Induction chemotherapy (IC) followed by radiotherapy (RT) versus cetuximab plus IC and RT in advanced laryngeal/hypopharyngeal cancer resectable only by total laryngectomy-final results of the larynx organ preservation trial DeLOS-II.

Background: The German multicenter randomized phase II larynx organ preservation (LOP) trial DeLOS-II was carried out to prove the hypothesis that cetuximab (E) added to induction chemotherapy (IC) and radiotherapy improves laryngectomy-free survival (LFS; survival with preserved larynx) in locally advanced laryngeal/hypopharyngeal cancer (LHSCC). Patients and methods: Treatment-naïve patients with stage III/IV LHSCC amenable to total laryngectomy (TL) were randomized to three cycles IC with TPF [docetaxel (T) and cisplatin (P) 75 mg/m2/day 1, 5-FU (F) 750 mg/m2/day days 1-5] followed by radiotherapy (69.6 Gy) without (A) or with (B) standard dose cetuximab for 16 weeks throughout IC and radiotherapy (TPFE). Response to first IC-cycle (IC-1) with ≥30% endoscopically estimated tumor surface shrinkage (ETSS) was used to define early responders; early salvage TL was recommended to non-responders. The primary objective was 24 months LFS above 35% in arm B. Results: Of 180 patients randomized (July 2007 to September 2012), 173 fulfilled eligibility criteria (A/B: larynx 44/42, hypopharynx 41/46). Because of 4 therapy-related deaths among the first 64 randomized patients, 5-FU was omitted from IC in the subsequent 112 patients reducing further fatal toxicities. Thus, IC was TPF in 61 patients and TP in 112 patients, respectively. The primary objective (24 months LFS above 35%) was equally met by arms A (40/85, 47.1%) as well as B (41/88, 46.6%). One hundred and twenty-three early responders completed IC+RT; their overall response rates (TPF/TP) were 94.7%/87.2% in A versus 80%/86.0% in B. The 24 months overall survival (OS) rates were 68.2% and 69.3%. Conclusions: Despite being accompanied by an elevated frequency in adverse events, the IC with TPF/TP plus cetuximab was feasible but showed no superiority to IC with TPF/TP regarding LFS and OS at 24 months. Both early response and 24 months LFS compare very well to previous LOP trials and recommend effective treatment selection and stratification by ETSS. Clinical trial information: NCT00508664.

Transformation of the structure of collagen. A time-resolved analysis of mechanochemical processes using synchrotron radiation.

Mechanochemically induced molecular transformations of collagen fibres were analysed using time-resolved small-angle diffraction spectra and histomechanical measurements. In particular, the influence of aqueous and methanolic perchlorate solutions was examined. According to a transformation continuing from the periphery towards the centre, the macroscopic contraction that is completed less than five minutes after incubation with perchlorate is caused by peripherally transformed fibrils only, whereas the centrally situated fibrils first undergo an accordion-like folding, but after more than 20 minutes are transformed similarly. The triple-helical transformation is preceded by a structure-breaking effect on structural water that can be monitored in time-resolved diffraction spectra. The combined loss of meridional low-angle reflections and cross-striated fibrils in micrographs is irreversible. By dialysis of colloidally dissolved collagen against a solution of ATP, however, segment-long spacing aggregates are obtained. Under isometric conditions, an instantaneous transformation of intermittent regions leads to an increase in the long period of adjacent, still structured regions of the same fibril that is correlated with a delayed increase in tension in the fibre. Increase of tension under isometric conditions as well as the flow-properties of a fibre relaxed in perchlorate are interpreted in terms of the parallel sliding of subunits of varying lengths, which has been demonstrated by diffraction analysis.

Development of the human submandibular salivary gland.

The development and morphogenetic timetable of the submandibular gland was studied in 37 human embryos and human fetuses. The medial paralingual groove constituted the anlage of the submandibular gland: Its anterior three-quarters gave rise to Wharton's duct, and its posterior quarter to the submandibular gland proper. The sublingual process of the submandibular gland originated from a lateral ectodermal bud of the anlage of the submandibular gland, in the posterior quarter of the medial paralingual groove.

Aberrant expression of epidermal growth factor receptor in aural cholesteatoma.

A monoclonal antibody recognizing an epitope of the external domain of the human epidermal growth factor (EGF) receptor was used in an alkaline phosphatase-antialkaline phosphatase (APAAP) technique to compare the distribution of this protein in normal human skin and aural cholesteatoma. EGF receptors appear to be highly expressed on the basal layer of the epidermis, in hair follicle apocrine sweat glands, and in the capillary system of normal skin. Cholesteatoma epithelium showed increased positive reactions in the suprabasal layers. A heterogeneity in the expression was found in different parts of the cholesteatoma. These results suggest the presence of an aberrant regulation and persistence of EGF receptors in cholesteatoma and confirm the hyperproliferative character of the cholesteatoma epithelium.

[The value of computer tomography in the diagnosis of congenital spinal malformations particularly diastematomyelia (author's transl)]. / Die Bedeutung der Computertomographie in der Diagnostik kongenitaler spinaler Missbildungen, insbesondere der Diastematomyelie.

The value of computer tomography (CT) in the diagnosis of diastematomyelia (DM) was studied in our own clinical material. Thirty-one patients with congenital spinal anomalies were examined; in 21 a DM could be demonstrated. CT has two major advantages in the radiological investigation of spinal abnormalities, such as spinal dysraphism and DM: 1. Cross sections of the spinal canal can be obtained without overlying opacities and 2. The demonstration of intraspinal soft tissues structures, corresponding to their attenuation characteristics and the relative differences in absorption of the x-rays.

Localization of transforming growth factor-beta-expressing cells and comparison with major extracellular components in aural cholesteatoma.

Transforming growth factor-beta (TGF-beta) plays an important role in the regulation of extracellular matrix (ECM) deposition by stimulating the synthesis of individual matrix proteins like tenascin and fibronectin. Cholesteatoma shows significant changes in the ECM, supporting the view of a disturbed cell-matrix interaction. The purpose of our present study was to evaluate the distribution of TGF-beta in comparison to the deposition of tenascin, fibronectin, and collagen as major components of the ECM in cholesteatoma (n = 12) by means of histochemistry and immunohistochemistry. We found TGF-beta in lymphocytes and fibrohistiocytes in the stroma of 7 cholesteatomas. In corresponding sections, a marked expression of tenascin and fibronectin was seen manifesting as a continuous band along the epidermal-stromal junction, extending to the deeper stroma. In addition, in those cases of TGF-beta expression, beginning collagen fibril formation was seen in adjacent deeper stroma layers, indicating beginning stromal fibrosis. These results suggest that TGF-beta may be involved in the stimulation of the synthesis of tenascin, fibronectin, and collagen. Furthermore, the enhanced expression of tenascin and fibronectin provides evidence for a deregulated cell-matrix interaction in cholesteatoma associated with the enhanced proliferative process of cholesteatoma formation.

Hyperproliferation-associated keratin expression in human middle ear cholesteatoma.

Cholesteatoma is characterized by the presence of a squamous epithelium invading the middle ear altering its growth properties. This epithelium is believed to have hyperproliferative properties. Keratin 16 is accepted as a molecular marker for hyperproliferative epithelia. Two monoclonal antibodies K8.12 (directed against keratin 13) and KS.1A3 (directed against keratin 13 and 16) were used in an alkaline phosphatase anti-alkaline-phosphatase (APAAP)-technique to compare the expression of both keratin 13 and keratin 16 in normal human skin and aural cholesteatoma. Furthermore, the cytokeratin expression was compared to that of normal skin and palatine tonsil using one-dimensional gel electrophoresis. For both monoclonal antibodies, normal ear skin was stained only in the basal layer. In contrast, in the cholesteatoma samples the immunostaining of the antibody KS-1A3 was done not only in the basal cell layer but also in the suprabasal cells of the stratum spinosum and stratum granulosum. Using gel-electrophoresis, the presence of cytokeratin 16 was demonstrated in the cholesteatoma samples only. These results support the hyperproliferative character of cholesteatoma epithelium.

Overexpression of tenascin in cholesteatoma and external auditory meatal skin compared to retroauricular epidermis.

In the present study the distribution of tenascin in cholesteatoma was immunohistochemically investigated. The results were compared with those in external auditory meatal skin and in retroauricular skin of healthy controls. The staining pattern was additionally correlated to the degree of cell proliferation as detected by the monoclonal antibody MIB-1 (Ki-67 antigen). Retroauricular skin showed a limited distribution of tenascin in the papillary dermis and a sparse reactivity of MIB-1 in only a few epithelial cells. External auditory meatal skin revealed a more pronounced reaction for tenascin and MIB-1. In contrast, cholesteatoma tissue exhibited an abundant and continuous expression of tenascin covering the whole stroma compartment. This coincided with a significant increase of MIB-1-positive cells in the basal and suprabasal epithelial layers. Doublestaining experiments revealed most prominent stromal tenascin-expression in areas with marked signs for epithelial proliferation. This suggests that tenascin is selectively increased in response to epidermal hyperproliferation. This matrix protein thus shows a quantitatively and qualitatively enhanced expression under pathological conditions. Moreover, the abundant reactivity for tenascin in the cholesteatoma provides evidence of a deregulated cell-matrix interaction involved in the hyperproliferative process of cholesteatoma formation.

[Thrombogenic and endothelial damage markers in patients with ischemic systolic impairment]. / Marcadores de hipercoagulabilidad y daño endotelial en pacientes con disfunción sistólica de origen isquémico.

INTRODUCTION: Anticoagulation is rarely indicated in patients with left ventricular dysfunction who show an increased risk for thromboembolism. In theory, the three arms of the Virchow' triad may be present: abnormal blood flow, endothelial damage and prothrombotic markers. The aim of this study was to identify the last two arms. PATIENTS AND METHOD: We studied 82 consecutive patients with demonstrated ischaemic heart disease and sinus rhythm, and compared them with a control group comprised of 32 healthy subjects matched for age and sex. None or the patients had had an acute coronary event or hemodynamic decompensation within the 3 months prior to inclusion in the study. The plasma concentration or von Willebrand factor and fibrin d-dimer and fibrinogen were determined as endothelial damage and prothrombotic markers, respectively. A fractional shortening less than 29% by echography was defined as ventricular systolic dysfunction. RESULTS: The patients showed significantly higher levels of von Willebrand factor with respect to the control group (109.2 31.9 vs 85.5 32.6%, p < 0.01), with no differences in fibrinogen and fibrin d-dimer values. Twenty-six patients fulfilled criteria of left ventricular systolic dysfunction. Patients with left ventricular dysfunction showed higher fibrinogen (386 118 vs 322 102 mg/dl, p = 0.03) and fibrin d-dimer (0.36 0.22 vs 0.26 0.10 g/ml; p = 0.04) levels, with no differences in von Willebrand factor levels. CONCLUSIONS: After acute coronary events, patients with ischaemic heart disease show markers of endothelial damage. However, patients with left ventricular dysfunction show a hypercoagulable state.

[Improvement in fibrinolytic function following anticoagulant treatment in chronic rheumatic atrial fibrillation]. / Mejoría de la función fibrinolítica tras el tratamiento anticoagulante en la fibrilación auricular reumática crónica.

INTRODUCTION AND OBJECTIVES: Patients with rheumatic atrial fibrillation are considered at high risk of systemic embolism and require oral anticoagulation. Fibrinolytic function has been little studied. We evaluated fibrinolytic activation markers before starting anticoagulation, at 1 and 6 months following the introduction of oral anticoagulation therapy. We analyzed the relationship with left atrial diameter and mitral area. METHODS: Tissue plasminogen activator (tPA), its inhibitor (PAI-1), plasmin-antiplasmin complexes (PAP) and D-dimer were measured in 13 patients with rheumatic atrial fibrillation. Basal levels were compared with those found in plasma of 20 healthy subjects matched by sex and age. Transthoracic echocardiography was made. RESULTS: A significant increase for PAI-1 and D-dimer levels were detected in patients with atrial fibrillation group (p < 0.05), with no differences in tPA and PAP concentrations. Significant correlation between left atrial diameter and basal t-PA levels was found. Levels of t-PA, PAI-1 and D-dimer decreased significantly under anticoagulation therapy, whereas PAP levels were significantly increased. CONCLUSIONS: Patients with rheumatic atrial fibrillation show a relative hypofibrinolytic state due to elevated PAI-1 levels with no increase in PAP concentration. At six months of anticoagulation therapy, an improvement of fibrinolytic function markers was observed. This is consistent with the prophylactic effect of oral anticoagulants therapy against thromboembolic risk.

[Set up of a protocol for heparin use in special patients]. / Establecimiento de un protocolo para el uso de la heparina en pacientes con características especiales.

Low-molecular weight (LMW) heparins bring a series of advantages as compared to non-fractionated heparin (NFH), such as safety, efficacy, bioavailability, fewer monitoring, and persistent anti-coagulant response. There exist, however, a concern about their use in particular patients that may require a special control, such as those with renal failure, age over 75 years, obesity, and pregnancy. The aim of this study was the set up between the department of Pharmacy, Hematology, and Internal Medicine of a consensus protocol for the follow-up ad monitoring of LMWH in patients requiring a special control. For this purpose, we carried out a bibliographical review of the different heparins used under de above mentioned conditions. Based on the evidence available and the consensus among the members of the working group, we established a protocol that contained recommendations on prophylaxis, management and monitoring by means of the determination of anti-Xa factor. Besides, we included some clues on the therapeutic figures of anti-Xa and administration schedules for obtaining anti-Xa values within the range. Enoxaparin was the selected heparin given the evidence and its availability at our center.

Las HBPMs (heparina de bajo peso molecular) tienen numerosas ventajas sobre la heparina no fraccionada (HNF) como seguridad, eficacia, biodisponibilidad, menor monitorización y una respuesta anticoagulante persistente. Pero, existe cierta preocupación en su manejo para determinados pacientes que requieren un control especial como en insuficiencia renal, mayores de 75 años, obesidad y embarazo. El objetivo de este estudio fue la realización de un protocolo consensuado entre los Servicios de Farmacia, Hematología y Medicina Interna, para el seguimiento y monitorización de HBPM en pacientes que requieren un especial control. Para ello, llevamos a cabo una revisión bibliográfica de las distintas heparinas en las situaciones comentadas. Basándonos en la evidencia disponible y en el consenso entre los miembros del grupo de trabajo, elaboramos el protocolo, recomendando unas dosis para profilaxis, tratamiento y monitorización, mediante la determinación del factor anti-Xa. Además, recogemos unas orientaciones sobre los valores terapéuticos del anti-Xa y unas pautas posológicas para la obtención de un anti-Xa en rango. La heparina seleccionada fue la enoxaparina, por su evidencia y disponibilidad en nuestro centro.

DNA replication stress response involving PLK1, CDC6, POLQ, RAD51 and CLASPIN upregulation prognoses the outcome of early/mid-stage non-small cell lung cancer patients.

Lung cancer is the leading cause of cancer deaths worldwide. Clinical staging classification is generally insufficient to provide a reliable prognosis, particularly for early stages. In addition, prognostic factors are therefore needed to better forecast life expectancy and optimize adjuvant therapeutic strategy. Recent evidence indicates that alterations of the DNA replication program contribute to neoplasia from its early stages and that cancer cells are frequently exposed to endogenous replication stress. We therefore hypothesized that genes involved in the replication stress response may represent an under-explored source of biomarkers. Expressions of 77 DNA replication-associated genes implicated in different aspects of chromosomal DNA replication, including licensing, firing of origins, elongation, replication fork maintenance and recovery, lesion bypass and post-replicative repair were determined in primary tumors and adjacent normal tissues from 93 patients suffering from early- or mid-stage non-small cell lung cancer (NSCLC). We then investigated a statistically significant interaction between gene expressions and survival of early-stage NSCLC patients.The expression of five genes, that is, POLQ, PLK1, RAD51, CLASPIN and CDC6 was associated with overall, disease-free and relapse-free survival. The expression levels are independent of treatment and stage classification. Except RAD51, their prognostic role on survival persists after adjustment on age, sex, treatment, stage classification and conventional proliferation markers, with a hazard ratio of 36.3 for POLQ (95%CI 2.6-517.4, P=0.008), 23.5 for PLK1 (95%CI 1.9-288.4, P=0.01), 20.7 for CLASPIN (95%CI 1.5-275.9, P=0.02) and 18.5 for CDC6 (95%CI 1.3-267.4, P=0.03). We also show that a five-gene signature including POLQ, PLK1, RAD51, CLASPIN and CDC6 separates patients into low- and high-risk groups, with a hazard ratio of 14.3 (95% CI 5.1-40.3, P<0.001). This 'replication stress' metamarker may be a reliable predictor of survival for NSCLC, and may also help understand the molecular mechanisms underlying tumor progression.

[Infusion systems for intra-arterial chemotherapy]. / Infusionssysteme für die intraarterielle Chemotherapie.

A short description of the standard devices with extra-corporal or intracorporal pumps is followed by a description of a new extracorporal pump. Since the new pump is independent of electric mains supply, it is more comfortable for the patient, who can walk around even during intraarterial infusion. However, as the flow rate of the pump is low, very thin catheters have to be used to avoid clotting caused by blood reflux within the catheter.

[Objective gustometry with adequate stimulation by registration of contingent negative variation]. / Objektive Gustometrie mit adäquaten Reizen durch Registrierung der Contingent Negative Variation.

Cortical evoked potentials require a steep and temporally reproduceable onset of sensory stimulation. As both items are difficult with gustatory stimuli, we tried to record the slow expectancy potential or "contingent negative variation" (CNV), which has turned out to be useful in objective olfactometry and speech audiometry. Sour, sweet, bitter or salty fluids were embedded in a water stream, which was sucked through a device well fitting to the tongue with holes at adequate locations. The negative vertex potential developped reproduceably between the recognition of taste and the acoustic signal, which could be expected only after one of the two alternatively offered gustatory stimuli. Clinical use of the method is demonstrated in a case of asymmetric loss of smell due to interruption of the chorda tympani during cholesteatoma operation. The CNV appeared only when stimulating the side of the tongue opposite to the ear operated on. In addition to the total or unilateral loss of taste, the ability to differentiate between two gustatory qualities can be diagnosed with an objective method, provided the patient cooperates by calm and attentive behaviour.

[Hyperproliferation-associated keratin 16 expression in cholesteatoma]. / Hyperproliferationsassoziierte Keratin 16-Expression im Cholesteatom.

The position of a cell in a stratified squamous epithelium correlates with its state of differentiation as well as with the expression of certain cytokeratins. By means of both immunohistochemistry using the APAAP method and high resolution one-dimensional gel electrophoresis, specimens of cholesteatoma epithelium and normal ear skin were investigated whether they contain keratin 16 (K 16) which is known to be a marker of hyperproliferation. It could be shown that K 16 is expressed in all layers of cholesteatoma epithelium. Further to prove this fact gel electrophoresis was used showing a protein band at the localization of keratin 16. Hence, cholesteatoma can be seen as a locally hyperproliferative disease.

[Autocrine growth mechanisms of cholesteatoma epithelium]. / Autokrine Wachstumsmechanismen des Cholesteatomepithels.

Transforming growth factor alpha (TGF alpha) and interleukin 1 alpha (IL-1 alpha) are known to be produced by normal human keratinocytes stimulating their proliferation. The distribution and expression of TGF alpha and IL-1 alpha were examined in specimens of middle ear cholesteatoma by means of immunohistochemical methods using a monoclonal antibody against TGF alpha and a polyclonal one against IL-1 alpha. Normal retroauricular skin was stained for comparison. Staining for TGF alpha was consistently stronger in cholesteatoma epithelium than in normal epidermis, and encompassed all epithelial cell layers. Immune cells occurring in the stroma of cholesteatoma also reacted positively for TGF alpha. The intensity of staining for IL-1 alpha was markedly stronger in cholesteatoma tissue than in normal epidermis. All cellular layers of the squamous epithelium of cholesteatoma stained strongly and uniformly for IL-1 alpha, whereas the keratin layer was negative for IL-1 alpha. In the connective tissue beneath the cholesteatoma epithelium intensely positive cells were scattered between negative stromal cells. These data are consistent with autocrine stimulation of the squamous epithelium of cholesteatoma by TGF alpha and IL-1 alpha as well as with a paracrine stimulation by immune cells. Both factors contribute to the unrestrained growth of cholesteatoma in the middle ear cavity.

[Interleukin-1-containing cells in cholesteatoma of the middle ear]. / Interleukin 1-enthaltende Zellen im Cholesteatom des Mittelohres.

Cholesteatoma of the middle ear and the adjacent temporal bone consists of hyperproliferative keratinizing squamous epithelium in the middle ear cavity, and is capable of destroying the bone. Interleukin-1 (IL-1), an autocrine growth factor for epithelial keratinocytes, is characterized by its capacity to initiate bone absorption. Using immunohistochemical methods, the distribution of two different species of interleukin, IL-1 alpha and IL-1 beta, in cholesteatoma tissue (Fig. 2), the skin of the external ear canal, and the retroauricular region was investigated (Fig. 1). Comparable amounts of both IL-1-species were found in all squamous epithelia examined, but interleukin in cholesteatoma epithelium was increased in comparison with normal epidermis. All cellular layers stained uniformly and equally strongly for IL-1 alpha and IL-1 beta, whereas the dead cells of the keratin layer were negative for both. Some intensely stained cells were found scattered in the connective tissue underlying the basal layer of the cholesteatoma (Fig 4). Using double staining techniques these cells were shown to be mainly macrophages (Fig 6). Our results suggest that IL-1 could be liberated from disintegrating keratinocytes and cells of the monocyte-/macrophage lineage, and stimulate the proliferation of the cholesteatoma epithelium in an autocrine manner, thus contributing to the increased bone destruction seen in cholesteatoma.

[Immunohistochemical identification of cholesteatoma-associated macrophage populations]. / Immunhistochemische Identifizierung Cholesteatom-assoziierter Makrophagen-Populationen.

Extensive bone resorption occurring in aural cholesteatoma is responsible for the severe complications of this disease. In the area of active bone destruction, typical multinucleated osteoclasts are rarely seen, but a heavy cellular infiltrate is found. In the present study we tried to characterize the immunophenotype and the functional state of the cells infiltrating the stroma and the epithelial layer of aural cholesteatoma, using a panel of monoclonal antibodies directed against cell type specific antigens. The results were compared with normal retroauricular skin. The vast majority of cells infiltrating the stroma was bone marrow derived and consisted of T-cells and macrophages. By means of the activation markers HLA-DR and Interleukin-2 receptor an immunologically activated state of the majority of infiltrating cells in cholesteatomas was shown. The great number of activated macrophages in cholesteatomas seems to be very important in the cholesteatomatous immunological process. Because of their various immunological functions (antigen presentation to T-lymphocytes, participation in ingestion and killing of different invading microorganisms and synthesizing a great number of substances involved in host defence and inflammation) these cells play a central role in human immunological system. Langerhans cells, however, did not appear to be involved in the immune process of cholesteatoma. The characteristics of the infiltrating cell population with the great number of phagocytic cells suggest an active immune process resulting in autoaggressive bone resorption.

Immunologically activated cells in aural cholesteatoma.

In this immunohistochemical study, we characterized the cells infiltrating the stroma of acquired aural cholesteatomas in detail, using a panel of monoclonal antibodies directed against immune cell type-specific antigens, HLA class II antigens, and interleukin-2 receptor. For all antibodies used, normal ear skin was stained for comparison. The vast majority of the infiltrating cells was CD45-positive, ie, derived from bone marrow. Reactivity with anti-CD3 and anti-CD6 antibodies revealed an abundant infiltration of T lymphocytes beneath the squamous epithelium of cholesteatoma. The B lymphocyte-specific anti-CD19 and anti-CD22 antibodies detected only occasional positive cells. Hence, the cellular infiltrate in the stroma of aural cholesteatoma is made up primarily of T cells with macrophages scattered between them. Expression of HLA-DR was almost as high as that of CD45, whereas CD25-positive cells were detected in lower amounts. We infer that the majority of T cells and macrophages in the stroma of cholesteatoma are in an immunologically activated state. The characteristics of the infiltrating cell population suggest an antigen-driven process in cholesteatoma.

[New aspects on the pathogenesis of cholesteatoma: the possible role of immune cell-induced keratinocyte hyperproliferation]. / Neue Aspekte zur Pathogenese des Cholesteatoms: Die mögliche Rolle einer durch Immunzellen induzierten Keratinozyten-Hyperproliferation.

Cholesteatoma of the middle ear consists of keratinising squamous epithelium in the middle ear cavity. Many points of the pathogenesis of cholesteatoma seem to be related to immune cell infiltrates in stroma and to epithelial cell migration and proliferation. In our study we found that the vast majority of cells infiltrating the stroma consisted of T-cells and macrophages, showing an immunologically activated state. Furthermore, the cholesteatoma epithelium showed an enhanced simultaneous expression of keratine-16 and Ki-67 positive cells. This expression was accompanied by the over-expression of transforming growth factor (TGF)-alpha, and its receptor, epidermal growth factor receptor (EGF-R) and interleukin-1, suggesting that the proliferation of keratinocytes could be stimulated in an autocrine manner. Finally, we conclude that the presence of immunologically activated immune cells in the stroma may be responsible for keratinocyte dysregulation in cholesteatoma epithelium.