Clinical outcome and prognostic factors in recurrent oral squamous cell carcinoma after primary surgical treatment: a retrospective study.

OBJECTIVES: Survival for patients with recurrent oral squamous cell carcinoma is usually poor, and the most effective treatment has not yet been clearly defined. The present study evaluates the outcome in radiotherapy-naïve patients after recurrence of oral squamous cell carcinoma with respect to different treatment modalities including surgery, radiation, chemoradiation, and palliative treatment. PATIENTS AND METHODS: In this retrospective study, we included all patients with primary oral squamous cell carcinoma who received exclusively surgical therapy between 2010 and 2020 and who suffered from locoregional recurrence in their follow-up. Patients with previous adjuvant therapy were excluded from this protocol. Clinical and pathological parameters were collected and statistically evaluated. Survival analysis was performed according to Kaplan-Meier. The primary endpoints were overall and progression-free survival in dependance of treatment strategy for recurrent tumors. RESULTS: Out of a total of 538 patients with surgically treated primary oral squamous cell carcinoma, 76 patients met the inclusion criteria. The mean follow-up was 38 ± 32 months. Patients who received surgically based therapy had a significantly better outcome in terms of disease-free survival (DFS) and overall survival (OS) (DFS p < 0.001; OS p < 0.001). The presence of regional metastases and a short disease-free interval (DFI) between primary and recurrent cancer were significant predictors for adverse outcomes (DFI p < 0.001). CONCLUSION: We recommend primary surgical therapy for radiotherapy-naïve patients with recurrent oral squamous cell carcinoma, supplemented by risk-adapted adjuvant therapy. CLINICAL RELEVANCE: Surgical therapy continues to play a central role in the treatment of radiotherapy-naïve patients with recurrent oral squamous cell carcinoma.

A six-gene expression signature related to angiolymphatic invasion is associated with poor survival in laryngeal squamous cell carcinoma.

PURPOSE: Angiolymphatic invasion serves as a histopathological risk factor for unfavorable survival in head and neck squamous cell carinoma. The aim of the study was to explore  the molecular mechanisms characterizing angiolymphatic invasion and therefore identify a gene expression signature related to angiolymphatic invasion. METHODS: Gene expression analysis of head and neck squamous cell carcinoma was carried out based on clinical and whole genome expression data provided by The Cancer Genome Atlas. Results were validated in an independent cohort of laryngeal squamous cell carcinoma and confirmed by immunohistochemistry staining. RESULTS: A gene expression signature consisting of six genes (SHH, SLC18A3, LCE3E, LCE2B, LCE3D and DSG-1) related to angiolymphatic invasion was identified. The gene expression profile identified a subset of patients with decreased overall survival (p = 0.02, log rank test), which was most prominent for patients with laryngeal squamous cell carcinoma (p = 0.004, log rank test). Furthermore, these patients showed a significant shorter progression-free survival (p = 0.002, log rank test). By use of this gene expression signature, patients at high risk of recurrence could be identified even if morphological changes were not yet recognizable. CONCLUSION: Angiolymphatic invasion is characterized by a distinct histopathological phenotype and specific gene expression signature. The newly identified signature might serve as a reliable predictor of outcome in laryngeal cancer and add additional benefit to histopathological evaluation.

Development of osteomyelitis following dental abscesses-influence of therapy and comorbidities.

OBJECTIVES: Bacterial osteomyelitis of the jaw is a severe disease potentially requiring extensive surgical treatment. We have evaluated the incidence rates of bacterial osteomyelitis following dental abscessation associated with primary or secondary tooth extraction. MATERIALS AND METHODS: A retrospective cohort study was designed and included patients with dental abscesses and surgical treatment including the extraction of focus teeth. Patients were either treated with primary removal during acute infection or secondary delayed extraction within an infection-free interval. The primary outcome variable was the occurrence of bacterial osteomyelitis following the abscess. Secondary outcomes were the influence of general disease, antibiotic therapy, and surgical technique. RESULTS: One hundred nine patients were enrolled in the study; 4 patients (3.7%) developed osteomyelitis. Primary tooth extraction was performed on all these patients (p = 0.017). Significant associations of diabetes (p = 0.001), the use of clindamycin (p = 0.025), and transcutaneous incision (p = 0.017) with the incidence of osteomyelitis were detected. CONCLUSIONS: More severe infections may be associated with a higher risk for the development of osteomyelitis following dental abscesses. A history of diabetes and clindamycin therapy might form further predisposing risk factors. Because of the low incidence and the small case number, our data need to be interpreted carefully. CLINICAL RELEVANCE: Osteomyelitis of the jaw is a rare but severe disease that may require extensive therapy and that impairs the quality of life of affected patients. The evaluation of risk factors to enable further reduction of incidence is therefore urgently required.

A novel bi-directional promoter system allows tunable recombinant protein production in Pichia pastoris.

BACKGROUND: The methylotrophic yeast Pichia pastoris is a well-studied host organism for recombinant protein production, which is usually regulated either by a constitutive promoter (e.g. promoter of glyceraldehyde-3-phosphate dehydrogenase; PGAP) or an inducible promoter (e.g. promoter of alcohol oxidase 1; PAOX1). Both promoter systems have several advantages and disadvantages; with one of the main disadvantages being their lack of tunability. Various novel promoter systems, which are either inducible or de-repressed, allowing higher degrees of freedom, have been reported. Recently, bi-directional promoter systems in P. pastoris with two promoter systems regulating recombinant expression of one or more genes were developed. In this study, we introduce a novel bi-directional promoter system combining a modified catalase promoter system (PDC; derepressible and inducible) and the traditional PAOX1, allowing tunable recombinant protein production. RESULTS: We characterized a recombinant P. pastoris strain, carrying the novel bi-directional promoter system, during growth and production in three dynamic bioreactor cultivations. We cloned the model enzyme cellobiohydralase downstream of either promoter and applied different feeding strategies to determine the physiological boundaries of the strain. We succeeded in demonstrating tunability of recombinant protein production solely in response to the different feeding strategies and identified a mixed feed regime allowing highest productivity. CONCLUSION: In this feasibility study, we present the first controlled bioreactor experiments with a recombinant P. pastoris strain carrying a novel bi-directional promotor combination of a catalase promoter variant (PDC) and the traditional PAOX1. We demonstrated that this bi-directional promoter system allows tunable recombinant protein expression only in response to the available C-sources. This bi-directional promoter system offers a high degree of freedom for bioprocess design and development, making bi-directional promoters in P. pastoris highly attractive for recombinant protein production.

Glyco-variant library of the versatile enzyme horseradish peroxidase.

When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library.

Treatment delay and tumor size in patients with oral cancer during the first year of the COVID-19 pandemic.

BACKGROUND: We set out to investigate how the ongoing coronavirus pandemic affected the size of tumors and the duration of treatment delay in patients with surgically treated oral squamous cell carcinoma. METHODS: Patients with surgically treated oral cavity squamous cell carcinoma were assessed retrospectively and divided into two groups depending on when they had first presented at our clinic. Patients presenting from 2010 to 2019, that is, before COVID-19 onset (n = 566) were compared to patients presenting in 2020 (n = 58). RESULTS: A total of 624 patients were included. Treatment delay was significantly longer in 2020 (median = 45 days) versus 2010-2019 (median = 35 days) (p = 0.004). We observed a higher pathological T classification in 2020 (p = 0.046), whereas pathological N classification was unchanged between groups (p = 0.843). CONCLUSIONS: While extraordinary efforts continue to be made in the context of the pandemic, it is imperative that this does not lead to significant disadvantages for many people with oral cancer.

Multiple Sequential Free Flap Reconstructions of the Head and Neck: A Single-Center Experience.

BACKGROUND: Free flap reconstruction is the standard of care in extensive defects of the head and neck area, and although most patients may be treated sufficiently with one flap, recurrence of a malignant tumor or failure of a previous reconstruction may make the use of a second (or more) flap necessary. The aim of this study was to evaluate the indications and success rates of multiple consecutive reconstructive procedures in a large cohort of patients. METHODS: Nine hundred ninety-six free flap reconstructions were retrospectively analyzed and cases of sequential reconstructions in the same patient were identified. Indications, success rates, perioperative procedures, and frequently used flaps were evaluated. RESULTS: Two hundred twenty cases of sequential microvascular reconstructions were identified, ranging from two to six flaps per patient. The overall flap success rate was 89.1 percent. A history of diabetes was identified as a risk factor for flap failure (p = 0.029). There was no association of flap loss with the number of reconstructive procedures per patient. CONCLUSIONS: The use of several free flaps in the same patient is a feasible option for patients suffering from recurrent tumors or to improve quality of life by a secondary reconstruction. A salvage free flap transfer to replace a lost transplant exhibits good success rates. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Full-Thickness Tumor Resection of Oral Cancer Involving the Facial Skin-Microsurgical Reconstruction of Extensive Defects after Radical Treatment of Advanced Squamous Cell Carcinoma.

Advanced tumors of the head and neck are challenging for the treatment specialist due to the need to synergize oncological and functional requirements. Free flap reconstruction has been established as the standard of care for defects following tumor resection. However, depending on the affected anatomic subsite, advanced tumors may impose specific difficulties regarding reconstruction, especially when full-thickness resection is required. This study aimed to evaluate reconstructive strategies and oncological outcomes in patients with full-thickness resection of the oral cavity. A total of 33 patients with extensive defects due to squamous cell carcinoma of the oral cavity were identified. Indications, reconstructive procedures, and clinical outcome were evaluated. Thirty-two patients (97%) presented locally advanced tumors (T3/T4). Complete tumor resection was achieved in 26 patients (78.8%). The anterolateral thigh flap was the most frequently used flap (47.1%), and the primary flap success rate was 84.8%. The cohort demonstrated a good local control rate and moderate overall and progression-free survival rates. Most patients regained full competence regarding oral alimentation and speech. Full-thickness tumor resections of the head and neck area may be necessary due to advanced tumors in critical anatomic areas. In many cases, radical surgical treatment leads to good oncological results. Free flap reconstruction has been shown to be a suitable option for extensive defects in aesthetically challenging regions.

Treatment delay in early-stage oral squamous cell carcinoma and its relation to survival.

The aim of this study was to investigate the impact of a prolonged treatment delay on survival in patients with primary oral squamous cell carcinoma. The investigators hypothesized that treatment delay affects survival, supposing a poor outcome in patients with prolonged treatment initiation. In addition, a critical treatment delay should be defined. Inclusion criteria were a histopathological diagnosis of primary squamous cell carcinoma of the oral cavity and a surgery-based treatment of the tumor. Patients with a history of previously diagnosed malignancies and patients with distant metastasis at the time of diagnosis were excluded from this protocol. Common clinical and histopathological data were assessed retrospectively. Treatment delay was analyzed for the interval between initial presentation and the date of surgery. A total of 484 patients could be included. Considering early-stage patients, the risk of death increases by 1.8% for each day that the treatment delay is prolonged if all other characteristics do not change (p = 0.0035). In patients with advanced disease, a prolonged treatment delay does not affect the risk of death (p = 0.9134). In terms of progression-free survival, treatment delay tends to be associated with a higher risk of recurrence in early-stage disease, but without being statistically significant (p = 0.0718). For patients with early-stage disease, a treatment delay of 20 days is critical regarding overall survival (p = 0.011). For patients with advanced-stage disease, no significant differences have been observed. As patients with early-stage oral squamous cell carcinoma profit from early treatment initiation, we suggest an acceptable maximum treatment delay of no more than 20 days in the surgical management of these patients.

Digital Pathology Scoring of Immunohistochemical Staining Reliably Identifies Prognostic Markers and Anatomical Associations in a Large Cohort of Oral Cancers.

Utilizing digital pathology algorithms for the objective quantification of immunohistochemical staining, this study aimed to identify robust prognostic biomarkers for oral cancer. Tissue microarrays with specimens of a large cohort of oral squamous cell carcinoma (n=222) were immunohistochemically stained to determine the expression of PD-L1, EGFR, and COX-2 and the amount of infiltrating NK cells and CD8-positive T cells. Immunoreactivity scores were assessed using both a classical manual scoring procedure and a digital semi-automatic approach using QuPath. Digital scoring was successful in quantifying the expression levels of different prognostic biomarkers (CD8: p<0.001; NK cells: p=0.002, PD-L1: p=0.026) and high levels of concordance with manual scoring results were observed. A combined score integrating EGFR expression, neck node status and immune cell signatures with a significant impact on overall and progression-free survival was identified (p<0.001). These data may contribute to the ongoing research on the identification of reliable and clinically relevant biomarkers for the individualization of primary and adjuvant treatment in oral cancer.

3D-printed individualized tooth-borne tissue retraction devices compared to conventional dental splints for head and neck cancer radiotherapy: a randomized controlled trial.

BACKGROUND: Despite modern treatment techniques, radiotherapy (RT) in patients with head and neck cancer (HNC) may be associated with high rates of acute and late treatment-related toxicity. The most effective approach to reduce sequelae after RT is to avoid as best as possible healthy tissues and organs at risk from the radiation target volume. Even small geometric changes can lead to a significant dose reduction in normal tissue and better treatment tolerability. The major objective of the current study is to investigate 3D printed, tooth-borne tissue retraction devices (TRDs) compared to conventional dental splints for head and neck RT. METHODS: In the current two-arm randomized controlled phase II trial, a maximum of 34 patients with HNC will be enrolled. Patients will receive either TRDs or conventional dental splints (randomization ratio 1:1) for the RT. The definition of the target volume, modality, total dose, fractionation, and imaging guidance is not study-specific. The primary endpoint of the study is the rate of acute radiation-induced oral mucositis after RT. The quality of life, local control and overall survival 12 months after RT are the secondary endpoints. Also, patient-reported outcomes and dental status, as well as RT plan comparisons and robustness analyzes, will be assessed as exploratory endpoints. Finally, mesenchymal stem cells, derived from the patients' gingiva, will be tested in vitro for regenerative and radioprotective properties. DISCUSSION: The preliminary clinical application of TRD showed a high potential for reducing acute and late toxicity of RT in patients with HNC. The current randomized study is the first to prospectively investigate the clinical tolerability and efficacy of TRDs for radiation treatment of head and neck tumors. TRIAL REGISTRATION: ClinicalTrials.gov; NCT04454697; July 1st 2020; https://clinicaltrials.gov/ct2/show/record/NCT04454697 .

Adsorptive filtration: A case study for early impurity reduction in an Escherichia coli production process.

Primary recovery of intracellular products from Escherichia coli requires cell disruption which leads to a massive release of process-related impurities burdening subsequent downstream process (DSP) unit operations. Especially, DNA and endotoxins challenge purification operations due to their size and concentrations. Consequently, an early reduction in impurities will not only simplify the production process but also increase robustness while alleviating the workload afterward. In the present work, we studied the proof of concept whether a nonwoven anion exchange filter material decreases soluble impurities immediately at the clarification step of E. coli DSP. In a first attempt, endotoxin burden was reduced by 4.6-fold and the DNA concentration by 3.6-fold compared to conventional depth filtration. A design of experiment for the adsorptive filtration approach was carried out to analyze the influence of different critical process parameters (CPPs) on impurity reduction. We showed that depending on the CPPs chosen, a DNA lowering of more than 3 log values, an endotoxin decrease of approximately 7 logs, and a minor HCP clearance of at least 0.3 logs could be achieved. Thus, we further revealed a chromatography column protecting effect when using adsorptive filtration beforehand.

Head motion during cone-beam computed tomography: Analysis of frequency and influence on image quality.

PURPOSE: Image artifacts caused by patient motion cause problems in cone-beam computed tomography (CBCT) because they lead to distortion of the 3-dimensional reconstruction. This prospective study was performed to quantify patient movement during CBCT acquisition and its influence on image quality. MATERIALS AND METHODS: In total, 412 patients receiving CBCT imaging were equipped with a wireless head sensor system that detected inertial, gyroscopic, and magnetometric movements with 6 dimensions of freedom. The type and amplitude of movements during CBCT acquisition were evaluated and image quality was rated in 7 different anatomical regions of interest. For continuous variables, significance was calculated using the Student t-test. A linear regression model was applied to identify associations of the type and extent of motion with image quality scores. Kappa statistics were used to assess intra- and inter-rater agreement. Chi-square testing was used to analyze the impact of age and sex on head movement. RESULTS: All CBCT images were acquired in a 10-month period. In 24% of the investigations, movement was recorded (acceleration: >0.10 [m/s2]; angular velocity: >0.018 [°/s]). In all examined regions of interest, head motion during CBCT acquisition resulted in significant impairment of image quality (P<0.001). Movement in the horizontal and vertical axes was most relevant for image quality (R2>0.7). CONCLUSION: Relevant head motions during CBCT imaging were frequently detected, leading to image quality loss and potentially impairing diagnosis and therapy planning. The presented data illustrate the need for digital correction algorithms and hardware to minimize motion artefacts in CBCT imaging.

Squamous cell carcinoma of the mandible - Patterns of metastasis and disease recurrence in dependence of localization and therapy.

INTRODUCTION: Oral squamous cell carcinomas exhibit distinct patterns of disease progression, depending on their localisation. This study aimed to evaluate clinicopathological data in patients with tumors of the mandibular alveolar process, to facilitate risk assessment and therapy planning. MATERIALS AND METHODS: A retrospective cohort study was designed including patients with squamous cell carcinoma of the mandibular gingiva. Clinical and pathological data were collected to determine the rate of cervical metastases and clinical outcomes depending on tumor stage, localization (anterior, intermediate and posterior) and the extent of tumor resection. RESULTS: 120 patients were included in the analysis. Rate of metastases was 42.6%. Tumors of the anterior part of the mandible exhibited significantly higher rates of bilateral metastases (anterior: 85.7%, intermediate: 15.8%, posterior: 4%, p < 0.001) and local recurrence (anterior: 25%, intermediate: 16.3%, posterior: 5.5%, p = 0.03) compared to posterior malignancies. CONCLUSION: Tumors of the anterior segment of the mandible are characterized by high rates of metastases and local recurrence. Therefore, we propose radical segmental resection and bilateral neck dissection in those patients.

IGF1 inclusion bodies: A QbD based process approach for efficient USP as well as early DSP unit operations.

E. coli is an attractive host organism for strong recombinant protein expression. It expresses products either as soluble protein or as inclusion bodies (IB). IBs are insoluble, mostly inactive aggregates. However, recent progress enabled the efficient refolding back into their bioactive structure. Targeted IB production processes have been designed based on their characteristic features such as high yields along with purity and their simple separation. More profound process knowledge is needed to reveal interacting parameters required for quality by design grounded process development. This enables strategies for simplifying and accelerating upstream as well as downstream procedures. We present a workflow for gathering deeper process knowledge by a design of experiment approach for improved IGF1 IB formation in relation to impurity concentration. An IB expression maximum of 19.8 mgIGF1·gDCW-1 was found at pH 6.5, 37 °C and an IPTG induction of >45 µmol gDCW-1 for 12 h. Subsequently, three refolding buffers were tested together with a nonwoven anion exchange adsorber filter module. Knowledge-based buffer selection enabled high impurity log reduction values (LRVEndotoxin = 4.9; LRVDNA = 4.8, LRVHCP = 0.1-1) as well as chromatography column guarding potential by using those adsorptive matrices. Furthermore, adsorptive filtration followed by tangential flow filtration proved to be a promising alternative for product concentration.

The prognostic significance of the lymph node ratio in oral cancer differs for anatomical subsites.

The aim of this study was to validate the prognostic significance of the lymph node ratio (LNR) in patients suffering from oral squamous cell carcinoma in regard to different anatomical subsites. A cohort of 430 patients was investigated to determine the rates of primary metastasis and local and regional disease recurrence. Correlation analysis of the LNR with relevant clinical and pathological parameters was performed. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the prognostic impact for different subsites. Significantly differing rates of primary metastasis and loco-regional disease recurrence were found for cancer of different anatomical subsites of the head and neck. Furthermore, ROC curve analysis suggested that LNR has prognostic relevance in subsets of cancer (tongue, P< 0.001; alveolar process, P= 0.04; maxilla, P= 0.03; buccal mucosa, P= 0.02). The LNR of cancer located in the soft palate (P= 0.6) and floor of the mouth (P= 0.11) showed little or no association with the clinical outcome. There is the need for a more sensitive consideration of the LNR as a factor in the assessment of risk and the treatment decision, as the anatomical subsite plays a crucial role in its impact on the clinical outcome.

Prospective feasibility analysis of salvage surgery in recurrent oral cancer in terms of quality of life.

OBJECTIVES: The goals of the present study were to prospectively analyze salvage surgery with microvascular reconstruction in recurrent squamous cell carcinoma of the oral cavity (OSCC) in terms of oncological outcome and quality of life. PATIENTS AND METHODS: From 2012 to 2015, 28 patients underwent salvage surgery due to recurrent OSCC or second primary OSCC without the option of curative re-irradiation. Endpoints were disease-specific survival and progression-free survival after 12 months. The survival was estimated by using the Kaplan-Meier blotting. Quality of life data (European Organization for Research and Treatment of Cancer - EORTC: QLQ-C30 and QLQ-H&N35) was assessed at baseline and subsequently every 3 months up to one year. RESULTS: Estimated 1-year-survival was 68.4% and progression-free survival was 38.5%. Overall quality of life was significantly reduced three months after salvage surgery [baseline (mean 64.15) versus time 1 (mean 53.04); p = 0.002]. However, the patients experienced a recovery within the first year [baseline (mean 64.15) versus time 4 (mean 70.33); p = 0.176]. Furthermore, the sensation of pain is significantly reduced after salvage surgery [baseline (mean 47.53) versus time 2 (mean 31.25); p = 0.036]. Microvascular reconstruction success rate was 93.1%. CONCLUSION: Salvage surgery is a curative treatment option in recurrent and intensively pretreated OSCC. Microvascular reconstruction is feasible with acceptable morbidity and high success rates. Quality of life can be preserved. Further studies combining checkpoint inhibition with salvage surgery are justified.

The occurrence of cervical metastases in squamous cell carcinoma of the tongue: Is there a rationale for bilateral neck dissection in early-stage tumors?

INTRODUCTION: The aim of this retrospective study was to evaluate the incidence of regional metastases in squamous cell carcinoma (SCC) of the tongue, in order to validate different neck management regime recommendations. MATERIALS AND METHODS: A cohort of 97 patients suffering from primary SCC of the tongue was analyzed in regard to the development of primary and late neck node metastases, considering tumor stage and affected side. Survival analysis was performed to determine the impact of different relevant clinical and pathological factors on overall and progression-free survival. RESULTS: Regional metastases occurred in 29 patients (29.9%). In early-stage tumors (T1 and T2), the rates of primary metastases were 28.6% and 22.4%, respectively. Bilateral cervical metastases are rare but were detected in early-stage cancer in several cases (T1: 2.1%; T2: 11.8%). CONCLUSION: The development of regional metastases in the ipsilateral and contralateral neck, even in early-stage SCC of the tongue, illustrates the importance of elective bilateral neck dissection in the treatment of affected patients.