Geometric Cuts by an Autonomous Laser Osteotome Increase Stability in Mandibular Reconstruction With Free Fibula Grafts: A Cadaver Study.

BACKGROUND: Nonunion and plate exposure represent a major complication after mandibular reconstruction with free fibula flaps. These drawbacks may be resolved by geometric osteotomies increasing intersegmental bone contact area and stability. PURPOSE: The aim of this study was to compare intersegmental bone contact and stability of geometric osteotomies to straight osteotomies in mandibular reconstructions with free fibula grafts performed by robot-guided erbium-doped yttrium aluminum garnet laser osteotomy. STUDY DESIGN, SETTING, SAMPLE: This cadaveric in-vitro study was performed on fresh frozen human skull and fibula specimens. Computed tomography (CT) scans of all specimens were performed for virtual planning of mandibular resections and three-segment fibula reconstructions. The virtual planning was implemented in a Cold Ablation Robot-guided Laser Osteotome. PREDICTOR/EXPOSURE/INDEPENDENT VARIABLE: For predictor variables, straight and geometric puzzle-shaped osteotomies were designed at resection of the mandible and corresponding fibula reconstruction. MAIN OUTCOME VARIABLES: The primary outcome variable was the stability of the reconstructed mandible investigated by shearing tests. Moreover, secondary outcome variables were the duration of the laser osteotomies, the contact surface area, and the accuracy of the reconstruction, both evaluated on postsurgical CT scans. COVARIATES: Covariables were not applicable. ANALYSES: Data were reported as mean values (± standard deviation) and were statistically analyzed using an independent-sample t-test at a significance level of α = 0.05. Root mean square deviation was tested for accuracy. RESULTS: Eight skulls and 16 fibula specimens were used for the study. One hundred twelve successful laser osteotomies (96 straight and 16 geometrical) could be performed. Geometric osteotomies increased stability (110.2 ± 36.2 N vs 37.9 ± 20.1 N, P < .001) compared to straight osteotomies. Geometric osteotomy of the fibula took longer than straight osteotomies (10.9 ± 5.1 min vs 5.9 ± 2.2 min, P = .028) but could provide larger contact surface (431.2 ± 148.5 mm2 vs 226.1 ± 50.8 mm2, P = .04). Heat map analysis revealed a mean deviation between preoperational planning and postreconstructive CT scan of -0.8 ± 2.4 mm and a root mean square deviation of 2.51 mm. CONCLUSION AND RELEVANCE: Mandibular resection and reconstruction by fibula grafts can be accurately performed by a Cold Ablation Robot-guided Laser Osteotome without need for cutting guides. Osteotomy planning with geometric cuts offers higher stability and an increased bone contact area, which may enhance healing of the reconstructed mandible.

Tumor gene signatures that correlate with release of extracellular vesicles shape the immune landscape in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCCs) evade immune responses through multiple resistance mechanisms. Extracellular vesicles (EVs) released by the tumor and interacting with immune cells induce immune dysfunction and contribute to tumor progression. This study evaluates the clinical relevance and impact on anti-tumor immune responses of gene signatures expressed in HNSCC and associated with EV production/release. Expression levels of two recently described gene sets were determined in The Cancer Genome Atlas Head and Neck Cancer cohort (n = 522) and validated in the GSE65858 dataset (n = 250) as well as a recently published single-cell RNA sequencing dataset (n = 18). Clustering into HPV(+) and HPV(-) patients was performed in all cohorts for further analysis. Potential associations between gene expression levels, immune cell infiltration, and patient overall survival were analyzed using GEPIA2, TISIDB, TIMER, and the UCSC Xena browser. Compared to normal control tissues, vesiculation-related genes were upregulated in HNSCC cells. Elevated gene expression levels positively correlated (P < 0.01) with increased abundance of CD4(+) T cells, macrophages, neutrophils, and dendritic cells infiltrating tumor tissues but were negatively associated (P < 0.01) with the presence of B cells and CD8(+) T cells in the tumor. Expression levels of immunosuppressive factors NT5E and TGFB1 correlated with the vesiculation-related genes and might explain the alterations of the anti-tumor immune response. Enhanced expression levels of vesiculation-related genes in tumor tissues associates with the immunosuppressive tumor milieu and the reduced infiltration of B cells and CD8(+) T cells into the tumor.

Adenoid cystic carcinoma of the salivary glands: a pilot study of potential therapeutic targets and characterization of the immunological tumor environment and angiogenesis.

BACKGROUND: Adenoid cystic carcinoma (ACC) is a rare type of cancer commonly occurring in salivary glands. It is characterized by slow but infiltrative growth, nerve infiltration and overall poor prognosis, with late recurrence and distant metastasis. The treatment of ACC is still limited to surgery and/or (adjuvant) radiotherapy. Till now no promising systemic therapy option exists. However, various studies deliver promising results after treatment with anti-angiogenetic agents, such as anti-EGFR-antibody Cetuximab or Tyrosinkinase inhibitor Lenvatinib. METHODS: By using of immunohistological methods we analyzed and compared the macrophage and lymphocyte populations, vascularization, and PD-L1-status in 12 ACC of the salivary glands. RESULTS: All cases showed a significant elevation of macrophages with M2 polarization and a higher vascularization in ACC compared to normal salivary gland tissue. The CD4/CD8 quotient was heterogenous. ACC does not show relevant PD-L1 expression. CONCLUSIONS: The predominant M2 polarization of macrophages in ACC could be responsible for elevated vascularization, as already been proved in other cancer types, that M2 macrophages promote angiogenesis.

Opposing MMP-9 Expression in Mesenchymal Stromal Cells and Head and Neck Tumor Cells after Direct 2D and 3D Co-Culture.

Bone marrow-derived mesenchymal stromal cells (BMSCs) respond to a variety of tumor cell-derived signals, such as inflammatory cytokines and growth factors. As a result, the inflammatory tumor microenvironment may lead to the recruitment of BMSCs. Whether BMSCs in the tumor environment are more likely to promote tumor growth or tumor suppression is still controversial. In our experiments, direct 3D co-culture of BMSCs with tumor cells from the head and neck region (HNSCC) results in strong expression and secretion of MMP-9. The observed MMP-9 secretion mainly originates from BMSCs, leading to increased invasiveness. In addition to our in vitro data, we show in vivo data based on the chorioallantoic membrane (CAM) model. Our results demonstrate that MMP-9 induces hemorrhage and increased perfusion in BMSC/HNSCC co-culture. While we had previously outlined that MMP-9 expression and secretion originate from BMSCs, our data showed a strong downregulation of MMP-9 promoter activity in HNSCC cells upon direct contact with BMSCs using the luciferase activity assay. Interestingly, the 2D and 3D models of direct co-culture suggest different drivers for the downregulation of MMP-9 promoter activity. Whereas the 3D model depicts a BMSC-dependent downregulation, the 2D model shows cell density-dependent downregulation. In summary, our data suggest that the direct interaction of HNSCC cells and BMSCs promotes tumor progression by significantly facilitating angiogenesis via MMP-9 expression. On the other hand, data from 3D and 2D co-culture models indicate opposing regulation of the MMP-9 promoter in tumor cells once stromal cells are involved.

BMSC-HNC Interaction: Exploring Effects on Bone Integrity and Head and Neck Cancer Progression.

In recent research, the tumor microenvironment has been shown to attract mesenchymal stromal cells (MSCs), which is of particular interest due to its implications for cancer progression. The study focused on understanding the interaction between bone marrow-derived MSCs (BMSCs) and head and neck cancer (HNC) cells. This interaction was found to activate specific markers, notably the osteogenic marker alkaline phosphatase and the oncogene Runx2. These activations corresponded with the release of collagenase enzymes, MMP9 and MMP2. To gain insights into bone resorption related to this interaction, bovine bone slices were used, supporting the growth of "heterogeneous spheroids" that contained both BMSCs and HNC cells. Through scanning electron microscopy and energy-dispersive X-ray (EDX) analysis, it was observed that these mixed spheroids were linked to a notable increase in bone degradation and collagen fiber exposure, more so than spheroids of just BMSCs or HNC cells. Furthermore, the EDX results highlighted increased nitrogen content on bone surfaces with these mixed clusters. Overall, the findings underscore the significant role of BMSCs in tumor growth, emphasizing the need for further exploration in potential cancer treatment strategies.

Expression of pH-Sensitive TRPC4 in Common Skin Tumors.

TRPCs (transient receptor potential classical or cation channels) play a crucial role in tumor biology, especially in the Ca2+ homeostasis in cancer cells. TRPC4 is a pH-sensitive member of this family of proteins. As solid tumors exhibit an inversed pH-gradient with lowered extracellular and increased intracellular pH, both contributing to tumor progression, TRPC4 might be a signaling molecule in the altered tumor microenvironment. This is the first study to investigate the expression profiles of TRPC4 in common skin cancers such as basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma (MM) and nevus cell nevi (NCN). We found that all SCCs, NCNs, and MMs show positive TRPC4-expression, while BCCs do only in about half of the analyzed samples. These data render TRPC4 an immunohistochemical marker to distinguish SCC and BCC, and this also gives rise to future studies investigating the role of TRPC4 in tumor progression, and especially metastasis as BCCs very rarely spread and are mostly negative for TRPC4.

Treatment of extracapsular fractures of the mandibular condylar process: A retrospective evaluation of 377 cases.

BACKGROUND/AIM: Mandibular condylar fractures represent 25%-35% of all mandibular fractures. Despite profound research, there is still a controverse debate about treating these fractures conservatively or by open reduction and internal fixation (ORIF). The aim of this study is to analyse the outcome after open and closed treatment of extracapsular mandibular condyle fractures regarding general characteristics, post-treatment malocclusion, facial nerve palsy (FNP), maximum mouth opening (MMO) and parotid complications. METHODS: A retrospective cohort of 377 fractures (350 open, 27 closed treatment) was reviewed by reference to clinical and radiological pre- and postoperative documentation. Follow-up period was 12 months. Pearsons' chi-square-test, correlations, Kruskal-Wallis test and t-test were carried out for statistical analysis. RESULTS: The dominant type of fracture was type II in Spiessl and Schroll classification (50.1%). In the open treated fractures, the most common approach was retromandibular transparotid (91.7%). Post-treatment malocclusion occurred in 18.0% and was significantly increased in bilateral fractures (p = .039), in luxation fractures (p = .016) and in patients with full dentition (p = .004). After open reduction and internal fixation (ORIF), temporary FNP was documented in 7.1% whereas a permanent paresis occurred in 1.7%. FNP was significantly associated with high fractures (p = .001), comminution (p = .028) and increased duration of surgery (p = .040). Parotid complications were significantly associated with revision surgery (p = .009). Post-treatment reduction of MMO mainly occurred in female patients (p < .001) as well as in patients with bilateral fractures (p < .001), high fractures (p = .030) and concomitant mandibular (p = .001) and midfacial fractures (p = .009). CONCLUSION: Malocclusion seems to be the most frequent long-term complication after open reduction and osteosynthesis of extracapsular mandibular condyle fractures. We suggest ORIF by a transparotid approach to be an appropriate treatment with a low complication rate regarding especially FNP for extracapsular fractures of the mandibular condyle.

The first quantitative histomorphological analyses of bone vitality and inflammation in surgical specimens of patients with medication-related osteonecrosis of the jaw.

BACKGROUND: The purpose of the study was to categorize the vitality and inflammation of resected bone of patients with medication-related osteonecrosis of the jaw (MRONJ) and to correlate the grade of inflammation with the surgical success. METHODS: This prospective study includes 44 patients with stage III MRONJ. Necrotic bone was resected in a block fashioned way. After demineralization and staining, histological analyses were performed by measuring the areas of necrotic, vital, and regenerative bone. Areas of chronic and acute inflammation were categorized as non, mild, moderate, and severe and were correlated with surgical success and parameters of inflammation in blood plasma (C-reactive protein and leukocytes). RESULTS: An average area of 59.0% was necrotic in the examined specimen. Vital bone was measured with an average area of 40.9%. The stage of chronic inflammation correlated with the amount of vital bone (P < .001) and the success of surgery (P = .002). If acute inflammation was dominant, chronic inflammation areas were found less while necrotic areas were observed more (P < .001). Also, the risk of relapses, wound healing disorders, and the level of C-reactive protein were elevated if acute inflammation was severe or moderate (P = .031). Areas of bone regeneration were seen only in 11.3% of vital bone areas and occurred independently of infection stages. CONCLUSION: If possible, surgery should be delayed in patients with signs of severe acute inflammation. Patients may profit from prolonged pre-operative antibiotic therapy to reduce the level of acute inflammation.

Buparlisib modulates PD-L1 expression in head and neck squamous cell carcinoma cell lines.

High expression of the immune checkpoint receptor PD-L1 is associated with worse patient outcome in a variety of human cancers, including head and neck squamous cell carcinoma (HNSCC). Binding of PD-L1 with its partner PD-1 generates an inhibitory signal that dampens the immune system. Immunotherapy, that is blocking the PD-1/PD-L1 checkpoint, has proven to be an effective tool in cancer therapy. However, not all patients are able to benefit from this immune checkpoint inhibition. Therefore, evidence is growing of intrinsic PD-L1 signaling in cancer cells. For example, intrinsic PD-L1 expression was associated with PI3K/Akt/mTOR signaling, which is part of diverse oncogenic processes including cell proliferation, growth and survival. In this study we demonstrate the effects of PI3K/Akt/mTOR pathway inhibition by buparlisib on PD-L1 expression in HNSCC cell lines. After buparlisib treatment for 72 h, PD-L1 was downregulated in total cell lysates of HNSCC cells. Moreover, flow cytometry revealed a downregulation of PD-L1 membrane expression. Interestingly, the buparlisib mediated effects on PD-L1 expression were reduced by additional irradiation. In PD-L1 overexpressing cells, the buparlisib induced inhibition of proliferation was neutralized. In summary, our findings imply that blocking the PI3K/Akt/mTOR pathway could be a good additional therapy for patients who show poor response to immune checkpoint therapy.

Staging more important than grading? Evaluation of malignancy grading, depth of invasion, and resection margins in oral squamous cell carcinoma.

OBJECTIVES: The present study evaluated the predictive value of staging and grading parameters concerning the presence of lymph-node metastases, overall survival (OS), and relapse-free survival (RFS) of patients with oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: HE-stains of 135 surgically treated (R0) primary OSCCs were analyzed using a both microscopic and software-based approach. Depth of invasion (DOI) and resection margins (RM) were measured, and each case was graded according to the malignancy grading system as described by Anneroth et al. and Bryne et al. on two different sites of the tumor (surface and invasion front; TS and IF). RESULTS: Parameters that could be identified as significant predictors of OS and RFS were UICC cancer stage (p = 0.009 and p = 0.012); pT-stage as defined in the 7th edition (p = 0.029 and 0.015) and, after restaging using DOI, 8th edition (p = 0.023 and p = 0.005) of the TNM classification of malignant tumors; the presence of lymphonodular metastases (LM) (p = 0.004 and p = 0.011); degree of keratinization (p = 0.029 and p = 0.042); and pattern of growth (p = 0.029 and p = 0.024) at the TS after applying a binary scale for both parameters. Also, when directly comparing the most extreme subgroups (scores 1 and 4) of lymphoplasmacytic infiltration at the IF, there was a significant difference in OS (p = 0.046) and RFS (p = 0.005). Invasion of blood vessels (p = 0.013) and perineural invasion (p = 0.023) were significantly associated with a lower OS. Age lower than 60 years (univariate p = 0.029, multivariate p = 0.031), infiltration of lymphatic vessels (p = 0.003), infiltration of nerves (p = 0.010), pT-stage (8th edition) (p = 0.014), degree of keratinization at the IF (p = 0.033), and nuclear polymorphism at the IF (p = 0.043) after conversion to a binary scale were found to be significant prognostic parameters regarding the presence of LM. DOI evolved as a significant predictor for OS (p = 0.006), RFS (p = 0.003), and LM (p = 0.032) in metric and grouped analysis. CONCLUSIONS: The current evaluation revealed depth of invasion as strongest histologic predictor of metastatic tumor growth, overall survival, and relapse-free survival in OSCC, confirming the current adaption of the T-classification. Other distinct histologic grading parameters investigated during this study can give valuable indications of a tumor's potential aggressiveness, but the exact site, mode, and procedure need further exploration. CLINICAL RELEVANCE: Integrating measurement of DOI also into the pretherapeutic staging process could aid in treatment planning.

Lymph node ratio as a predictor for outcome in oral squamous cell carcinoma: a multicenter population-based cohort study.

OBJECTIVES: Recently, multiple studies addressed the importance of lymph node ratio (LNR) in specifying patients' risk of disease recurrence in various malignancies. The present study examines the prognostic significance of LNR in predicting outcome of oral squamous cell carcinoma (OSCC) patients after surgical treatment with curative intent. METHODS: Here, we describe a retrospective population-based cohort with 717 patients previously diagnosed with OSCC. Histopathologically verified lymph node metastasis was diagnosed in 290 patients. Among these patients, we evaluated the impact of LNR on overall survival (OAS) and recurrence-free survival (RFS) in uni- as well as multivariate analysis. RESULTS: A median cutoff (0.055) in LNR was found to significantly predict outcome in OSCC patients. Five-year OAS was 54.1% in patients with a low LNR, whereas a high LNR was associated with a 5-year OAS of 33.3% (p < 0.001). Similar results were detected for RFS with a 5-year survival rate of 49.8% (LNR low) and 30.3% (LNR high) (p = 0.002). Results were confirmed in multivariate Cox regression which substantiated the importance of LNR in predicting survival in OSCC patients. CONCLUSIONS: LNR was shown to be an independent prognostic factor for outcome of OSCC in a population-based cohort in uni- as well as multivariate analysis. Hereby, a LNR ≥ 0.055 predicted a shorter OAS and RFS in our cohort. CLINICAL RELEVANCE: Besides established histopathological factors, LNR can be used as a reliable predictor of outcome in OSCC and might therefore be further applied in evaluating adjuvant treatment after resection in curative intention.

Differential Expression of PD-L1 during Cell Cycle Progression of Head and Neck Squamous Cell Carcinoma.

The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable effects in advanced cancer patients including HNSCC. In this context, irradiation is currently being investigated as a synergistic treatment modality to immunotherapy. However, the majority of HNSCC patients still show little improvement or even hyperprogression. Interestingly, there is increasing evidence for additional cell-intrinsic functions of PD-L1 in tumor cells. In previous studies, we showed that PD-L1 has a strong influence on proliferation, migration, invasion, and survival after irradiation. We demonstrated that cellular expression and localization of PD-L1 differed depending on sensitivity to irradiation. Here, we show that PD-L1 is also differentially expressed during cell cycle progression of HNSCC. Furthermore, cellular localization of PD-L1 also changes depending on a particular cell cycle phase. Moreover, distinct observations occurred depending on the general differentiation status. Overall, the function of PD-L1 cannot be generalized. Rather, it depends on the differentiation status and microenvironment. PD-L1 expression and localization are variable, depending on different factors. These findings may provide insight into why differential response to PD-1/PD-L1 antibody therapy can occur. Detailed understanding of cell-intrinsic PD-L1 functions will further allow antibody-based immunotherapy to be optimized.

Radiotherapy for osteoarthritis of the ankle and tarsal joints-analysis of 66 joints.

PURPOSE: Osteoarthritis of the ankle and tarsal joints is less common than osteoarthritis of the knee or hip, but the associated disability is at least as severe as that of the other major joints of the lower limb. The results for total arthroplasty are still not satisfactory. For this reason, arthrodesis is still the gold standard of non-joint-conserving surgery. For the reason of functionality, joint-conserving therapies play a major role in treatment of ankle and tarsal osteoarthritis. Low-dose radiotherapy has a long history of treatment of osteoarthritis. The aim of this survey was to examine the results of low-dose radiotherapy for osteoarthritis of the ankle and tarsal joints. MATERIALS AND METHODS: The analysis was performed on patients of three German radiotherapy institutions and included 66 irradiated joints. Pain was documented with the numeric rating scale (NRS). Evaluation of the NRS was done before and directly after each radiation therapy course as well as for the follow-up of 24 months. The median age of the patients was 68 years, with 24.5% male and 75.5% female patients. The upper ankle was treated in 37.9%, the lower ankle in 27.3% and the tarsal joints in 34.8%. RESULTS: We could find a significant response to radiotherapy. For the whole sample, the median pain was 7 on the NRS before radiotherapy, 5 after 6 and 12 weeks, and 4 after 12 months. The percentage of patients with 0 or 1 on the NRS was 19.6% 12 months after radiotherapy. An improvement of joint mobility could be detected in 56.7% of the cases. All investigated subgroups had a significant reduction in pain. CONCLUSION: Radiotherapy of ankle and tarsal osteoarthritis is an effective treatment without showing side effects. All analysed subgroups show a good response to radiotherapy for at least 24 months.

A prospective clinical pilot study on the effects of a hydrogen peroxide mouthrinse on the intraoral viral load of SARS-CoV-2.

OBJECTIVES: SARS-CoV-2 is mainly transmitted by inhalation of droplets and aerosols. This puts healthcare professionals from specialties with close patient contact at high risk of nosocomial infections with SARS-CoV-2. In this context, preprocedural mouthrinses with hydrogen peroxide have been recommended before conducting intraoral procedures. Therefore, the aim of this study was to investigate the effects of a 1% hydrogen peroxide mouthrinse on reducing the intraoral SARS-CoV-2 load. METHODS: Twelve out of 98 initially screened hospitalized SARS-CoV-2-positive patients were included in this study. Intraoral viral load was determined by RT-PCR at baseline, whereupon patients had to gargle mouth and throat with 20 mL of 1% hydrogen peroxide for 30 s. After 30 min, a second examination of intraoral viral load was performed by RT-PCR. Furthermore, virus culture was performed for specimens exhibiting viral load of at least 103 RNA copies/mL at baseline. RESULTS: Ten out of the 12 initially included SARS-CoV-2-positive patients completed the study. The hydrogen peroxide mouthrinse led to no significant reduction of intraoral viral load. Replicating virus could only be determined from one baseline specimen. CONCLUSION: A 1% hydrogen peroxide mouthrinse does not reduce the intraoral viral load in SARS-CoV-2-positive subjects. However, virus culture did not yield any indication on the effects of the mouthrinse on the infectivity of the detected RNA copies. CLINICAL RELEVANCE: The recommendation of a preprocedural mouthrinse with hydrogen peroxide before intraoral procedures is questionable and thus should not be supported any longer, but strict infection prevention regimens are of paramount importance. TRIAL REGISTRATION: German Clinical Trials Register (ref. DRKS00022484).

Implant survival or implant success? Evaluation of implant-based prosthetic rehabilitation in head and neck cancer patients-a prospective observational study.

OBJECTIVES: Definition of implant success is unclear in prosthetic implant-based rehabilitation of head neck cancer patients. MATERIALS AND METHODS: Fifty-two patients with 309 inserted implants were included in this prospective observational study. Implant survival (in situ and loaded) and implant success (modified Albrektsson criteria) at 2-year follow-up were evaluated under the influence of patient- and implant-specific variables. RESULTS: Thirty-nine patients with 234 implants finished the study. Overall implant survival after 2 years was 92.3% (216/234) with an osseointegration rate of 94% (220/234). Implant success was 78.6% (184/234). Main reasons for failure were "bone resorption > 1.7mm" (n = 27, 11.5%) and "implant not in situ or not loaded" (n = 18, 7.7%). Smoking (OR 3.1, p = 0.034), bone grafts (OR 2.4, p = 0.021) and radiation dose > 60 Gy (OR 3.8, p = 0.025) revealed as significant predictors for implant failure. CONCLUSION: Implant survival differs significantly from implant success in head and neck cancer patients. Implant success is mainly determined by radiographic peri-implant bone resorption. CLINICAL RELEVANCE: Dealing with head and neck cancer patients a higher amount of peri-implant bone resorption must be taken into account and warrants for intensified implant monitoring.

PD-L1 Influences Cell Spreading, Migration and Invasion in Head and Neck Cancer Cells.

The programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis blockade has been implemented in advanced-stage tumor therapy for various entities, including head and neck squamous cell carcinoma (HNSCC). Despite a promising tumor response in a subgroup of HNSCC patients, the majority suffer from disease progression. PD-L1 is known to influence several intrinsic mechanisms in cancer cells, such as proliferation, apoptosis, migration and invasion. Here, we modulated PD-L1 expression in three HNSCC cell lines with differential intrinsic PD-L1 expression. In addition to an alteration in the epithelial-to-mesenchymal transition (EMT) marker expression, we observed PD-L1-dependent cell spreading, migration and invasion in a spheroid spreading assay on four different coatings (poly-L-lysine, collagen type I, fibronectin and Matrigel®) and a chemotactic transwell migration/invasion assay. Furthermore, the overexpression of PD-L1 led to increased gene expression and small interfering ribonucleic acid (siRNA) knockdown and decreased gene expression of Rho-GTPases and related proteins in a RT2 Profiler™ PCR Array. Rac1 and Rho-GTPase pulldown assays revealed a change in the activation state concordantly with PD-L1 expression. In summary, our results suggest a major role for PD-L1 in favoring cell motility, including cell spreading, migration and invasion. This is presumably caused by altered N-cadherin expression and changes in the activation states of small Rho-GTPases Rho and Rac1.

Influence of early elective tracheostomy on the incidence of postoperative complications in patients undergoing head and neck surgery.

BACKGROUND: The incidence of postoperative complications after head and neck surgery is high. This study evaluated the influence of early elective tracheostomy on the incidence of postoperative pneumonia and delirium. METHODS: We reviewed the data of all patients who had undergone removal of an oropharyngeal tumor and microsurgical tissue transfer at our department in a two year period. Pearson's Chi-squared test and the Fischer's exact t-test were then used to measure the influence of patients' preexisting conditions and risk factors and of early elective tracheostomy on the incidence of postoperative complications. RESULTS: In total, 47 cases were analyzed. Patients with an endotracheal tube were ventilated for a longer time (3.4 days vs. 1.5 days) and were transferred to the regular ward later (after 6.9 days vs. 4.7 days) than patients with tracheostomy. Only 1 (2.1%) of the patients with a tracheostomy developed pneumonia in contrast to 5 intubated patients (10.6%) and only 2 patients with a tracheostomy developed postoperative delirium (9.5%) in contrast to 8 intubated patients (30.8%). CONCLUSION: Early primary tracheostomy in patients undergoing resection of oropharyngeal cancer seems to have numerous benefits, such as lower complication rates with regard to pneumonia and postoperative delirium and shorter duration of both mechanical ventilation and intensive care unit (ICU) stays. Further studies have to evaluate if these benefits also influence morbidity and mortality rates.

Predictive value of FHIT, p27, and pERK1/ERK2 in salivary gland carcinomas: a retrospective study.

OBJECTIVES: The aim of this study was to investigate the predictive value of the biomarkers FHIT, p27, and pERK1/ERK2 in salivary gland carcinomas. MATERIAL AND METHODS: Immunohistochemical staining of FHIT, p27, and pERK1/ERK2 of 265 patients with salivary gland carcinomas was conducted, and associations with clinico-histopathological data, overall survival, and disease-specific survival were examined. RESULTS: Expression of FHIT (quick score 98.7 vs. 206.4) and p27 (QS 187.3 vs. 244.8) was significantly lower in carcinomas compared to non-tumor control tissue. Loss of FHIT frequently occurred in ACC (55.2%), SDC (68.2%), and SCC (100%). In the totality of tumors, loss of FHIT expression was found in 46.7% (106/227) and was significantly associated with advanced T stage and UICC stage, high-grade histology, loss of p27, PI3K, and survivin. FHIT positivity went along with significantly better overall and disease-specific survival. Negativity of p27 occurred in 28.7% (70/244) of tumors, particularly in SDC (54.4%) and SCC (50%). In the totality of tumors, p27 was associated with advanced patient age, high-grade histology, PI3K, survivin as well as better overall and disease-specific survival (p < 0.05). Positive pERK1/ERK2 expression correlated with positive survivin expression but did not affect overall survival in the totality of tumors. In mucoepidermoid carcinomas, pERK1/ERK2 expression was associated with low-grade malignancy, positive nuclear survivin, and better disease-specific survival. CONCLUSIONS: Loss of FHIT and p27 characterizes aggressive tumor growth and unfavorable prognosis in salivary gland cancer. CLINICAL RELEVANCE: The results may help to stratify patient-specific therapies according to individual tumor characteristics.

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.

OBJECTIVES: Head 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 METHODS: Proliferation (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 12 Gray, Gy). RESULTS: Cell 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 (2 Gy) 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. CONCLUSION: PCI 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 RELEVANCE: HNSCC use HSP to escape radiation-induced apoptosis and certain subtypes might increase proliferation after low-dose irradiation.

Biological predictors of radiosensitivity in head and neck squamous cell carcinoma.

OBJECTIVES: The 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 METHODS: The 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. RESULTS: Complete tumor response was associated with increased patient age (p = 0.007), N0-status (p = 0.022), M0-status (p = 0.007), and p16-positivity (p = 0.022). High PD-L1 was associated with M0-status (p = 0.026) and indicated tumor response to irradiation (p = 0.057); survivin expression showed higher rates of response failure (p = 0.073). Low PD-1 was associated with increased T-stage (p = 0.029) and local recurrence (p = 0.014). High PD-1 was strongly correlated with PD-L1-positive tumor infiltrating lymphocytes (p < 0.001). Low PD-L1 showed a significant correlation with high c-Met expression (p = 0.01). Significant predictors for unfavorable univariate survival were incomplete tumor response (DSS, p < 0.001), single radiotherapy (DSS, p = 0.002), M1-status (DSS, p < 0.001), decreased radiation dose (DSS, p = 0.014), high survivin (DSS, p = 0.045), and high c-Met (OS, p < 0.05). Survivin and c-Met also showed prognostic significance in multivariate survival analysis. CONCLUSIONS: P16 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 RELEVANCE: The findings may improve patient-specific therapy according to individual tumor characteristics.