Clinicopathological Factors as Predictors for Establishment of Patient Derived Head and Neck Squamous Cell Carcinoma Organoids.

INTRODUCTION: Patient derived organoids (PDOs) are 3D in vitro models and have shown to better reflect patient and tumor heterogeneity than conventional 2D cell lines. To utilize PDOs in clinical settings and trials for biomarker discovery or drug response evaluation, it is valuable to determine the best way to optimize sample selection for maximum PDO establishment. In this study, we assess patient, tumor and tissue sampling factors and correlate them with successful PDO establishment in a well-documented cohort of patients with head and neck squamous cell carcinoma (HNSCC). METHODS: Tumor and non-tumorous adjacent tissue samples were obtained from HNSCC patients during routine biopsy or resection procedures at the University Medical Center Utrecht. The tissue was subsequently processed to establish PDOs. The sample purity was determined as the presence of epithelial cells in the culture on the day of organoid isolation as visualized microscopically by the researcher. PDO establishment was recorded for all samples. Clinical data was obtained from the medical records and was correlated to PDO establishment and presence of epithelial cells. RESULTS: Organoids could be established in 133/250 (53.2%) primary tumor site tissues. HNSCC organoid establishment tended to be more successful if patients were younger than the median age of 68 years (74/123 (60.2%) vs. 59/127 (46.5%), p = 0.03). For a subset of samples, the presence of epithelial cells in the organoid culture on the day of organoid isolation was recorded in 112/149 (75.2%) of these samples. When cultures were selected for presence of epithelial cells, organoid establishment increased to 76.8% (86/112 samples). CONCLUSION: This study found a trend between age and successful organoid outgrowth in patients with HNSCC younger than 68 years and emphasizes the value of efficient sampling regarding PDO establishment.

p-mTOR, p-ERK and PTEN Expression in Tumor Biopsies and Organoids as Predictive Biomarkers for Patients with HPV Negative Head and Neck Cancer.

BACKGROUND: Survival rates of head and neck squamous cell carcinoma (HNSCC) have only marginally improved in the last decades. Hence there is a need for predictive biomarkers for long-time survival that can help to guide treatment decisions and might lead to the development of new therapies. The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway is the most frequently altered pathway in HNSCC, genes are often mutated, amplificated and overexpressed causing aberrant signaling affecting cell growth and differentiation. Numerous genetic alterations of upstream and downstream factors have currently been clarified. However, their predictive value has yet to be established. Therefore we assess the predictive value of p-mTOR, p-ERK and PTEN expression. METHODS: Tissue microarrays (TMA's) of HPV-negative patients with oropharyngeal (n = 48), hypopharyngeal (n = 16) or laryngeal (n = 13) SCC, treated with primary chemoradiation (cisplatin/carboplatin/cetuximab and radiotherapy), were histologically stained for p-mTOR, PTEN and p-ERK. Expression was correlated to overall survival (OS), disease free survival (DFS) and locoregional control (LRC). Also p-mTOR was histologically stained in a separate cohort of HNSCC organoids (n = 8) and correlated to mTOR-inhibitor everolimus response. RESULTS: High p-mTOR expression correlated significantly with worse OS in multivariate analysis in the whole patient cohort [Hazar Ratio (HR) 1.06, 95%CI 1.01-1.11, p = 0.03] and in the cisplatin/carboplatin group with both worse OS (HR 1.09, 95%CI 1.02-1.16, p = 0.02) and DFS (HR 1.06, 95%CI 1.00-1.12, p = 0,04). p-ERK expression correlated significantly with DFS in univariate analysis in the whole patient cohort (HR 1.03, 95%CI 1.00-1.05, p = 0.04) and cisplatin/carboplatin group (HR 1.03, 95%CI 1.00-1.07, p = 0.04). PTEN-expression did not correlate with OS/DFS/LRC. Better organoid response to everolimus correlated significantly to higher p-mTOR expression (Rs = - 0.731, p = 0.04). CONCLUSIONS: High p-mTOR expression predicts and high p-ERK expression tends to predict worse treatment outcome in HPV negative HNSCC patients treated with chemoradiation, providing additional evidence that these markers are candidate prognostic biomarkers for survival in this patient population. Also this study shows that the use of HNSCC organoids for biomarker research has potential. The role of PTEN expression as prognostic biomarker remains unclear, as consistent evidence on its prognostic and predictive value is lacking.

Zygomaticomaxillary complex fracture repair with intraoperative CBCT imaging. A prospective cohort study.

The aim of this study was to evaluate the value of intraoperative conebeam computed tomography (CBCT) imaging in the treatment of zygomaticomaxillary complex (ZMC) fractures. A prospective single center cohort study was performed. Included were consecutive patients who underwent surgery for a unilateral ZMC fracture. An intraoperative CBCT scan was performed after reduction of the ZMC fracture. Revision reduction was performed of the ZMC and/or orbital floor (OF) on indication. The preoperative and postoperative asymmetry of the outer surface of the ZMC was measured on digital 3D-models of CBCT scans, using a mirroring and surface-based matching technique. The postoperative asymmetry of the ZMC in the study group was compared to the asymmetry of the ZMC in the control group with healthy individuals. A total of 38 patients with a unilateral ZMC fracture were included. The mean postoperative asymmetry in the study group (1.67 mm, SD 0.89) was less than the mean preoperative asymmetry (2.69 mm, SD 0.95) (paired samples T-test p < 0.01) but showed no statistically significant difference with the mean asymmetry in the healthy control group (1.40 mm, SD 0.54) (independent samples T-test p = 0.31). Revision reduction of the ZMC and/or OF fracture had been performed in 11 cases after malalignment was noted on the intraoperative CBCT. The indication for intraoperative revision reduction was associated with comminuted ZMC fractures and/or fractures with indication for OF reduction (Pearson Chi Square p < 0.01). Within the limitations of the study, intraoperative CBCT imaging seemed to have a positive influence on ZMC fracture treatment, especially in the case of comminuted ZMC fractures and/or fractures with indication for OF treatment.

A Novel Method for Quantitative Three-Dimensional Analysis of Zygomatico-Maxillary Complex Symmetry.

OBJECTIVE: To develop a reliable and accurate method to quantify the symmetry of the zygomaticomaxillary complex (ZMC). METHODS: Virtual three-dimensional models were created from 53 computed-tomography scans: 15 healthy cases without maxillofacial disorders and 38 patients with ZMC fractures requiring surgical treatment.Asymmetry of the ZMC was measured using a mirroring and surface-based matching technique that uses the anterior cranial fossa as reference to determine the symmetrical position of the ZMC. The measure for ZMC asymmetry was defined as mean surface distance (MSD) between the ZMC-surface and the symmetrical position.Reliability of the method was tested in the 15 healthy cases. Inter-and intra-observer correlation coefficients (Ce) and variabilities were assessed. Accuracy was assessed by comparing ZMC asymmetry between healthy and ZMC fracture cases, and by assessing correlation of ZMC fracture severity with ZMC asymmetry. RESULTS: The average MSD of the 15 healthy cases was 1.40 ± 0.54 mm and the average MSD of the 38 ZMC fracture cases was 2.69 ± 0.95 mm ( P < 0.01). Zygomaticomaxillary complex asymmetry correlated with fracture severity ( P = 0.01). Intra-rater CC was 0.97 with an intra-rater variability of 0.09 ± 0.11 mm. Inter-rater Ce was 0.95 with an inter-rater variability of 0.12 ± 0.13 mm. CONCLUSIONS: Our method is reliable and accurate for quantitative three-dimensional analysis of ZMC-symmetry. It takes into account asymmetry caused by the shape of the ZMC as well as asymmetry caused by the position of the ZMC. CLINICAL RELEVANCE: This method is useful for the evaluation of ZMC asymmetry associated with congenital and acquired disorders of craniofacial skeleton, for surgical planning and for evaluation of postoperative results.

Predictive Value of EGFR-PI3K-AKT-mTOR-Pathway Inhibitor Biomarkers for Head and Neck Squamous Cell Carcinoma: A Systematic Review.

BACKGROUND: Understanding molecular pathogenesis of head and neck squamous cell carcinomas (HNSCC) has considerably improved in the last decades. As a result, novel therapeutic strategies have evolved, amongst which are epidermal growth factor receptor (EGFR)-targeted therapies. With the exception of cetuximab, targeted therapies for HNSCC have not yet been introduced into clinical practice. One important aspect of new treatment regimes in clinical practice is presence of robust biomarkers predictive for therapy response. METHODS: We performed a systematic search in PubMed, Embase and the Cochrane library. Articles were included if they investigated a biomarker for targeted therapy in the EGFR-PI3K-AKT-mTOR-pathway. RESULTS: Of 83 included articles, 52 were preclinical and 33 were clinical studies (two studies contained both a preclinical and a clinical part). We classified EGFR pathway inhibitor types and investigated the type of biomarker (biomarker on epigenetic, DNA, mRNA or protein level). CONCLUSION: Several EGFR-PI3K-AKT-mTOR-pathway inhibitor biomarkers have been researched for HNSCC but few of the investigated biomarkers have been adequately confirmed in clinical trials. A more systematic approach is needed to discover proper biomarkers as stratifying patients is essential to prevent unnecessary costs and side effects.