the head and neck during the initial course of chemoradiation or radiotherapy alone.

Previous studies have shown that radiation of hypoxic tumors can result in reoxygenation phenomenon. The relevance of this phenomenon for prognosis is unclear. This study analyzes whether the presence of hypoxia, or the extent to which reoxygenation occurs during the initial phase of primary chemoradiation or radiotherapy, can predict the clinical outcome in advanced tumors of the head and neck.

Immunomodulating Effects of Surgical Intervention in Tumors of the Head and Neck

The immunomodulating effect of primary surgical intervention in 33 patients with squamous cell carcinoma of the oral cavity, pharynx, and larynx was analyzed prospectively. An operation time of longer than 7 hours was significantly associated with a decrease of total lymphocyte counts, CD4+ T lymphocytes, and CD8+ T lymphocytes. The CD4/CD8 ratio as a marker for the downregulation of the cellular immune response was slightly decreased but still in the normal range. CD4+ lymphocyte counts increased within 7 days, and CD8+ lymphocytes increased 4 weeks after the operation. The in vitro stimulation of the lymphocytes was impaired for 1 to 4 weeks. Release of interleukins, interferon-γ, and tumor necrosis factor-α remained low despite the surgical trauma. The decreased lymphocyte counts, especially CD4+ and CD8+ lymphocytes, were significantly associated with duration of operation and volume of blood loss. Extension of trauma, age, type of anesthesia, and type of intensive care intervention were not associated with specific immunomodulating effects. However, these factors might be responsible for suppression of the immune system, which is expressed by lymphocyte depletion, lymphocyte dysfunction, and impaired upregulation of cytokine secretion.