Transnasal Videoendoscopy for Preoperative Airway Risk Stratification: Development and Validation of a Multivariable Risk Prediction Model.

BACKGROUND: Transnasal flexible videoendoscopy (TVE) of the larynx is a standard of care for the detection and staging of pharyngolaryngeal lesions in otorhinolaryngology. Patients frequently present with existing TVE examinations before anesthesia. Although these patients are considered high risk, the diagnostic value of TVE for airway risk stratification is currently unknown. How can captured images or videos be used for anesthesia planning, and which lesions are most concerning? This study aimed to develop and validate a multivariable risk prediction model for difficult airway management based on TVE findings and to determine whether the discrimination of the Mallampati score can be improved by adding this new TVE model. METHODS: This retrospective single-center development and validation study assessed 4021 patients who underwent 4524 otorhinolaryngologic surgeries at the University Medical Centre Hamburg-Eppendorf between January 1, 2011, and April 30, 2018, with electronically stored TVE videos and included 1099 patients who underwent 1231 surgeries. TVE videos and anesthesia charts were systematically reviewed in a blinded fashion. The Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis was used for variable selection, model development, and cross validation. RESULTS: The prevalence of difficult airway management was 24.7% (304/1231). Lesions at the vocal cords, epiglottis, or hypopharynx were not selected by the LASSO regression, while lesions at the vestibular folds (ß-coefficient 0.123), supraglottic region (ß-coefficient 0.161), arytenoids (ß-coefficient 0.063), and viewing restrictions on the rima glottidis that cover ≥50% of the glottis area (ß-coefficient 0.485) and pharyngeal secretion retention (ß-coefficient 0.372) were relevant risk factors for difficult airway management. The model was adjusted for sex, age, and body mass index. The area under the receiver operating characteristic curve (95% confidence interval) of the Mallampati score was 0.61 (0.57-0.65) and 0.74 (0.71-0.78) of the TVE model combined with Mallampati ( P < .001). CONCLUSIONS: Stored images and videos from TVE examinations can be reused for the purpose of predicting risk associated with airway management. Vestibular fold, supraglottic, and arytenoid lesions are most concerning, especially if they are accompanied by secretion retention or restrict the glottic view. Our data indicate that the TVE model improves discrimination of the Mallampati score and might, therefore, be a useful addition to traditional bedside airway risk examinations.

Analyzing tyrosine kinase activity in head and neck cancer by functional kinomics: Identification of hyperactivated Src family kinases as prognostic markers and potential targets.

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.

Patient derived ex vivo tissue slice cultures demonstrate a profound DNA double-strand break repair defect in HPV-positive oropharyngeal head and neck cancer.

BACKGROUND: HPV-positive head and neck squamous cell carcinoma of the oropharynx (OPSCC) are more sensitive towards radiation than HPV-negative OPSCC. Two main theories exist regarding the underlying mechanism. Stronger lymphocyte infiltration points to an enhanced immunogenicity, whereas data from HPV-positive HNSCC cell lines suggest an enhanced cellular radiosensitivity based on a defect in DNA double-strand break (DSB) repair. The critical limitation of the latter theory is that the evidence was largely derived from a small number of established HPV-positive HNSCC cell lines. METHODS AND MATERIALS: Fresh patient-derived OPSCC samples were cut in 400 µm sections and cultured on cell culture inserts. Slice cultures were irradiated, in part combined with ATM inhibition, and fixed and frozen after 2 and 24 h. DSBs were analyzed by quantification of 53BP1 foci in nuclei co-stained with the SCC marker p63 via immunofluorescence microscopy. RESULTS: Ex vivo OPSCC tumor slice cultures maintained stable oxygenation and proliferation characteristics for at least 3 days. Areas of p63-positivity in immunofluorescence microscopy matched histologically confirmed tumor cell areas in serial sections, indicating the suitability of p63 as a tumor cell marker. p63-positive nuclei in HPV-positive OPSCC tissues (n = 14) showed profoundly elevated numbers of residual radiation-induced DSBs as compared to those from HPV-negative OPSCC (n = 12) (3 Gy: on average 4.9 vs. 1.2 foci per nucleus; p < 0.0001). Within the HPV-positive subgroup, samples derived from patients with a smoking history of less than 10 pack years demonstrated higher residual DSBs as compared to those derived from patients with 10 or more pack years (3 Gy: on average 6.5 vs. 3.2 foci per nucleus; p = 0.0105). Additional ATM inhibition resulted in a substantial increase in residual foci in all 4 HPV-negative samples tested but strikingly only in 2 out of 11 HPV-positive samples. CONCLUSIONS: In summary, our data provide robust, cell line-independent experimental evidence for an intrinsic DSB repair deficiency in HPV-positive OPSCC, strongly suggesting a meaningful contribution to the enhanced clinical radiosensitivity. The reduced effectiveness of ATM inhibition indicates a defect in the ATM-orchestrated DNA damage response. Lower numbers of residual 53BP1 nuclear foci in the ex vivo assay may identify HPV-positive patients with effective DSB repair who should potentially be excluded from de-intensification approaches.

Kinomic comparison of snap frozen and ex vivo-cultured head and neck tumors.

OBJECTIVES: The use of primary tumor tissue in experimental and pre-clinical cancer research is becoming increasingly important. Especially the use of tissue slice cultures of tumor specimen, so called ex vivo cultures or tumor explants, promises functional analysis under approximate physiological conditions. This includes screening and testing of targeted therapeutics directed against deregulated protein kinases. However, it is unclear if ex vivo cultures indeed represent the in situ situation especially with respect to very sensitive and transient molecular processes such as kinase dependent signaling. We now asked here, if and to what extent ex vivo culturing affects kinase activity. MATERIALS AND METHODS: We analyzed the activity of protein tyrosine kinases (PTK) using functional kinome profiling of either snap frozen or ex vivo-cultured tumor tissue samples of head and neck cancer patients. RESULTS: Although we observed a quantitative decline in overall kinase activity after 24 h or 48 h of ex vivo cultivation, we most importantly noticed that the signaling characteristics were conserved in most samples; approximately two thirds of all ex vivo-cultured samples displayed a signaling pattern which was qualitatively comparable to the parental tumor. We could also demonstrate kinase inhibition by treatment of ex vivo slice cultures with the multi-kinase inhibitor staurosporine, although higher concentrations were needed compared to cell cultures. CONCLUSION: We here demonstrate that the tyrosine kinase dependent signaling is conserved under exvivo culturing conditions in the majority of samples, which highlights the power of this method in experimental and pre-clinical cancer research.