Tobias Ettl

AKT and MET signalling mediates antiapoptotic radioresistance in head neck cancer cell lines

OBJECTIVESnInduction of apoptosis is a major mechanism of radiosensitivity in different types of cancer. In contrast, EGFR/PI3K/AKT signalling and recently the presence of so-called cancer stem cells are discussed as reasons for radioresistance.nnnMATERIALS AND METHODSnThe study investigates mechanisms of apoptosis, key oncogenes of the PI3K/AKT pathway and the presence of cancer cells with stem cell properties during irradiation in two cell lines (PCI-9A, and PCI-15) of head and neck squamous cell carcinoma. WST-1-tests, qRT-PCR, western blots and FACS analysis were performed for analysis.nnnRESULTSnThe two cell lines presented different degrees of cell death upon irradiation. The radiosensitive cell line PCI-9A showed increased apoptosis after irradiation measured by expressed cleaved caspases 3 and 7 while the radioresistant cell line PCI-15 upregulated antiapoptotic Survivin and BCL2A1 mRNA. Besides, increased PI3K/AKT- and ERK1/2-signalling was associated with radioresistance accompanied by loss of PTEN function through phosphorylation on S380. Blockade of pAKT increased radiation-induced cell death, and moreover, led to an upregulation of pMET in the radioresistant cell line. The percentage of ALDH-positive tumour cells was markedly decreased after irradiation in the radiosensitive cell line.nnnCONCLUSIONSnFunctional apoptosis is mandatory for sensitivity to irradiation in head neck cancer cells. Upregulation of the AKT-pathway seems to be one reason for poor radioresponse. Activated MET may also predict radioresistance, possibly through ERK1/2 signalling. Moreover MET may indicate the presence of cancer stem cells facilitating radioresistance as shown by increased ALDH expression.

Positive frozen section margins predict local recurrence in R0-resected squamous cell carcinoma of the head and neck

OBJECTIVESnThe purpose of this study was to analyse the impact of surgical margins on tumour recurrence and survival of patients with carcinomas of the head and neck.nnnMATERIAL AND METHODSnA cohort of 156 patients with primary squamous cell carcinoma of the head and neck treated by local resection with negative margins and neck dissection between 2004 and 2012 was investigated. Margin status in frozen sections and permanent paraffin tissues were analysed and compared to clinical and histopathological parameters as well as to tumour recurrence (local, regional and distant) and disease-specific survival (DSS).nnnRESULTSnClose margins (<5mm) on permanent sections were correlated to high-grade differentiation (p=0.070), lymphangiosis (p=0.009) and positive neck nodes (p=0.025) implicating regional and distant recurrence (p=0.001) as well as unfavorable DSS (p=0.002). Positive margins on initial frozen section analysis revised into negative margins during further surgery were the strongest predictor for local recurrence in uni- and multivariate analysis (p<0.001, hazard ratio 3.34). However, positive frozen section margins were not significantly predictive for DSS (p=0.150). Significant predictors for DSS in univariate analysis were local recurrence (p=0.026), T-stage (p=0.02), N-stage (p<0.001), grading (p=0.02) and lymphangiosis (p=0.001). Multivariate DSS analysis revealed lymph node metastasis (p=0.005) and local recurrence (p=0.017) as significant negative predictors.nnnCONCLUSIONnClose margins on permanent sections are associated with aggressive tumour characteristics, regional and distant metastasis implicating worse DSS. The accuracy of frozen section analysis seems limited as positive frozen section margins revised into negative margins bear a high risk of local recurrence.

The induction of cellular senescence in dental follicle cells inhibits the osteogenic differentiation

Dental stem cells such as human dental follicle cells (DFCs) have opened new promising treatment alternatives for today’s dental health issues such as periodontal tissue regeneration. However, cellular senescence represents a restricting factor to cultured stem cells, resulting in limited lifespan and reduced cell differentiation potential. Therefore, this study evaluated if and how DFCs exhibit features of cellular senescence after being expanded in cell culture. The cell proliferation of DFCs decreased, while the cell size increased during prolonged cell culture. Moreover, DFCs expressed the senescence-associated β-galactosidase after a prolonged cell culture. The onset of senescence inhibited both the induction of osteoblast markers RUNX2 and osteopontin and the biomineralization of DFCs after stimulation of the osteogenic differentiation. In conclusion, we showed that a prolonged cell culture induces cellular senescence and inhibits the osteogenic differentiation in DFCs.

Long-term outcome and subjective quality of life after surgical treatment of lower lip cancer

ObjectivesSquamous cell carcinoma of the lower lip represents a common type of cancer with a favourable prognosis. Different types of defect reconstruction after ablative surgery are described for maintenance of patients’ quality of life.Materials and methodsThe study retrospectively evaluates the outcome of 105 patients treated for lower lip cancer. Oncological data as well as individual quality of life were investigated.ResultsCervical lymph node metastases rarely occurred (7.6xa0%) and were correlated to larger-sized tumours (pu2009=u20090.041). Only five patients (4.8) died related to the tumour disease, especially after lymph node metastasis and tumour recurrence (pu2009<u20090.001). The 5-year rates for recurrence-free, overall and disease-specific survival were 84.5, 61.2 and 93.9xa0%. Tumour recurrence correlated with a resection margin of less than 0.75xa0cm (pu2009=u20090.089). With view to postoperative quality of life, the modified Bernard-Fries technique showed the most unfavourable results, particularly referring to sensibility, paraesthesia, lip pursing and mouth opening compared to a stair-step and/or Abbe reconstructions.ConclusionsSquamous cell carcinoma of the lower lip shows a very favourable prognosis. Neck dissection seems often negligible particularly in small tumours. Patients’ functional quality of life after lip surgery depends on the size of the primary and the used technique. Stair-step and/or Abbe reconstructions should be preferred to a Bernard-Fries technique in comparable defect sizes.Clinical relevanceProper treatment of lower lip cancer provides favourable prognosis and preserves patients’ functional and aesthetic quality of life.

Can dental panoramic radiographic findings serve as indicators for the development of medication-related osteonecrosis of the jaw?

OBJECTIVESnThe purpose of this case-control study was to find a correlation between certain imaging findings in dental panoramic radiographs and the risk for developing a medication-related osteonecrosis of the jaw (MRONJ) in patients taking antiresorptive therapy (AT).nnnMETHODSnRandomized and blinded dental panoramic radiographs of 60 patients undergoing antiresorptive drug treatment (36 patients with MRONJ, 24 patients without MRONJ) and of 60 patients without AT were analyzed by 3 experts for the following signs: sequestrum, osteosclerosis, difference in sclerosing of alveolar process and body of mandible, visible alveolar socket, enhancement and loss of lamina dura, enhancement of the oblique ridge, enhancement of the mandibular canal, proliferative periostitis and osteolytic processes at the cortex.nnnRESULTSnSigns were seen significantly more often in patients undergoing AT than in the control group (CG) (osteosclerosis p-valueu2009=u20090.019, visible alveolar socket p-valueu2009=u20090.001, enhancement of lamina dura p-valueu2009<u20090.001, enhancement of the mandibular canal p-valueu2009=u20090.025, proliferative periostitis p-valueu2009=u20090.05 and osteolytic processes at the cortex p-valueu2009<u20090.001). While there is no significant difference between the CG and the group of patients with AT without manifest MRONJ for any sign, the significance increases when taking the group of patients under AT with manifest MRONJ into consideration. In addition, if medication was administered for malignant reasons, the signs visible alveolar socket, enhancement of the lamina dura and the enhancement of the mandibular canal were seen significantly more often.nnnCONCLUSIONSnThe radiographic findings mentioned above are not indicators for the development of MRONJ, as they are seen only in patients with manifest osteonecrosis. However, these findings could be important to assess the dimension and potency of a MRONJ.

Biological predictors of radiosensitivity in head and neck squamous cell carcinoma

ObjectivesThe aim of this study is to investigate the influence of prognostic biomarkers on radiosensitivity and survival of advanced head and neck squamous cell carcinomas treated by primary (chemo)radiation.Material and methodsThe clinicopathological data and immunohistochemical staining of p16, c-Met, survivin, PD-1, and PD-L1 of 82 primarily (chemo)irradiated patients with head and neck squamous cell carcinoma were analyzed. Associations with local and locoregional radiation response, overall survival (OS), disease-free (DFS), and disease-specific survival (DSS) were assessed.ResultsComplete tumor response was associated with increased patient age (pxa0=xa00.007), N0-status (pxa0=xa00.022), M0-status (pxa0=xa00.007), and p16-positivity (pxa0=xa00.022). High PD-L1 was associated with M0-status (pxa0=xa00.026) and indicated tumor response to irradiation (pxa0=xa00.057); survivin expression showed higher rates of response failure (pxa0=xa00.073). Low PD-1 was associated with increased T-stage (pxa0=xa00.029) and local recurrence (pxa0=xa00.014). High PD-1 was strongly correlated with PD-L1-positive tumor infiltrating lymphocytes (pxa0<xa00.001). Low PD-L1 showed a significant correlation with high c-Met expression (pxa0=xa00.01). Significant predictors for unfavorable univariate survival were incomplete tumor response (DSS, pxa0<xa00.001), single radiotherapy (DSS, pxa0=xa00.002), M1-status (DSS, pxa0<xa00.001), decreased radiation dose (DSS, pxa0=xa00.014), high survivin (DSS, pxa0=xa00.045), and high c-Met (OS, pxa0<xa00.05). Survivin and c-Met also showed prognostic significance in multivariate survival analysis.ConclusionsP16 and PD-L1 indicate radiosensitivity, whereas survivin and c-Met implicate radioresistance in primarily (chemo)irradiated head and neck squamous cell carcinomas. The role of the PD-1/PD-L1 immune checkpoints in radiation response and survival merits further investigation.Clinical relevanceThe findings may improve patient-specific therapy according to individual tumor characteristics.

Contrast-enhanced ultrasonography as a new method for assessing autonomization of pedicled and microvascular free flaps in head and neck reconstructive surgery

OBJECTIVEnEvaluating vascular autonomization of pedicled and microvascular free flaps for soft tissue reconstruction in the head and neck area by means of postoperative quantitative measurement of dynamic contrast values obtained with contrast-enhanced ultrasound.nnnMETHODSn8/18 patients underwent lip reconstruction with a pedicle flap, 10 patients reconstruction of other parts of the head with a microvascular free transplant. Ultrasound examinations were conducted within the 1st postoperative week and 4 weeks after surgery. After the intravenous bolus of the ultrasound contrast agent, examinations were carried out for 30u200asec without compression followed by 30u200asec with compression of the vascular pedicle in bolus and flash kinetics. Digital cine loops were analyzed off-line with a quantification software (VueBox™) to determine the Rise Time (RT) between flap tissue with and without compression.nnnRESULTSnMeasurements showed increasing autonomous perfusion, independent of the vascular pedicle. No transplant was lost, but 4/10 patients with a microvascular flap and 1/8 patients with a pedicle flap developed postoperative complications. RT values for the pedicled and microvascular flaps obtained under compression differed significantly between the 1st and the 4th week (pu200a=u200a0.025).nnnCONCLUSIONSnReliable neovascularization was achieved 4 weeks postoperatively. CEUS showed to be a useful method for assessing the degree of autonomization of pedicle and microvascular free flaps.

NHE1-expression at wound margins increases time-dependently during physiological healing.

Wound repair is an orchestrated process, encompassing the phases of inflammation, proliferation and tissue remodeling. In this context, sodium hydrogen exchanger 1 (NHE1) is crucial to epidermal barrier integrity and acidification. Recently, we found that extracellular pH (pHe) on wound surfaces is dramatically increased initially after barrier disruption, and that pHe decreases gradually during physiological healing. Additionally, we have shown that spatial NHE1‐patterns account for pHe‐gradients on surfaces of chronic wounds. Here, we show that NHE1‐expression is very low at margins initially after wounding and that it increases massively during the time‐course of physiolgical healing. This finding is in accordance with the decrease of pHe on wound surfaces, which we reported on in previous works. Thus, we show that NHE1 is an interesting target when it comes to modification of surface pHe on wounds, both acute and chronic, and that NHE1 is time‐dependently regulated in physiological healing.

Accuracy in orthognathic surgery─comparison of preoperative plan and postoperative outcome using computer-assisted two-dimensional cephalometry by the Onyx Ceph® system

PURPOSEnThis retrospective study analyzes deviations between preoperative planning and postoperative outcome in orthognathic surgery using 2D Onyx Ceph®-cephalometric analyzing and planning system.nnnMATERIALS AND METHODSnA total of 100 patients with a mean age 25.1 of years were included in this study. In 33 patients a bilateral sagittal split osteotomy and in seven patients a Le Fort I osteotomy was performed. A total of 60 patients were treated by a bimaxillary approach. Onyx Ceph® was used as cephalometric planning software (Onyx Ceph®), followed by mock operations. Postoperative cephalograms were obtained after 3.3 days and compared to preoperative planning cephalograms for sagittal (SNA, SNB, ANB) and vertical (ArGoMe, ML-NSL, NL-NSL) angle measurements. Real and absolute mean deviation were documented.nnnRESULTSnAbsolute mean deviation (degrees) between postoperative and planned jaw movement was lower for the sagittal parameters SNA (0.58), SNB (1.15) and ANB (1.05) compared to the vertical parameters NL-NSL (1.47), ML-NSL (1.96) and ArGoMe (3.20). SNA, SNB and ANB showed constant deviations independent from the extent of jaw movement. With regard to the vertical parameters ML-NSL, ArGoMe and NL-NSL the extent of the postoperative rotational jaw movement was not as much as planned, particularly for vertical shifts of more than 4°.nnnCONCLUSIONnBy using the 2D Onyx Ceph® cephalometric software for orthognathic surgery, the deviations between planned and actual movements are within an acceptable and predictable range. Planning of extensive vertical alterations may result in greater deviations after surgery.

A comparison of cell survival and heat shock protein expression after radiation in normal dermal fibroblasts, microvascular endothelial cells, and different head and neck squamous carcinoma cell lines

ObjectivesHead and neck squamous cell carcinoma (HNSCC) shows increased radioresistance due to the manipulation of homeostatic mechanisms like the heat shock response. This study intended to comparatively analyze effects of ionizing radiation on different HNSCC cell lines (PCI) and normal human dermal fibroblasts (NHFs) and human dermal microvascular endothelial cells (HDMECs) to uncover differences in radiation coping strategies.Materials and methodsProliferation (BrdU assay), apoptosis (caspase 3/7) and intracellular protein expression of heat shock protein (HSP)-70, and phosphorylated and total HSP27, determined by enzyme-linked immunosorbent assay (ELISA), were analyzed after exposure to increasing doses of ionizing radiation (2, 6, and 12xa0Gray, Gy).ResultsCell count decreased dose-dependently, but PCI cell lines consistently showed higher numbers compared to NHF and HDMEC. Likewise, high doses reduced cell proliferation, but low-dose radiation (2xa0Gy) instead increased proliferation in PCI 9 and 52. Apoptosis was not detectable in PCI cell lines. Basic HSP70 expression was high in PCI cells with little additional increase by irradiation. PCI cells yielded high basic total HSP27 concentrations but irradiation dose-dependently increased HSP27 in HDMEC, NHF, and PCI cells. Phosphorylated HSP27 concentrations were highest in NHF.ConclusionPCI cell lines showed higher resistance to dose-dependent reduction in cell number, proliferation, and protection from apoptosis compared to NHF and HDMEC. In parallel, we observed a high basic and radiation-induced expression of intracellular HSP70 leading to the assumption that the radioresistance of PCI cells is conferred by HSP70.Clinical relevanceHNSCC use HSP to escape radiation-induced apoptosis and certain subtypes might increase proliferation after low-dose irradiation.