Cytological and Immunocytological Monitoring of Oropharyngeal Dysplasia and Squamous Cell Carcinomas.

BACKGROUND/AIM: Due to the high recurrence rates of squamous cell carcinoma of the head and neck (SCCHN) and de-novo local secondary carcinomas, a close monitoring of patients is mandatory. In doubtful cases, a clearance by histological biopsy is necessary. This, however, bears potential complications. We analyzed the application of combined cytology and immunocytology in cytological brush smears for diagnosing pre-malignant and malignant lesions of the oral/oropharyngeal cavity. MATERIALS AND METHODS: Brush biopsies of 30 subsequently histologically-confirmed oral/oropharyngo-/laryngeal cavity cancer cases (all then in a recurrence status) and normal mucosa were obtained for routine cytology and immunocytology for cytokeratin-8 (CK-8). Additionally 20 samples with inflammatory lesions were investigated. RESULTS: Our results showed a high rate for positive prediction of oral/oropharyngo-/laryngeal dysplasia/cancer cases. Accordingly, 82% of all subsequently confirmed cases were detected by cytology alone (sensitivity). The specificity, however, of cytology was distinctly lower since several doubtful cases contained only inflammatory lesions (specificity 85%). The addition of CK-8-immunocytology did not increase the sensitivity, since the rate of detected cases by immunocytology was comparable to routine cytology (79%); however, the addition of immunocytology significantly increased the specificity (up to 90%). CONCLUSION: Routine cytology is a simple, non-invasive and cost-effective method for routine control and screening of dysplastic oral/oropharyngo-/laryngeal lesions. In doubtful cases, the addition of CK-8-immunocytology is very helpful for the distinction of reactive from neoplastic cases.

Expression of the Epithelial Cell Adhesion Molecule and Cytokeratin 8 in Head and Neck Squamous Cell Cancer: A Comparative Study.

The epithelial cell adhesion molecule (EpCAM) is a well-known and widely accepted tumor-associated antigen in head and neck squamous cell carcinoma (HNSCC). In contrast, little is known about cytokeratin 8 (CK8), an intermediary filament protein, recently associated with HNSCC. Studies demonstrated an aberrant expression on the cell surface of different carcinomas of both antigens. We performed an immunohistochemical study on the expression pattern of CK8 in comparison to EpCAM on cryosections, followed by microscopic quantitative and semi-qualitative analyses. Both antigens showed heterogenous expression both in individual carcinomas and between different carcinoma types. Furthermore, the expression of CK8 is clearly dependent on the degree of histological tumor cell differentiation. With increasing de-differentiation, the amount of CK8 expression increased, which was not seen for EpCAM. The expression of EpCAM was high on all carcinomas independent of their anatomical localization. Regarding CK8, there seems to be a correlation between the expression grade and the anatomical site. The application of CK8 may provide additional supplementary information on HNSCC.

Cytokine changes in response to radio-/chemotherapeutic treatment in head and neck cancer.

BACKGROUND: Radiation and systemic chemotherapy are standard treatment strategies for advanced or metastatic head and neck cancer. However, little is known about the implications and changes in the tumor microenvironment, including the T-helper (TH)1/TH2 balance in response to these treatment regimens. The aim of the current study was to unravel the effects of chemotherapeutic drugs and radiation on cytokine changes. MATERIALS AND METHODS: In this study, the effect of radiation and chemotherapeutic treatment (5-fluorouracil and cisplatin) on eight cell lines was determined. Before and after exposure, cytokine levels in culture supernatants of cell lines were evaluated using the Bio-Plex Assay (Bio-Rad) and the Human TH1/TH2 Cytometric Bead Array (Becton Dickinson). Results were correlated with parallel measurements for cellular proliferation assessed by cytotoxicity assay. RESULTS: Seven out of eight cell lines of primary tumors or metastases demonstrated an enhanced level of the cytokines interleukin (IL)-1ß, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α), after sub-lethal radiation doses. Under treatment with low concentrations of 5-fluorouracil and cisplatin, all examined cell lines showed an increasing secretion of the cytokines IL-6 and G-CSF. In contrast, sub-lethal doses of both cytostatic drugs revealed a dose-dependent decrease in secretion IL-1ß. Regarding GM-CSF and TNF-α, we demonstrated an increase in secretion by the primary tumors under low doses of 5-fluorouracil and cisplatin, whereas the metastases showed a sharp drop of GM-CSF and TNF-α secretion. Chemotherapeutic treatment led to no changes of the IL-8 cytokine profile. CONCLUSION: The results suggest complex cytokine changes of the tumor microenvironment and more aberrant expression profiles under treatment with radiation and the chemotherapeutic drugs 5-fluorouracil and cisplatin.

Biodistribution of 131I-labeled anti-CK8 monoclonal antibody in HNSCC in xenotransplanted SCID mice.

BACKGROUND: A new promising approach to improve the outcome of head and neck squamous cell carcinoma (HNSCC) is the application of radio-labeled antibodies directed against tumor-associated antigens. Cytokeratin 8 (CK8), an intermediate filament forming protein, is shown to be de novo expressed in dysplastic lesions as well as in HNSCC. Therefore like the epithelial cell adhesion molecule CK8 seems to be a suitable anchor molecule for targeted radioimmunotherapy (RIT). The aim of this study was to investigate the biodistribution of a radio-labeled Cytokeratin 8-specific monoclonal antibody (mAb) in a SCID (severe combined immunodeficiency disease) mouse model. MATERIALS AND METHODS: The mAb against CK8 was labeled with (131)I and biodistribution was tested in established HNSCC xenografts in SCID mice. The biodistribution of the mAb in the tumor and different organs was determined with a gamma counter and was calculated as % injected dose/gram tissue. RESULTS: Initially, after systemic administration of (131)I-anti CK8 monoclonal antibody high activity was seen in all the organs. Over time the general activity decreased, whereas activity accumulated in the tumor. This activity decayed compared to the other tissues with a two- to threefold prolonged radioactive half-life. CONCLUSION: Specific antibody-antigen-binding is probably responsible for the prolonged radioactive half-life in the tumor and the resulting cumulative activity due to enrichment of the (131)I-anti CK8 mAb, so that Cytokeratin 8 seems to be a suitable anchor molecule for radioimmunotherapy in HNSCC.

Comparison of two different local anaesthetic infiltrations for postoperative pain relief in tonsillectomy: a prospective, randomised, double blind, clinical trial.

In previous studies, it was shown that the post-tonsillectomy wound infiltration of bupivacaine can reduce postoperative pain. The objective of this study is to determine whether the postoperative wound infiltration with a mixture of bupivacaine, mepivacaine and adrenaline is more effective than the sole application of bupivacaine. A prospective, double-blind, randomized, control study included 30 patients scheduled for "cold steel" tonsillectomy. All patients obtained post-tonsillectomy infiltration of 6.25 mg bupivacaine alone on one side and 3.75 mg bupivacaine, 25 mg mepivacaine and 0.0125 mg epinephrine on the other side (intra-individual study design). Intake of analgesics and postoperative pain was assessed 0-6 days after surgery by visual analogue scale in inactivity and during swallowing by the nurse staff. Bleeding, dysphagia, pain, aspiration or extraordinary pain sensation were registered by the patient. The pain scores did not differ between the groups. All patients received systemic painkillers; 6 (20%) patients needed intravenous analgesics. Postoperative haemorrhage occurred in two patients without correlation to a certain local anaesthetic. Two patients developed sinus tachycardia for 2.5 min after epinephrine infiltration. Because of cost-effectiveness and complication rates, we recommend only post-tonsillectomy wound infiltration of bupivacaine. The injection should be placed in superficial muscle and connective tissue. A stringent systemic analgesia regime is indispensable for pain relief after tonsillectomy.

Application methods of local anaesthetic infiltrations for postoperative pain relief in tonsillectomy: a prospective, randomised, double-blind, clinical trial.

Perioperative local anaesthetics are often used to reduce the postoperative pain in tonsillectomy. There exist three different ways of applying local anaesthetics: (1) pre-incisional peritonsillar; (2) post-tonsillectomy wound infiltration; (3) post-tonsillectomy packing with soaked gauze. The objective of the study is the evaluation of differences of pain reduction comparing the three different techniques of application. The study design mainly includes intra-individual, prospective and double-blinded. One hundred and eighty patients (3-45 years) with recurrent tonsillitis were included. The charts of 156 were eligible for analysis. Bupivacaine was applied on both sides randomized in different ways. Pain on each side was registered for 6 days on the ward by a blinded nurse. When directly compared with the other two application methods, the post-tonsillectomy injection of bupivacaine provides significantly better results during the monitored time period. Postoperative bleeding was observed in 11 (7.3%) cases without any correlation to an application procedure. No other adverse effects were observed. In conclusion, post-tonsillectomy infiltration of the wounds with bupivacaine is superior to pre-incisional infiltration technique as well as post-tonsillectomy packing of the wounds with 0.5% bupivacaine-soaked gauze swab.

Biodistribution and radioimmunotherapy of SCCHN in xenotransplantated SCID mice with a 131I-labelled anti-EpCAM monoclonal antibody.

BACKGROUND: The mortality from squamous cell carcinoma of the head and neck (SCCHN) remains high and almost unchanged throughout the last decades. Therefore, new therapeutic strategies are urgently needed. One promising approach is the application of radio-labeled antibodies directed against tumor-associated antigens. EpCAM is a transmembrane protein, which is overexpressed on almost all SCCHN, making it a suitable anchor molecule for targeted radioimmunotherapy (RIT). The aim of this study was to establish an animal model to investigate the biodistribution and the therapeutic effect of a radio-labeled EpCAM-specific monoclonal antibody (mAb). MATERIALS AND METHODS: The mAb C215 was labeled with 131I and tested for its antitumor effect against established SCCHN xenografts in SCID mice. Initially, the biodistribution of the mAb in the tumor and different organs was determined with a gamma counter and was calculated as % injected dose/gram tissue. For therapeutic approaches 5, 15 or 25 MBq 131I-labeled mAb was injected as a single bolus into tumor-bearing mice. Control animals received either sodium chloride or the unlabeled mAb. The tumor growth and body weight of the animals were measured at various times after administration of the antibody. RESULTS: Initially, high activity was seen in all organs after systemic administration of 13I-C215. Over time general activity decreased whereas an accumulation of activity was seen in the tumor. Tumor growth was delayed in the groups receiving either 15 MBq or 25 MBq 131I-C215 relative to control groups and the 5 MBq group. However, animals in the high-dose groups suffered from treatment-related toxicity, which led to body weight loss of more than 20%. CONCLUSION: Our data demonstrate that the EpCAM-specific radio-labeled mAb C215 is a promising tool to target SCCHN leading to significant tumor control. Further studies are necessary to increase efficacy and reduce toxicity of this new therapeutic approach.

Immune restoration in head and neck cancer patients after in vivo COX-2 inhibition.

PURPOSE: To determine the immunomodulatory effects of in vivo COX-2 inhibition on leukocyte infiltration and function in patients with head and neck cancer. EXPERIMENTAL DESIGN: Patients with squamous cell carcinoma of the head and neck preoperatively received a specific COX-2 inhibitor (rofecoxib, 25 mg daily) orally for 3 weeks. Serum and tumor specimens were collected at the start of COX-2 inhibition (day 0) and again on the day of surgery (day 21). Adhesion to peripheral blood monocytes to ICAM-1 was examined. Percentages of tumor-infiltrating monocytes (CD68, CCR5) and lymphocytes (CCR5, CD4, CD8 and CD25) were determined by immunohistochemistry. RESULTS: Monocytes obtained from untreated cancer patients showed lower binding to ICAM-1 compared to monocytes of healthy donors but significantly regained adhesion affinity following incubation in sera of healthy donors. Conversely, sera of cancer patients inhibited adhesion of healthy donors' monocytes. Tumor monocyte adhesion to ICAM-1 was increased (P<0.001) after 21 days of COX-2 inhibition, and concomitant increases in tumor infiltrating monocytes (CD68+), lymphocytes (CD68- CCR5+, CD4+ and CD8+) and activated (CD25+) T cells were observed. CONCLUSIONS: Short-term administration of a COX2 inhibitor restored monocyte binding to ICAM-1 and increased infiltration into the tumor of monocytes and Th1 and CD25+ activated lymphocytes. Thus, in vivo inhibition of the COX-2 pathway may be useful in potentiating specific active immunotherapy of cancer.

Human ceruminous gland: ultrastructure and histochemical analysis of antimicrobial and cytoskeletal components.

The ceruminous glands in the skin of the human external auditory canal are modified apocrine glands, which, together with sebaceous glands, produce the cerumen, the ear wax. Cerumen plays an important role in the protection of the ear canal against physical damage and microbial invasion. We studied the morphology of the glandular cells by light and electronmicroscopy. Antimicrobial and cytoskeletal components of the ceruminous glands were investigated by immunohistochemical methods. Numerous antimicrobial proteins and peptides are present in the ceruminous glandular cells: beta-defensin-1, beta-defensin-2, cathelicidin, lysozyme, lactoferrin, MUC1, secretory component of IgA. These data indicate a crucial role in the innate host defense against diverse pathogens. The apocrine secretion mechanism is a special mode of secretion by which the apical part of the cell cytoplasm surrounded by a membrane is pinched off. We could show that the presence of actin filaments, CK 19 and CK 7, seems to play a role in the pinching-off mechanism. Finally, we showed the secretion of lipid vesicles from the ceruminous gland. We could extend the number of detected antimicrobial peptides and proteins in human ceruminous glandular cells that protect the surface of the external auditory meatus. In addition, we detected proteins involved in the apocrine secretion mode of the ceruminous gland.

Limited suitability of EpCAM for molecular staging of tumor borders in head and neck cancer.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is expressed in most normal epithelia, but is absent from squamous stratified epithelia. However, a de novo expression can be observed in squamous epithelia during carcinogenesis. MATERIALS AND METHODS: In order to evaluate EpCAM as a molecular marker to indicate borders of high risk for the development of local recurrences, its expression was examined in the marginal zone of malignancies. Specimens of squamous cell carcinoma of the head and neck (SCCHN), of the histologically tumor-free defined resection margin and of healthy epithelia of 20 patients were examined by RT-PCR in order to identify the expression of EpCAM in these three different areas. Additionally, immunohistochemistry was perfonned on biopsies from 10 patients in order to confirm these findings and to investigate a potential correlation between EpCAM expression and the degree of dysplasia. RESULTS: By RT-PCR, high expression of EpCAM was found in the tumor. An inverse correlation was observed between EpCAM expression and the distance from the tumor, with no expression being detectable in healthy oral mucosa. In 70% of the cases, EpCAM was expressed in the marginal zone, which had been defined as tumor-free by routine histopathological assessment. Additional immunohistology revealed no correlation between EpCAM expression and the grade of dysplasia. CONCLUSION: Our data provide evidence that EpCAM is restricted as a marker for redefining the real tumor margin by RT-PCR. To complement routine histology, immunohistochemical staining with EpCAM is limited due to its expression in hyperplastic tissue without dysplastic changes. Both observations limit the reliable use of EpCAM for the molecular definition of the critical tumor border and resection margins.

Liposomal transfection of squamous carcinoma cells of the head and neck with IL-2 and B7 plasmids inducing an autologous immune response in vitro.

New treatment strategies need to be developed to face the increasing incidence and mortality of squamous cell carcinoma of the head and neck (SCCHN), as the overall survival rate remains poor, with minor therapeutic progress having been achieved over the past forty years. One major goal could be to restore a damaged immune system by intratumoral injection of IL-2-genes that permanently provide non-toxic IL-2-protein concentrations at the tumor site, sufficient to activate cellular immunity in vivo. We showed that the transfection of SCCHN cell lines with IL-2-plasmids, encapsulated in DOTMA/Col, in vitro resulted in the synthesis of bioactive IL-2-protein for up to 30 days by the tumor cells themselves. The transcription of secondary cytokines (IL-6, IL-8, GM-CSF, TNF-alpha) and the expression of immunomodulatory surface molecules (MHC Class II, ICAM1) were enhanced. The IL-2-modified tumor cells were effectively lysed by autologous peripheral blood lymphocytes (PBLs). The immune response was enhanced by B7.1-gene-cotransfection and/or preactivation of PBLs with exogenous IL-2. We demonstrated that in vitro liposome-mediated IL-2-gene-transfection of SCCHN cells is an effective method to stimulate an autologous immune response and is, therefore, promising for clinical application.

Generation of an autologous cell system for immunotherapy of squamous cell carcinoma of the head and neck.

BACKGROUND: To date, there is no tumor antigen known to be sufficiently specific for diagnosis, therapy monitoring and immunotherapy of squamous cell carcinoma of the head and neck (SCCHN). The aim of our study was to generate an autologous immune response against SCCHN in vitro for further characterization of SCCHN-specific tumor markers and adoptive immunotherapy. MATERIALS AND METHODS: As sources for tumor antigens (Ags) for the restimulation of autologous immune cells, cell lines from solid SCCHN were established and characterized. Forty-five percent of 40 tumors of different SCCHN specimens were maintained for more than 20 cell generations in culture. RESULTS: One primary cell line, SCCHN-GHD, newly established from a hypopharynx carcinoma, was further characterized as a telomerase-positive, immortalized cell line with epithelial cell characteristics. It was found to be tumorigenic in SCID mice. CONCLUSION: This new SCCHN-GHD cell line is competent as a target for lysis by autologous immune cells and for the restimulation of autologous tumor-specific immune cells. Subsequent characterization of tumor antigens will be performed.

Prednisolone reduces TNF-alpha release by PBMCs activated with a trifunctional bispecific antibody but not their anti-tumor activity.

BACKGROUND: New adjuvant immunological therapies, that selectively redirect effector cells towards tumors, are currently under development. These strategies include trifunctional bispecific antibodies (trAb) as promising tools for the elimination of disseminated tumor cells and micrometastases. To date, these chimeric molecules have demonstrated their antitumor potential mainly in vitro. Here, trAb-activated peripheral blood mononuclear cells (PBMCs) displayed considerable antitumor activity, accompanied by the release of cytokines, which contributed to the antitumor activity but, on the other hand, may evoke serious limiting side-effects in vivo, demanding therapeutic interventions. MATERIALS AND METHODS: The antitumor activity and cytokine release by trAb-activated PBMCs were studied in co-cultures with multicellular tumor spheroids (MTS), which represent a three-dimensional in vitro model for solid tumors, especially non-vascularized micrometastases. The glucocorticoid prednisolone was tested for its influence on the release of TNF-alpha and the activity of PBMCs. RESULTS: It was shown that PBMCs, which were stimulated with a trifunctional bispecific antibody, BiUII, displayed an excellent antitumor activity, resulting in complete disintegration of the MTS. Also, it was demonstrated that prednisolone significantly reduced the release of TNF-alpha, without impairing the antitumor activity of BiUII-activated PBMCs. In contrast, unspecific killing was reduced, as demonstrated with an identical trAb (Bi48), which recognizes an antigen absent from the target cells. CONCLUSION: The in vivo application of bispecific antibodies for adjuvant tumor therapies may be limited by the manifest activation of immune effectors, accompanied by overwhelming cytokine release. Glucocorticoids, like prednisolone, may effectively reduce cytokine release without impairing the antitumor activity of trAb-activated immune cells.

Soluble CD44v6 is not a sensitive tumor marker in patients with head and neck squamous cell cancer.

BACKGROUND: In some epithelial tumors, isoforms of CD44 are overexpressed and soluble isoforms detectable in serum samples are elevated. In squamous cell cancer of the head and neck (SCCHN) the alteration of CD44 isoforms could be associated with poor prognosis. A comprehensive study was undertaken to examine the value of CD44v6 as a tumor marker for SCCHN. PATIENTS AND METHODS: Serum samples of SCCHN and healthy smokers were analyzed for soluble CD44v6 by ELISA. The expression of CD44 isoforms was determined by immunohistochemical staining of healthy and dysplastic tissue. RESULTS: There was no significant difference between the serum levels of sCD44v6 in SCCHN and healthy smokers. Nor was there a correlation between the serum level of sCD44v6 and UICC stage, TNM stage or histological grading. In tissue of primary SCCHN, expression of CD44v6 was found as a strong, specific staining of the lower epithelial layers. Similar amounts of CD44v6-positive-labelled tumor cells were found in invasive carcinoma. CONCLUSION: Soluble CD44v6 is not a valuable tumor marker for SCCHN since the soluble form appears to be present in healthy smokers and does not reflect the stage of the disease.

Subglottic tracheal stenosis as primary manifestation of a marginal zone B-cell lymphoma of the larynx.

BACKGROUND: More than 90% of laryngeal tumors are squamous cell carcinomas. Primary hematopoetic neoplasms of the larynx are rare, being mainly extramedullary plasmocytoma and non-Hodgkin's lymphoma (NHL). These are mainly located in the supraglottic and glottic area, with only a few reported in the subglottic region. CASE REPORT: We report on a 58-year-old man, who presented at our clinic with severe dyspnea. On microlaryngoscopy, a subglottic stenosis at the level of the cricoid cartilage was found. The biopsy revealed the diagnosis of a MALT-type lymphoma (marginal zone B-cell lymphoma). The tracheostomy was followed by locoregional radiotherapy. CONCLUSION: This is the first report of a subglottic MALT-type lymphoma causing a tracheal stenosis. The preferred treatment is locoregional radiotherapy including the draining lymph nodes.

Cytokeratin 8 associates with the external leaflet of plasma membranes in tumour cells.

We reported the identification of tumour-associated antigens from head and neck carcinomas, including cytokeratin 8 (CK8). These antigens were isolated based on the humoral immune response they elicit in vivo using the antibody-mediated identification of antigens technology. Unlike healthy squamous epithelium, tumour cells displayed CK8 at the plasma membrane. However, the actual presence of CK8 at the plasma membrane is still a matter of debate. Here, we have analyzed the expression of CK8 in detail using confocal laser scanning microscopy and circumstantiated its localization at the plasma membrane of carcinoma cells. Healthy human tissues were devoid of CK8 at the membrane, with the exception of hepatocytes. Moreover, membrane-associated CK8 molecules experienced a re-distribution throughout mitosis, which was associated with phosphorylation at serine 73. Phosphorylated CK8 redistributed into dense speckles and relocated to the plasma membrane upon cytokinesis. Thus, CK8 possesses genuine extracellular epitopes on tumour cells, which may represent valuable targets for therapy.

Allogenic antibody-mediated identification of head and neck cancer antigens.

Recently, we described a new target-identification technology, autoantibody-mediated identification of antigens (AMIDA). AMIDA takes advantage of autologous serum autoantibodies to identify disease-associated antigens. Here, we evaluated the allogenic variant of AMIDA (allo-AMIDA), using permanent cancer cell lines as an antigen-pool rather than primary biopsy samples. Twelve different proteins were retrieved exclusively with antibodies from cancer patients, but not from healthy donors. The expression of three of these antigens, e-FABP, hnRNP H, and Grb2, was evaluated in more detail. All three proteins were strongly overexpressed in primary carcinomas and metastases thereof, as compared to healthy epithelium. Additionally, serum reactivity against e-FABP was detected in 20% of cancer patients but only 2% of healthy volunteers. In summary, we demonstrate that permanent cancer cell lines represent a reliable source for tumour-associated antigens. Moreover, we show that allo-AMIDA is suitable for the identification of tumour-specific antigens overcoming the limitations of autologous screening techniques.