Christian Mozet

Current aspects of targeted therapy in head and neck tumors

This review focuses on the current and upcoming options of targeted therapy (biologicals) in head and neck squamous cell carcinoma (HNSCC) with special regard to conceptual integration in future strategies. Epidermal growth factor receptor (EGFR) is the most prominent candidate for therapeutic targeting because of its more than 90% expression rate in HNSCC and influence on the regulation of proliferation, apoptosis, metastasis, angiogenesis and cell differentiation. The point of view of head and neck surgeons is mainly adjusted to reach a balance between targeted, minimal ablative surgery and the evidence-based demand of oncologic accurate surgery with clear margins and, if needed, adjuvant or primary systemic chemoradiation. Therefore, the long-term effects of chemoradiation regimens, such as dysphagia, aspiration and laryngeal immobility caused by fibrosis, are just beginning to be studied and are becoming one of the major problems in the ongoing treatment of HNSCC. In this context, molecular targeting biologicals with a different toxicity profile and hopefully less late damage to functionally important tissues may open new strategies in primary and adjuvant treatment of HNSCC. Besides cetuximab and other EGFR targeting mAbs, this review focuses on receptor and non-receptor tyrosine kinase inhibitors, which further might play a role in the future treatment of HNSCC. To complete the current picture, the problem of multi drug resistance in cancer progenitor cells, targeting members of several relevant pathways and novel agents like pemetrexed and enzastaurin, are discussed in a broader sense of targeted therapy.

Effect of age and hypoxia/reoxygenation on mRNA expression of antioxidative enzymes in rat liver and kidneys

The influence of a short-time isobaric hypoxia as well as reoxygenation on markers of oxidative stress (MDA, total SOD, GSH) and on the mRNA expression of the antioxidative enzymes (Cu/Zn-and Mn-SOD, catalase, GSH reductase and GSH peroxidase) has been studied in liver and kidneys of young (6 months) and old (22-25 months) Wistar rats. In livers of old animals, the concentration of GSH, the activity of SOD, and the mRNA expression of the antioxidative enzymes (except Mn-SOD) points to a restricted protection against oxidative stress or a lower production of ROS compared to young animals. Hypoxia resulted in a significant decrease of enzyme gene expression in both age groups. Reoxygenation caused an increase in mRNA of Cu/Zn-SOD and GPX in livers of young and of Mn-SOD in livers of old animals. In kidneys, gene expression of Cu/Zn-SOD, GSH reductase, and GPX was significantly higher in old animals compared to young animals. Whereas hypoxia caused a decrease of gene expression in the livers, it lead to a significant increase of Cu/Zn-SOD, catalase, and GSH reductase mRNA in kidneys of young rats. A reduced gene expression was observed after reoxygenation. In old kidneys, the expression of all enzymes except for catalase progressively declined in the hypoxic and reoxygenation groups. These data show that gene expression of antioxidative enzymes is affected by age and significantly differs between liver and kidney.

Single Tissue Samples from Head and Neck Squamous Cell Carcinomas are Representative Regarding the Entire Tumor’s Chemosensitivity to Cisplatin and Docetaxel

Background: In multimodal therapy concepts for advanced head and neck squamous cell carcinoma (HNSCC), a valid predictive assay for the quick detection of efficient chemotherapeutic agents is desirable. Questionable so far was whether tissue samples of about 100 mg correctly reflect the chemoresponse of a whole HNSCC. This was proven using an ex-vivo colony-forming assay. Materials and Methods: Of 14 HNSCC, 3 biopsies each were taken from separate sites, minced, and collagenase digested. HNSCC digests were added to microtiter plates containing serial dilutions of chemotherapeutic agents or medium as control. After 72-h incubation, wells were washed and cultures methanol-fixed before Giemsa-staining. Epithelial colonies were counted. Results: 11/14 HNSCC (78.6%) showed sufficient colony formation allowing reliable cut-off detection. Cut-off concentrations (complete chemotherapeutically suppressed colony formation) between 3.3 μM and >50 μM cisplatin, and 0.55 μM and 17.6 μM docetaxel were detected. Inhibition of colony formation to 50% of colonies detected in controls (IC50) was found between 0.2 μM and 17.9 μM cisplatin or 1.5 μM and 13.7 μM docetaxel. Cut-off concentrations and IC50 of the HNSCC fragments showed a strong correlation (docetaxel: r > 0.80, p < 0.005; cisplatin: r > 0.67, p < 0.044), while being only insignificantly different in the t-test for paired samples (docetaxel: p > 0.163; cisplatin: p > 0.167). Conclusion: In most cases, tissue samples of about 100 mg allow a representative assessment of chemoresponse of HNSCC.

Simvastatin suppresses head and neck squamous cell carcinoma ex vivo and enhances the cytostatic effects of chemotherapeutics.

PurposeSimvastatin (Sim) is approved as lipid-controlling drug in patients with cardiovascular risk to reduce hypercholesterolemia. Recent publications indicate possible inhibitory effects of Sim on tumor cell lines, and epidemiological data suggest activity in cancer patients. Still, its therapeutic efficacy, particularly in head and neck squamous cell carcinoma (HNSCC), remains to be elucidated. This study analyzes the effects of Sim on HNSCC cell lines (KB, HN5, FaDu) and on a larger set of primary HNSCC cells by employing a short-time ex vivo colony formation test (FLAVINO assay). Possible additive or synergistic effects of Sim combinations with established chemotherapeutics are determined as well.MethodsBiopsies of 49 HNSCC were tested in the FLAVINO assay with Sim alone or in combination with cisplatin (Cis) or docetaxel (DTX). Cell lines were studied for reference. Epithelial HNSCC cells were stained by Cy2-labeled anti-cytokeratin antibodies facilitating the detection of colony formation (CF) by immunofluorescence. Drug combinations were analyzed regarding their interaction.ResultsSim alone acted suppressive on tested cell lines and increased the cytostatic efficacy of Cis and DTX. 18/49 HNSCC qualified for FLAVINO-based dose-response analyses, and Sim significantly suppressed CF in 18/18 primary HNSCC. Moreover, Sim increased cytotoxic effects of Cis and DTX, primarily in an additive mode of action.ConclusionsThe ex vivo tumor cell inhibition of Sim and its additive effects upon combination with established cytostatics provide the basis for epidemiological and clinical studies on statins, potentially directed toward co-medication in future treatment regimens.

Cardioprotective effect of EGb 761 on myocardial ultrastructure of young and old rat heart and antioxidant status during acute hypoxia

Background and aims: Acute hypoxia is a threatening clinical case of emergency and may result in ultrastructural damage, with complete loss of cellular and organ functions. However, little is known about the differences in hypoxia tolerance between young and old myocardia and the protective effects of radical scavenging agents in acute hypoxic stress situations. Methods: We investigated the age-dependent differences of the myocardial ultrastructure and antioxidative status (superoxide-dismutase (SOD) activity and malondialdehyde (MDA) content) of young (6 months) and old (22–24 months) Wistar rats (Crl (Wi)Br) after acute respiratory hypoxia of 20 min at 5% v/v O2 in N2O mixture, and the protective effect of Ginkgo biloba extract (EGb 761). Results: Ultrastructural-morphometric and biochemical age analysis only revealed a decrease in the sarcoplasma volume fraction, an increase in homogeneous intramitochondrial areas, significant higher SOD activity and lower MDA levels in the group of old rats. Pretreatment with EGb 761 led to a significant decrease in MDA content in both control groups. Acute hypoxic stress increased the volume fractions of sarcoplasmatic reticulum, t-tubules, vacuoles, and lipid droplets, and caused mitochondrial swelling, with a more significant increase in degenerated and homogeneous intramitochondrial areas in the old group. SOD activity decreased only in the old hypoxic group; MDA content fell in both. Pretreatment with EGb 761 reduced ultrastructural-morphometric hypoxic damage in both groups, significantly below the levels of control. Young rat myocardium showed significantly higher SOD activity after hypoxia than untreated or older specimens. Conclusions: Better hypoxia tolerance is demonstrated by the young myocardium, and an obvious hypoxia-protective effect of EGb 761 in both age groups.

Overexpression of the Hedgehog Signalling Pathway in Head and Neck Squamous Cell Carcinoma

Background: Overexpression of the Hedgehog (HH) signalling pathway has been described in several malignancies and is associated with a poor prognosis. HH signalling blockade reduces tumour growth in vitro and in vivo. We aimed to determine whether head and neck squamous cell carcinomas (HNSCCs) express HH proteins in comparison to healthy mucosa. Patients and Methods: Formalin-fixed and paraffin-embedded tissue sections of 10 patients with HNSCC were stained with fluorescence-labelled antibodies for cytokeratin and HH proteins (SHH, PTCH1/2, SMO, Gli1-3) and photographs were taken with a laser scanning microscope. The pixel count and colour intensity were analysed in RGB (red/green/blue) colour mode, and expression levels were compared to healthy mucosa. Results: Image analysis in RGB mode provided objective evidence for the over-expression of HH signalling components in HNSCC, particularly with regard to the transcription factors Gli1 (10-fold) and SHH (5-fold) in comparison with healthy mucosa. The lowest levels were found for Gli3 in HNSCC. Conclusions: We postulate pivotal roles of Gli1 and SHH expression in the carcinogenesis of HNSCC. HH pathway overexpression appears to be involved in the initiation of tumour growth and spread due to its stem cell-modulating properties. Detection of HH pathway components, and especially Gli1 and SHH, in HNSCC might offer a promising target for further anticancer research in HNSCC.

Current therapy options in recurrent head and neck cancer

Recurrent disease is one of the main reasons for the persistently poor prognosis of squamous cell carcinoma of the head and neck (HNSCC; European 5-year survival, 42%). The main treatment option for primary and secondary malignancy as well as recurrent disease is surgical therapy. If R0 resection (resection margin >5 mm) for a primary tumor is not viable, survival probability is reduced by 50%. In recurrent or secondary tumors with R1- or -2 resection or in the presence of non-resectable metastases, a palliative situation results in more than 80% of cases. In the case of surgery following radiotherapy or radiochemotherapy, attention should be paid to the criteria for salvage surgery (tissue perfusion, fibrosis, wound healing) and the procedure adapted to focus on functionality. In the case of relapse, primary surgery can potentially be supplemented with adjuvant therapy protocols such as (re-) irradiation, as well as possibly with chemotherapeutic agents or targeted therapies. Interdisciplinary collaboration and case discussions should take place in the context of a tumor board.

Nitric Oxide Synthase (NOS2/3) Expression in Head and Neck Squamous Cell Carcinomas in Correlation with Clinical Patterns

Background: Increased nitric oxide synthase (NOS) expression has been demonstrated in a number of carcinomas and is discussed to play a key role in tumor progression. The aim of this immunohistochemical study was to examine the protein expression rates of endothelial (e)NOS and inducible (i)NOS in head and neck squamous cell carcinomas (HNSCCs) and oral mucosa and to correlate the results with clinicopathologic factors (TN stage). Patients and Methods: Protein expression patterns of NOS were studied immunohistochemically (score 0–7) in 58 patients with HNSCC and 7 mucosa samples, and the results were correlated with tumor stages. Results: In oral mucosa, iNOS was only expressed in the basal epithelial layers and in macrophages, eNOS in endothelial cells and lymphocytes. In contrast, both NOS isoforms were expressed in HNSCC with preference at the tumor margins. 64% of tumor specimens demonstrated a positive eNOS immunoreactivity (score ≥3), 55% a positive iNOS immunoreactivity. NOS protein expression rates reached higher scores in tumors of patients with lymph node metastasis (N > 0; iNOS protein expression rate p < 0.05). Conclusions: HNSCCs are able to express both NOS protein isoforms in relevant amounts, and we presume that synthesized NO is able to support angiogenetic patterns and facilitate tumor progression and lymphatic spread.

The Effect of Ginkgo biloba extract (EGb 761) on parameters of oxidative stress in different regions of aging rat brains after acute hypoxia

Background and aims: Neurodegenerative processes of aging seem to be associated with oxidative stress by reactive oxygen species (ROS). This study investigates the influence of age and of acute respiratoric hypoxia on parameters of oxidative stress in different brain regions of Wistar rats and the protective effects of Ginkgo extract (EGb 761) as a radical scavenger. Methods: Biopsies of frontal and temporal cortices, the cerebellum, and the brainstem of young and old rats (each group n=6–8: normoxic — hypoxic; unprotected — EGb-protected) were analyzed for malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, glutathione (GSH) content, and creatine kinase (CK) activity. Experimental hypoxia: downregulation of oxygen partial pressure to 5 vol. % for 20 minutes. EGb administration: daily 100 mg/kg of body weight in drinking water for 3 months. Results: Effects of age: While most oxidative stress parameters in the temporal cortex, the cerebellum, and the brainstem are increased, this is not the case in the frontal cortex; after additional hypoxia SOD and GSH are diminished in the temporal cortex and the brainstem of old rats. EGb treatment causes contradictory alterations in young, old, and hypoxic brain regions. Minor effects are seen in old hypoxic brains, while there are some protective effects in old normoxic brainstems and cerebellums. Conclusions: The old brain appears to adapt appropriately to chronic oxidative stress and to the specific conditions of short-term hypoxia. EGb’s protective effect is especially notable in the brainstem and the cerebellum.

Aktuelle Therapieoptionen bei rezidivierenden Kopf-Hals-Tumoren

Recurrent disease is one of the main reasons for the persistently poor prognosis of squamous cell carcinoma of the head and neck (HNSCC; European 5-year survival, 42%). The main treatment option for primary and secondary malignancy as well as recurrent disease is surgical therapy. If R0 resection (resection margin >5 mm) for a primary tumor is not viable, survival probability is reduced by 50%. In recurrent or secondary tumors with R1- or -2 resection or in the presence of non-resectable metastases, a palliative situation results in more than 80% of cases. In the case of surgery following radiotherapy or radiochemotherapy, attention should be paid to the criteria for salvage surgery (tissue perfusion, fibrosis, wound healing) and the procedure adapted to focus on functionality. In the case of relapse, primary surgery can potentially be supplemented with adjuvant therapy protocols such as (re-) irradiation, as well as possibly with chemotherapeutic agents or targeted therapies. Interdisciplinary collaboration and case discussions should take place in the context of a tumor board.