Serum vascular endothelial growth factor in patients with head and neck squamous cell carcinoma.

Vascular endothelial growth factor (VEGF) is a key pro-angiogenic cytokine expressed by most human tumours. Two isoforms, VEGF121 and VEGF165, are soluble and can be assayed in serum. Serum VEGF has been shown to be significantly raised in patients with solid tumours and shows some promise as a potentially useful tumour marker. Serum levels of VEGF were assayed in 52 patients with untreated head and neck squamous cell carcinoma (HNSCC) and 104 healthy controls. Serum VEGF is significantly raised in patients with HNSCC (P < 0.001), but there was no association with either tumour stage or specifically the presence of nodal metastases. Sixteen patients (31%) had a higher serum VEGF than 95th centile of controls, suggesting that serum VEGF measurement is of little practical use as an initial diagnostic tool. The finding that patients with HNSCC have significantly raised serum VEGF probably relates to enhanced platelet aggregation in these patients.     

Analytical and clinical evaluation of CYFRA 21-1 by electrochemiluminescent immunoassay in head and neck squamous cell carcinoma

This paper attempts to evaluate the clinical usefulness of CYFRA 21-1 as a serum tumour marker in patients with head and neck squamous cell carcinoma (HNSCC). The serum concentration of CYFRA 21-1 was measured utilizing a new electrochemiluminescent immunoassay (ECLIA) in 142 patients with HNSCC before and after treatment, 68 patients with benign tumours of the head and neck, and 50 healthy controls. Serum levels of CYFRA 21-1 in patients with HNSCC were significantly higher than those of benign tumours and healthy controls (p < 0.001). The diagnostic sensitivity and specificity of CYFRA 21-1 for HNSCC were 62 per cent and 100 per cent, respectively. The positive rates of CYFRA 21-1 increased with progression of HNSCC, serum CYFRA 21-1 levels were related to the tumour stage expressed by primary tumour (T) and nodal status (N) (p < 0.001), but not related to patient age, gender, smoking and drinking habit, or histopathological grade (p > 0.05). Post-treatment levels of CYFRA 21-1 in HNSCC decreased significantly (p < 0.001). Among 38 patients with clinical or radiological evidence of a recurrence during follow-up, 78.9 per cent (30 of 38) showed an increase in CYFRA 21-1. The analytical ECLIA performance for serum CYFRA 21-1 provides a new means of clinical assessment for HNSCC. The results of ECLIA suggest that the serum marker CYFRA 21-1 is valuable not only for diagnosis but also for close monitoring of patients with HNSCC.     

Serum vascular endothelial growth factor in patients with head and neck squamous cell carcinoma

Vascular endothelial growth factor (VEGF) is a key pro-angiogenic cytokine expressed by most human tumours. Two isoforms, VEGF₁₂₁ and VEGF₁₆₅, are soluble and can be assayed in serum. Serum VEGF has been shown to be significantly raised in patients with solid tumours and shows some promise as a potentially useful tumour marker. Serum levels of VEGF were assayed in 52 patients with untreated head and neck squamous cell carcinoma (HNSCC) and 104 healthy controls. Serum VEGF is significantly raised in patients with HNSCC (P < 0.001), but there was no association with either tumour stage or specifically the presence of nodal metastases. Sixteen patients (31%) had a higher serum VEGF than 95th centile of controls, suggesting that serum VEGF measurement is of little practical use as an initial diagnostic tool. The finding that patients with HNSCC have significantly raised serum VEGF probably relates to enhanced platelet aggregation in these patients.     

Growth of xenografted head and neck cancer in nude mice pre-treated with whole body irradiation.

In an attempt to enhance the primary acceptance rate of human squamous cell carcinoma of the head and neck (HNSCC), nude mice (BALB/c) were given whole body irradiation (WBI) prior to heterotransplantation of tumour specimens. A total of 27 fresh HNSCC biopsies were transplanted, with a take rate of 15%. No difference in primary tumour acceptance rate was found between irradiated and non-irradiated mice. Only one of the accepted tumours established growth both in irradiated and non-irradiated mice. In a second experiment, three previously established tumour lines of HNSCC were re-transplanted to irradiated and non-irradiated mice. As compared with non-irradiated mice, the growth rate was lower in all tumours transplanted to irradiated mice, the difference being statistically significant in two out of the three tumour lines. The results of the study show that attempted immunosuppression by WBI of nude mice prior to tumour implantation does not improve the growth conditions of HNSCC. These findings further emphasise the complexity of the transplantation barrier against human tumour xenografts in nude mice.     

Recombinant erythropoietin beta enhances growth of xenografted human squamous cell carcinoma of the head and neck after surgical trauma

CONCLUSION: Treatment of anaemia with recombinant human erythropoietin beta (rHuEpo) in patients with squamous cell carcinoma of the head and neck (HNSCC) undergoing curative radiotherapy does not improve cancer control. In fact, incompletely resected patients with HNSCC receiving radiation in combination with rHuEpo showed poorer loco-regional progression-free survival than patients receiving radiation in combination with placebo. It could be hypothesized that the effects of rHuEpo on tumour cell growth might only be manifested in vivo and after cell trauma, and that treating anaemia with rHuEpo might contribute to poor outcome after incomplete surgical resection. OBJECTIVE: The aim of the present study was to examine the effect of rHuEpo alone and in combination with surgical trauma on the growth of human squamous cell carcinoma in vivo, xenografted onto nude mice. MATERIALS AND METHODS: The surgical trauma was inflicted through subcutaneous transection of the tumour with a needle. Immunohistochemical staining verified expression of the EPO receptor in tumour cells. RESULTS: rHuEpo alone had no effect on the growth of xenografted HNSCC. However, a significant increase in tumour growth was observed after surgical trauma in combination with rHuEpo compared with surgery alone (p = 0.0008).     

Recombinant erythropoietin β enhances growth of xenografted human squamous cell carcinoma of the head and neck after surgical trauma

Conclusion. Treatment of anaemia with recombinant human erythropoietin β (rHuEpo) in patients with squamous cell carcinoma of the head and neck (HNSCC) undergoing curative radiotherapy does not improve cancer control. In fact, incompletely resected patients with HNSCC receiving radiation in combination with rHuEpo showed poorer loco-regional progression-free survival than patients receiving radiation in combination with placebo. It could be hypothesized that the effects of rHuEpo on tumour cell growth might only be manifested in vivo and after cell trauma, and that treating anaemia with rHuEpo might contribute to poor outcome after incomplete surgical resection. Objective. The aim of the present study was to examine the effect of rHuEpo alone and in combination with surgical trauma on the growth of human squamous cell carcinoma in vivo, xenografted onto nude mice. Materials and methods. The surgical trauma was inflicted through subcutaneous transection of the tumour with a needle. Immunohistochemical staining verified expression of the EPO receptor in tumour cells. Results. rHuEpo alone had no effect on the growth of xenografted HNSCC. However, a significant increase in tumour growth was observed after surgical trauma in combination with rHuEpo compared with surgery alone (p?=?0.0008).     

Vascular endothelial growth factor expression correlates with p53 mutation and angiogenesis in squamous cell carcinoma of the head and neck

Vascular endothelial growth factor (VEGF) has potent angiogenic activity and has been identified in a wide variety of malignancies, including head and neck squamous cell carcinoma (HNSCC). The tumour-suppressor gene p53 has been thought to regulate VEGF. Cryostat sections of 33 head and neck squamous cell carcinomas (HNSCC) were immunostained for VEGF using a standard streptavidin-biotin complex procedure. To evaluate angiogenesis, microvascular density was counted by staining endothelial cells immunohistochemically using anti-vWF monoclonal antibody. The p53 gene status was analysed using a PCR-SSCP analysis and direct sequencing. VEGF positive staining was detected in 18 (55%) out of 33 tumours. VEGF immunoreactivity did not correlate with the main clinicopathological characteristics of the patients (localization, T-stage, N-status, histological grading). Statistical analysis gave a clear correlation between the tumour vascularity and the VEGF protein expression (p = 0.0036). VEGF negative tumours showed a lower mean number of microvessels per microscopic field (60.3 +/- 15.5) than VEGF positive tumours (79.6 +/- 22.9). P53 mutations were identified in 12 (36.4%) of 33 tumours. The association of p53 mutations and VEGF expression level was significant (0.027). The higher microvessel density in VEGF positive tumours supports the importance of VEGF for tumour angiogenesis in HNSCC. Our results support the hypothesis of a p53 regulation on the angiogenic process through a VEGF up-regulation.     

Molecular and cellular processes underlying the hallmarks of head and neck cancer

The hallmarks of cancer were updated by Hanahan and Weinberg in 2011. Here we discuss the updated hallmarks in relation to what is known of the molecular and cellular processes underlying the development of head and neck squamous cell carcinoma (HNSCC). Several mechanisms are described, and recent surveys of HNSCC suggest a limited number of mutations, from which more mechanisms may emerge. There are also epigenetic changes to the control of normal processes. More than one mechanism underlies each hallmark. Processes essential to the development of HNSCC need not be essential to the proliferation of the fully developed tumour. Attention is paid to the emerging hallmarks, deregulation of cellular energy metabolism and evasion of immune destruction, and enabling characteristics, genome instability and mutation and tumour-promoting inflammation. HNSCC may adapt to hypoxia, suppress HLA expression, and express Toll-like receptors to facilitate inflammation, which support the proliferation of the tumour.     

Investigation of interleukin 10, 12 and 18 levels in patients with head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. It is the most common neoplasm arising in the upper aerodigestive tract. Interleukin (IL) 12 and IL-18 are cytokines which have a major anti-tumour activity via stimulation of a T-helper type 1 (Th1) immune response. Interleukin 10, a potent antagonist of IL-12, is a cytokine which possesses immunosuppressive activity mainly produced via T-helper type 2 (Th2) cells. Studies of other types of cancer have shown that the level of IL-12 in serum or tissues is suppressed and/or the IL-10 level is increased, suggesting that there is an impaired cell-mediated anti-tumour response. The aim of this study was to measure pre-operative serum cytokine concentrations in HNSCC patients in order to detect any changes in IL-10, IL-12 and IL-18, compared with non-tumour controls. The relationship between cytokine levels and standard clinicopathological features, including tumour site, tumour stage and presence of nodal metastasis, was also examined. Fifty-seven patients with primary HNSCC were prospectively recruited, together with 40 non-tumour control patients with a similar age and sex distribution. Serum cytokine levels were measured using commercial quantitative enzyme-linked immunosorbent assay. The HNSCC patients had significantly lower IL-12 levels (median; interquartile range) than controls (42.8 pg/ml, 26.2-61.6 vs 52.3 pg/ml, 37.5-113.7; p=0.018). Also, patients were more likely to have detectable IL-10 levels than were controls, as IL-10 was positive in 27/55 patients but in only 9/39 controls (p=0.011). Furthermore, IL-10 detectability varied according to primary site, being more commonly observed in hypopharyngeal and laryngeal tumours, and IL-10 was more likely to be detected with advanced tumour stage (T3 and T4). No differences in IL-18 levels were observed between patients and controls (p=0.169). These results suggest (in agreement with studies on other solid malignancies) that HNSCC causes a significant change in the serum levels of specific Th1 and Th2 cytokines, producing an in vivo environment that is unlikely to promote an effective cell-mediated anti-tumour response.     

Clinical and biological factors affecting response to radiotherapy in patients with head and neck cancer: a review.

OBJECTIVE: The main aim of this article was to review the clinical and biological factors that have been shown to influence the response of the head and neck squamous cell carcinoma (HNSCC) to primary radiotherapy and briefly discuss how some of these factors could be exploited to improve outcome. DESIGN: Medline based search covering 1982-2006 to identify the HNSCC literature where the effect of clinical and biological factors on locoregional control and overall survival were investigated. RESULTS: Clinical factors are routinely used in management decisions. Nevertheless, identically staged tumours receiving the same treatment may have different outcomes. Biological factors such as hypoxia, proliferation and radio-sensitivity play an important role in radiation response. However, these are not currently used in practise because tests that are clinically reliable and feasible are not available. CONCLUSION: High-quality translational research will allow us to develop biological tests that can be used in routine clinical practise to tailor individual treatment, with the ability to improve patient outcome further by modifying the underlying tumour biology.     

The value of chest CT-scanning for staging of progressed or recurrent head and neck squamous cell carcinomas (HNSCC)

AIM: At time of diagnosis, up to 17 % of HNSCC present with distant metastasis or a second primary tumour. Distant metastases of these tumours most commonly occur in the lung, requiring a particularly precise evaluation of this organ within the staging process. It was the aim of this study to compare the radiological findings of plain chest X-rays with the results of CT-scans of the chest in regard to their sensitivity for metastasis detection. PATIENTS AND METHODS: The staging examinations of 47 patients (f: 13, m: 34, mean age: 61.6 y) with progressed (T3, T4, N+) or recurrent HNSCC were prospectively analysed and results of chest X-rays as well as CT-scans of the chest compared. RESULTS: Only one plain chest X-ray showed a possible metastasis, which was excluded by the following CT-scan. In none of the other 46 patients did X-ray reveal findings of metastatic disease or second primary tumours. CT-scans of the chest showed tumorous lesions in 8/47 (17 %) patients. Three of these tumours were confirmed as neoplastic by biopsy, in another case radiological signs and clinical symptoms permitted definite assumption of malignancy (4/47 : 8.5 %). Histologically, only one of the latter four tumours could be identified as metastatic. In the remaining three cases we found second primary tumours. A follow up CT-scan of one of the remaining four cases showed normal results. In 3 cases the aetiology of the CT-findings remained unclear. CONCLUSION: Cervical lymph node metastases, tumour-size and recurrence of HNSCC are known risk factors for metastatic disease in HNSCC. Chest X-ray as staging procedure in patients with progressed or recurrent HNSCC may not be able to identify metastases or a second primary tumour of the lung. We therefore recommend a CT-scan of the chest as a routine procedure in such patients to optimise the pre-operative staging.     

Head and neck cancer in the South West of England: influence of socio-economic status

The incidence of a number of cancers is affected by socio-economic status. We hypothesized that the incidence of head and neck squamous cell carcinoma (HNSCC) would fall with increasing affluence, that this pattern would be similar for all sites, and that more second primary tumours would appear in deprived groups. Data on all HNSCC registered between 1985 and 1991 in the South West of England was collected. Tumours of the lip and skin were excluded. The measure of socio-economic status chosen was the Carstairs Index.^{1 1 THorne M., GOddard E., HIckman M. & HUnter P. (1992)} testing was applied. There were 1570 cases of HNSCC, 72% in men, 28% in women. Carstairs index could be determined for 1467 cases. Overall, socio-economic status was inversely related to the development of HNSCC. In men, the most deprived group had a significantly higher incidence of oral carcinoma than all other groups (P < 0.01), whereas the incidence of laryngeal carcinoma showed a gradual rise with increasing deprivation. In women, the trend was less clear. Seventy-two (4.9%) went on to develop a second primary, of which 35% were lung and 12% bladder. Socio-economic status did not affect the development of a second primary tumour in either sex. Thus, in the South West of England, socio-economic status affects the incidence of HNSCC, but there are different patterns in different tumour sites. However, socio-economic status does not affect the incidence of a second primary. The association of HNSCC with carcinoma of the bladder is a new finding.     

Retroviral p15E-related serum factors and recurrence of head and neck cancer

Surgical removal of head and neck squamous cell carcinoma (HNSCC) restores the defective monocyte polarization found in patients with HNSCC. Since HNSCC contain p15E-like low molecular weight factors < 25 kD (LMWF) capable of suppressing N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced monocyte polarization, it is likely that HNSCC removal eradicates the production site of p15E-like factors. This report describes a prospective follow-up study on the levels of bioactive p15E-like serum factors for a period of 2 years in nine patients with HNSCC who had no recurrence and 11 patients with HNSCC who showed residual or recurrent disease after treatment. In the group of patients without recurrent disease p15E-like bioactivity gradually decreased and eventually became negative. In patients with recurrent/residual disease p15E-like bioactivity remained high or even became positive before or at the time of diagnosing tumour recurrence. This study strongly supports the concept that HNSCC tumours are the production site of p15E-like immuno-suppressive factors and indicates that serum p15E-like factors may be used for future studies on early serum markers for recurrent/residual disease developing in the first year after treatment.     

Evaluation of the primary tumour and the metastatic lymph node with or without extracapsular spread by means of argyrophillic nucleolar regions (AgNOR)

Though TNM staging is the most popular clinical system for defining the prognosis of Head and Neck Squamous Cell Carcinoma (HNSCC), different clinical trials of the tumours with the same TNM stage have caused doubts about this system. Extra Capsular Spread (ECS) in a metastatic lymph node is one of the recently defined prognostic factors in HNSCC. The hypothesis of this study was to assess the relationship between the primary tumour of laryngeal and oral cavity squamous cell carcinoma and the metastatic lymph nodes with and without ECS. The argyrophillic Nucleolar Organiser Regions (AgNOR) count was used as an index of the grade of malignancy of the neoplastic tissue. As a result, significantly higher AgNOR counts were obtained in the metastatic lymph nodes with ECS than in the primary tumours, while the lowest AgNOR counts were found in the metastatic lymph nodes without ECS. However, when primary tumours of the metastatic lymph nodes with ECS and the primary tumours of the metastatic lymph nodes without ECS were compared, no significant difference was found.     

Activity of differentiation-inducing agents and conventional drugs in head and neck cancer xenografts

Three putative differentiation inducing agents and five conventional drugs which have been shown to be active in patients with squamous cell carcinoma of the head and neck (HNSCC), were tested for activity against HNSCC transplanted in nude mice. Drugs were administered at a maximum tolerated dose level. By testing the effect of conventional drugs the value of the nude mouse xenograft model for testing new drugs was assessed. Bleomycin as well as cisplatin showed antitumour effects in nude mice, although toxicity and thereby the effectiveness of cisplatin varied during the 5-year period in which the experiments were performed. Bleomycin caused responses in 4 out of 13 tumour lines and cisplatin, when administered at a high dose, was active in 2 out of 5 tumours. 5-Fluorouracil and cyclophosphamide were only moderately active, while methotrexate was inactive. These data indicate that the model might be of value in the detection of new anticancer drugs although it may have a tendency to underestimate the activity of some drugs. We used the nude mouse xenograft model to test the antitumour activity of three differentiation inducing agents, the polar-planar solvents hexamethylene bisacetamide (HMBA) and N,N-dimethylformamide (DMF) and the antimetabolite 5-aza-2'-deoxycytidine (5-aza-dCyd). HMBA appeared to be inactive. In contrast, DMF was active in 1 out of 4 tumour lines while 5-aza-dCyd was active in 2 out of 5 lines tested. Furthermore, the whole panel of differentiation inducers and conventional drugs was tested for antitumour activity against three HNSCC tumour lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

鉴定头颈部鳞癌中异常甲基化的差异表达基因及其通路

目的 基于头颈部鳞癌(HNSCC)的生物标记物及其通路尚不明确的现状,研究旨在分析和鉴定HNSCC中异常甲基化的差异表达基因,探讨其关键基因和潜在通路。 方法 从GEO数据库下载基因表达的数据集GSE107591和甲基化数据集GSE33202。通过R软件筛选异常甲基化基因和差异表达基因,两者取交集后获得低甲基化高表达基因(Hypo-HGs)和高甲基化低表达基因(Hyper-LGs)。利用Enrichr对两组基因进行功能富集分析。蛋白互作(PPI)网络由STRING构建并在Cytoscape中可视化,最后利用生存分析来鉴定出关键基因。此外,还进行了免疫组化分析,利用CMap寻找可能逆转HNSCC基因表达的候选小分子。 结果 共鉴定出28个低甲基化高表达基因,GO富集分析显示其主要参与T细胞趋化性的正调节,表皮发育、细胞-基底连接组件及调节T细胞趋化性等方面。Wiki通路分析的结果表明,其主要参与典型和非典型TGF-β信号传导、血液凝结级联反应、α6β4信号通路、补体和凝血级联反应及癌症中的衰老和自噬途径。同时,发现了24个高甲基化低表达基因,主要富集于血管生成及发育的调节,对干扰素-γ反应的负调节,对干扰素-γ介导的信号通路的负调节和上皮发育的生物学过程。Wiki通路分析显示其主要参与哺乳动物含黄素单加氧酶(FMOs)的催化循环,HIF1A和PPARG调节糖酵解以及苯和黄曲霉毒素B1的代谢。此外,鉴定出与HNSCC预后相关的关键基因,分别是SERPINE1、PLAU、MMRN1、LAMB3、LAMC2、PDPN和CXCL13。 结论 通过生物信息学分析并鉴定出HNSCC中异常甲基化差异表达的基因和作用途径,为揭示HNSCC发病机制提供了重要的分子学基础。包括SERPINE1、PLAU、MMRN1、LAMB3、LAMC2、PDPN和CXCL13在内的关键基因可能作为基于甲基化的异常生物标志物,为未来寻找HNSCC诊断和治疗靶点提供了新的思路。     

How we do it: Chemo‐electroporation in the head and neck for otherwise untreatable patients

? Chemo‐electroporation therapy with bleomycin is a locoregional treatment modality for head and neck and skin cancer, with the potential to preserve function. ? In our institution, chemo‐electroporation therapy is used for patients that can no longer be treated by surgery or radiotherapy, or for whom surgical treatment would be very extensive and thus declined by the patient. ? This paper describes in detail the technique of bleomycin‐electroporation therapy. The literature is reviewed and preliminary results of the clinical trial are presented. ? The main focus of the trial is to determine the safety, effectiveness, and burden of bleomycin‐electroporation therapy for the patient. ? All 17 tumours responded to therapy. Local tumour control was reached in 14 of the 17 (82.4 %) tumours. ? Based on the outcome of the clinical trial, bleomycin‐electroporation therapy has the potential to become a valuable addition to the late‐stage treatment options for patients with head and neck or skin tumours.     

Circulating angiogenic cytokines as tumour markers and prognostic factors in head and neck squamous cell carcinoma

This pilot study investigated the potential use of three circulating angiogenesis-related cytokines, basic fibroblast growth factor (bFGF), angiogenin (ANG) and endostatin, as tumour markers and prognostic factors in patients with head and neck squamous cell carcinoma (HNSCC). A total of 30 patients with HNSCC treated with curative intent and 15 healthy controls were studied. Serum (bFGF and ANG) and plasma (endostatin) was assayed by enzyme-linked immunoabsorbance assay (ELISA). None of the cytokines was raised in HNSCC patients when compared with controls. Serum bFGF was not associated with any clinico-pathological or outcome parameters, although there was a trend towards higher levels in more advanced and aggressive tumours. Lower serum angiogenin (sANG) levels were associated with loco-regional disease recurrence (P = 0.036). Using a cut-off level of 400 pg/mL, a low level of sANG predicted tumour recurrence with a relative risk of 4.0 (95% CI: 0.7-24.0). Plasma endostatin was associated with higher histological grade (P = 0.01) and with both disease recurrence (P = 0.045) and death from disease (P = 0.021). Plasma endostatin above a cut-off point of 70 ng/mL could predict tumour recurrence with a relative risk of 4.7 (95% CI: 1.1-19.7). These data suggest that plasma endostatin and sANG have potential roles as prognostic factors and require further investigation.     

Conservative management of vestibular schwannomas: third review of a 10‐year prospective study

• ? Seventy‐two patients with a unilateral vestibular schwannoma have been treated conservatively for a median of 121 months. They have been followed prospectively by serial clinical examination, MRI scans and audiometry.
• ? Twenty‐five patients (35%, 95% CI: 24–47) failed conservative management and required active intervention during the study. No factors predictive of tumour growth or failure of conservative management could be identified. Seventy‐five per cent of failures occurred in the first half of the 10‐year study.
• ? The median growth rate for all tumours at 10 years was 1 mm/year (range ?0.53–7.84). Cerebellopontine angle tumours grew faster (1.4 mm/year) than intracanalicular tumours (0 mm/year, P < 0.01); 92% had growth rates under 2 mm/year.
• ? Hearing deteriorated substantially even in tumours that did not grow, but did so faster in tumours that grew significantly (mean deterioration in pure tone average at 0.5, 1, 2 and 3 kHz was 36 dB; speech discrimination scores deteriorated by 40%).
• ? Patients who failed conservative management had clinical outcomes that were not different from those who underwent primary treatment without a period of conservative management.

     

Expression of hypoxia inducible factor 1 alpha and Von Hippel Lindau protein in human middle ear cholesteatoma

OBJECTIVE: To evaluate the role of hypoxia in cholesteatoma progression. STUDY DESIGN: Immunohistochemical analysis of paraffin-embedded human specimens. METHODS: Thirteen middle ear cholesteatomas and 10 samples of normal human external ear canal skin were stained immunohistochemically for the presence of hypoxia inducible factor 1 alpha (HIF 1 alpha) and Von Hippel Lindau protein. Specimens were then analyzed semiquantitatively. RESULTS: Staining for both antibodies could be detected in all cholesteatomas (perimatrix and matrix), as well as in the samples of normal human ear canal skin. Cholesteatoma specimens showed statistically significant increased staining when compared with normal human skin and mucosa. The age of patients and relapse surgery affected immunohistochemical staining of HIF 1 alpha and Von Hippel Lindau protein. CONCLUSIONS: Elevated staining intensities for HIF 1 alpha and Von Hippel Lindau protein in cholesteatoma tissue indicated that cholesteatoma may be hypoxic. In addition, our data indicated that relapse cholesteatomas yield a higher degree of hypoxia than ears without surgery.