Validation of automated positive cell and region detection of immunohistochemically stained laryngeal tumor tissue using digital image analysis.

Objectives: This study aimed to validate a digital image analysis (DIA) workflow for automatic positive cell detection and positive region delineation for immunohistochemical hypoxia markers with a nuclear (hypoxia-inducible factor 1α [HIF-1α]) and a cytoplasmic (pimonidazole [PIMO]) staining pattern. Materials and methods: 101 tissue fragments from 44 laryngeal tumor biopsies were immunohistochemically stained for HIF-1α and PIMO. QuPath was used to determine the percentage of positive cells and to delineate positive regions automatically. For HIF-1α, only cells with strong staining were considered positive. Three dedicated head and neck pathologists scored the percentage of positive cells using three categories (0: <1%; 1: 1%-33%; 2: >33%;). The pathologists also delineated the positive regions on 14 corresponding PIMO and HIF-1α-stained fragments. The consensus between observers was used as the reference standard and was compared to the automatic delineation. Results: Agreement between categorical positivity scores was 76.2% and 65.4% for PIMO and HIF-1α, respectively. In all cases of disagreement in HIF-1α fragments, the DIA underestimated the percentage of positive cells. As for the region detection, the DIA correctly detected most positive regions on PIMO fragments (false positive area=3.1%, false negative area=0.7%). In HIF-1α, the DIA missed some positive regions (false positive area=1.3%, false negative area=9.7%). Conclusions: Positive cell and region detection on biopsy material is feasible, but further optimization is needed before unsupervised use. Validation at varying DAB staining intensities is hampered by lack of reliability of the gold standard (i.e., visual human interpretation). Nevertheless, the DIA method has the potential to be used as a tool to assist pathologists in the analysis of IHC staining.

Correlation and colocalization of HIF-1α and pimonidazole staining for hypoxia in laryngeal squamous cell carcinomas: A digital, single-cell-based analysis.

OBJECTIVE: Tumor hypoxia results in worse local control and patient survival. We performed a digital, single-cell-based analysis to compare two biomarkers for hypoxia (hypoxia-inducible factor 1-alpha [HIF-1α] and pimonidazole [PIMO]) and their effect on outcome in laryngeal cancer patients treated with accelerated radiotherapy with or without carbogen breathing and nicotinamide (AR versus ARCON). MATERIALS AND METHODS: Immunohistochemical staining was performed for HIF-1α and PIMO in consecutive sections of 44 laryngeal cancer patients randomized between AR and ARCON. HIF-1α expression and PIMO-binding were correlated using digital image analysis in QuPath. High-density areas for each biomarker were automatically annotated and staining overlap was analyzed. Kaplan-Meier survival analyses for local control, regional control and disease-free survival were performed to predict a response benefit of ARCON over AR alone for each biomarker. RESULTS: 106 Tissue fragments of 44 patients were analyzed. A weak, significant positive correlation was observed between HIF-1α and PIMO positivity on fragment level, but not on patient level. A moderate strength correlation (r = 0.705, p < 0.001) was observed between the number of high-density staining areas for both biomarkers. Staining overlap was poor. HIF-1α expression, PIMO-binding or a combination could not predict a response benefit of ARCON over AR. CONCLUSION: Digital image analysis to compare positive cell fractions and staining overlap between two hypoxia biomarkers using open-source software is feasible. Our results highlight that there are distinct differences between HIF-1α and PIMO as hypoxia biomarkers and therefore suggest co-existence of different forms of hypoxia within a single tumor.

Feasibility of assessment of skeletal muscle mass on a single cross-sectional image at the level of the fourth thoracic vertebra.

BACKGROUND: Skeletal muscle mass (SMM) determined on computed tomography (CT) is emerging as a novel imaging biomarker. Cross-sectional area (CSA) of SMM at the level of the third lumbar vertebra (L3) on abdominal imaging is considered the clinical reference standard for measuring SMM. In certain patient groups, such as those with oncological or non-oncological lung disease like COVID-19, a chest CT may be available while an abdominal CT is not. The purpose of this study was to investigate whether determining SMM on a chest CT is a feasible alternative to abdominal CT. RESEARCH QUESTION: What is the correlation between SMM measurements at the level of L3 and the level of the fourth thoracic vertebra (Th4)? STUDY DESIGN AND METHODS: In this study we retrospectively analyzed abdominal and thoracic series of whole-body CT-scans of trauma patients (N = 47) and head and neck cancer patients (N = 194). All abdominal muscles were delineated on a single axial slice at the level of L3. The erector spinae, levator scapulae, rhomboideus minor and major and pectoralis minor and major muscles were delineated on a single axial slice at the level of Th4. CSA of the muscles at Th4 and the L3 level were compared using linear regression, and a multivariate linear regression model was established. RESULTS: Muscle CSA at level Th4 strongly correlates with L3 muscle CSA (r = 0.791, p < 0.05). A multivariate model incorporating the patient characteristics arm positioning, age, sex, and weight achieved a stronger correlation (r = 0.856, p < 0.05). INTERPRETATION: Skeletal muscle CSA measured at the level of Th4 is a feasible alternative to measurements at L3. This allows diagnosing low SMM using clinically available thoracic CT-scans. SMM measurements at the level of Th4 may become a prognostic or triage tool when faced with mechanical ventilator shortage.

HIF-1a expression and differential effects on survival in patients with oral cavity, larynx, and oropharynx squamous cell carcinomas.

BACKGROUND: Hypoxia is a negative prognostic factor in head and neck squamous cell carcinomas. Under hypoxia, the hypoxia-inducible factor (HIF)-1a transcription factor is overexpressed. We investigated whether there were site differences in HIF-1a expression and its effect on patient outcomes per subsite. DESIGN/METHOD: A total of 941 patients with HNSCC in the squamous cell carcinoma of the oropharynx (OPSCC, n = 302), oral cavity (OSCC, n = 391), or larynx (LSCC, n = 248) were included. Expression of HIF-1a in tissue samples was investigated using immunohistochemistry. Overall survival (OS), disease-free survival (DFS), and locoregional control (LRC) were analyzed. RESULTS: HIF-1a expression was higher in OSCC than in LSCC and OPSCC. High HIF-1a expression led to worse prognosis in OPSCC (OS P = .029, DFS P = .085) and LSCC (OS P = .041, DFS P = .011) and better in OSCC (OS P = .055, DFS P = .012). There was no association between HIF-1a and LRC. CONCLUSIONS: High HIF-1a expression is related to poor outcome in OPSCC and LSCC and better outcome in OSCC.

Influence of tumor and microenvironment characteristics on diffusion-weighted imaging in oropharyngeal carcinoma: A pilot study.

OBJECTIVES: Diffusion weighted imaging (DWI) is a frequently performed MRI sequence in cancer patients. While previous studies have shown the clinical value of the apparent diffusion coefficient (ADC) for response prediction and response monitoring, less is known about the biological background of ADC. In the tumor microenvironment, hypoxia and increased proliferation of tumor cells contribute to resistance to (radio-)therapy, while high T-cell influx is related to better prognosis. We investigated the correlation between these three tissue characteristics and ADC in 20 oropharyngeal squamous cell carcinoma patients. MATERIALS AND METHODS: 20 patients with oropharyngeal squamous cell carcinoma (OPSCC) who underwent 1.5 T MRI, including DWI were included in this pilot study. Corresponding formalin-fixed paraffin-embedded tumor tissues were immunohistochemically analyzed for protein expression of hypoxia-inducible factor 1a (HIF-1a), Ki-67 and CD3. Expression of these markers was correlated with ADC. RESULTS: ADC negatively correlated with Ki-67 expression (p = .024) in tumor cells. There was a significant negative correlation between ADC and CD3-positive cell count (p = .009). No correlation was observed between HIF-1a expression and ADC. CONCLUSION: This study suggests that ADC reflects characteristics of tumor cells as well as the surrounding microenvironment. Interestingly, high tumor proliferation (a negative prognostic factor) and high T-cell influx (a beneficial prognostic factor) are both associated with a lower ADC. Further studies should be performed to correlate ADC to these histological characteristics in relation to previously known factors that affect ADC, to gain further knowledge on the role of DW-MRI in diagnostics and personalized medicine.

Low skeletal muscle mass is a predictive factor for chemotherapy dose-limiting toxicity in patients with locally advanced head and neck cancer.

OBJECTIVES: Low skeletal muscle mass (SMM) or sarcopenia is emerging as an adverse prognostic factor for chemotherapy dose-limiting toxicity (CLDT) and survival in cancer patients. Our aim was to determine the impact of low SMM on CDLT in patients with locally advanced head and neck squamous cell carcinoma (LA-HNSCC) treated with primary radiochemotherapy (RCT). PATIENTS AND METHODS: Consecutive patients diagnosed with LA-HNSCC and treated with primary RCT between 2007 and 2011 in our center were included. Clinical variables were retrospectively retrieved and SMM was measured at the level of the third cervical vertebra using pre-treatment head and neck CT-scans. After determining a cut-off value for low SMM, multivariate analysis was performed to identify prognostic factors for CDLT. RESULTS: Of 112 patients included, 30.4% experienced CDLT. The optimal cut-off value for low SMM as a predictor of CDLT was ≤43.2cm2/m2. Using this cut-off, 54.5% patients had low SMM. Patients with low SMM experienced CDLT more frequently than patients with normal SMM (44.3% vs. 13.7%, p<0.001) and received a higher dose of chemotherapy/kg lean body mass (estimated from SMM, p=0.044). At multivariate analysis, low SMM was independently inversely associated with CDLT (OR 0.93, 95%CI: 0.88-0.98). Patients experiencing CDLT had a lower overall survival than patients who did not (mean 36.6vs. 54.2months, p=0.038). CONCLUSION: Low SMM is an independent risk factor for CDLT in LA-HNSCC patients treated with primary RCT. Pre-therapeutic estimation of SMM using routine CT-scans of the head and neck region may identify patients at risk of CDLT.

Feasibility of using head and neck CT imaging to assess skeletal muscle mass in head and neck cancer patients.

OBJECTIVES: Patients with head and neck cancer (HNC) have a higher risk of malnutrition and sarcopenia, which is associated with adverse clinical outcome. As abdominal CT-imaging is often used to detect sarcopenia, such scans are rarely available in HNC patients, possibly explaining why no studies investigate the effect of sarcopenia in this population. We correlated skeletal muscle mass assessed on head and neck CT-scans with abdominal CT-imaging. METHODS: Head and neck, and abdominal CT-scans of trauma (n=51) and HNC-patients (n=52) were retrospectively analyzed. On the head and neck CT-scans, the paravertebral and sternocleidomastoid muscles were delineated. On the abdominal CT-scans, all muscles were delineated. Cross-sectional area (CSA) of the muscles at the level of the C3 vertebra was compared to CSA at the L3 level using linear regression. A multivariate linear regression model was established. RESULTS: HNC-patients had significantly lower muscle CSA than trauma patients (37.9 vs. 45.1cm2, p<0.001, corrected for sex and age). C3 muscle CSA strongly predicted L3 muscle CSA (r=0.785, p<0.001). This correlation was stronger in a multivariate model including sex, age and weight (r=0.891, p<0.001). DISCUSSION: Assessment of skeletal muscle mass on head and neck CT-scans is feasible and may be an alternative to abdominal CT-imaging. This method allows assessment of sarcopenia using routinely performed scans without additional imaging or additional patient burden. Identifying sarcopenic patients may help in treatment selection, or to select HNC patients for physiotherapeutic or nutritional interventions to improve their outcome.

Oropharyngeal squamous cell carcinomas differentially express granzyme inhibitors.

OBJECTIVES: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs) have an improved prognosis compared to HPV-negative OPSCCs. Several theories have been proposed to explain this relatively good prognosis. One hypothesis is a difference in immune response. In this study, we compared tumor-infiltrating CD3+, CD4+, CD8+ T-cells, and granzyme inhibitors (SERPINB1, SERPINB4, and SERPINB9) between HPV-positive and HPV-negative tumors and the relation with survival. METHODS: Protein expression of tumor-infiltrating lymphocytes (TILs) (CD3, CD4, and CD8) and granzyme inhibitors was analyzed in 262 OPSCCs by immunohistochemistry (IHC). Most patients (67%) received primary radiotherapy with or without chemotherapy. Cox regression analysis was carried out to compare overall survival (OS) of patients with low and high TIL infiltration and expression of granzyme inhibitors. RESULTS: HPV-positive OPSCCs were significantly more heavily infiltrated by TILs (p < 0.001) compared to HPV-negative OPSCCs. A high level of CD3+ TILs was correlated with a favorable outcome in the total cohort and in HPV-positive OPSCCs, while it reached no significance in HPV-negative OPSCCs. There was expression of all three granzyme inhibitors in OPSCCs. No differences in expression were found between HPV-positive and HPV-negative OPSCCs. Within the group of HPV-positive tumors, a high expression of SERPINB1 was associated with a significantly worse overall survival. CONCLUSION: HPV-positive OPSCCs with a low count of CD3+ TILs or high expression of SERPINB1 have a worse OS, comparable with HPV-negative OPSCCs. This suggests that the immune system plays an important role in the carcinogenesis of the virally induced oropharynx tumors.

Fibroblast growth factor receptor 3 protein is overexpressed in oral and oropharyngeal squamous cell carcinoma.

Fibroblast growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor receptor tyrosine kinase family. It has been identified as a promising therapeutic target in multiple types of cancer. We have investigated FGFR3 protein expression and FGFR3 gene copy-numbers in a single well-documented cohort of oral and oropharyngeal squamous cell carcinoma. Tissue microarray sets containing 452 formalin-fixed paraffin-embedded tissues were immunohistochemically stained with an anti-FGFR3 antibody and hybridized with a FGFR3 fluorescence in situ hybridization probe. FGFR3 protein expression was correlated with clinicopathological and survival data, which were retrieved from electronic medical records. FGFR3 mRNA data of 522 head and neck squamous cell carcinoma (HNSCC) were retrieved from The Cancer Genome Atlas (TCGA). Fibroblast growth factor receptor 3 (FGFR3) protein was overexpressed in 48% (89/185) of oral and 59% (124/211) of oropharyngeal squamous cell carcinoma. Overexpression of FGFR3 protein was not related to overall survival or disease-free survival in oral (HR[hazard ratio]: 0.94; 95% CI: 0.64-1.39; P = 0.77, HR: 0.94; 95% CI: 0.65-1.36; P = 0.75) and oropharyngeal squamous cell carcinoma (HR: 1.21; 95% CI: 0.81-1.80; P = 0.36, HR: 0.42; 95% CI: 0.79-1.77; P = 0.42). FGFR3 mRNA was upregulated in 3% (18/522) of HNSCC from the TCGA. The FGFR3 gene was gained in 0.6% (1/179) of oral squamous cell carcinoma but no amplification was found in oral and oropharyngeal squamous cell carcinoma. In conclusion, FGFR3 protein is frequently overexpressed in oral and oropharyngeal squamous cell carcinoma. Therefore, it may serve as a potential therapeutic target for FGFR3-directed therapies in oral and oropharyngeal squamous cell carcinoma.

A systematic review of the effect of different crimping techniques in stapes surgery for otosclerosis.

OBJECTIVES/HYPOTHESIS: To evaluate the effect of crimping techniques in stapes surgery for otosclerosis patients measured by hearing outcomes on pure-tone audiometry. DATA SOURCES: PubMed, EMBASE, and the Cochrane Library. METHODS: A systematic search was conducted. Studies comparing the effect of different crimping methods on pure-tone audiometric results in patients undergoing stapes surgery for otosclerosis were included. Relevance and risk of bias were assessed. Absolute risks and risk differences, means and mean differences, and 95% confidence intervals were extracted or calculated for the primary and secondary outcomes, which were air-bone gap closure to 10 dB or less, mean postoperative air-bone gap, and postoperative sensorineural hearing loss. RESULTS: Twenty-two studies with moderate or high risk of bias were included for data extraction. Air-bone gap closure to 10 dB or less was assessed in 17 studies and mean postoperative air-bone gap in 20 studies. The hearing outcomes did not consistently favor one crimping method. However, the differences that were statistically significant were consistently in favor of heat crimping over manual and no crimping (difference in air-bone gap closure to 10 dB or less ranged between 22% and 42% in these studies and difference in mean postoperative air-bone gap between 2.8 dB and 7.4 dB) and in favor of manual crimping over no crimping (30% difference in air-bone gap closure to 10 dB or less and difference in mean postoperative air-bone gap between 2.6 dB and 6.0 dB). CONCLUSION: Moderate to high risk of bias and inconsistent results characterize the current evidence. Laryngoscope, 126:1207-1217, 2016.

Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review.

Primary human tumor culture models allow for individualized drug sensitivity testing and are therefore a promising technique to achieve personalized treatment for cancer patients. This would especially be of interest for patients with advanced stage head and neck cancer. They are extensively treated with surgery, usually in combination with high-dose cisplatin chemoradiation. However, adding cisplatin to radiotherapy is associated with an increase in severe acute toxicity, while conferring only a minor overall survival benefit. Hence, there is a strong need for a preclinical model to identify patients that will respond to the intended treatment regimen and to test novel drugs. One of such models is the technique of culturing primary human tumor tissue. This review discusses the feasibility and success rate of existing primary head and neck tumor culturing techniques and their corresponding chemo- and radiosensitivity assays. A comprehensive literature search was performed and success factors for culturing in vitro are debated, together with the actual value of these models as preclinical prediction assay for individual patients. With this review, we aim to fill a gap in the understanding of primary culture models from head and neck tumors, with potential importance for other tumor types as well.

Clinical implications of hypoxia biomarker expression in head and neck squamous cell carcinoma: a systematic review.

Awareness increases that the tumor biology influences treatment outcome and prognosis in cancer. Tumor hypoxia is thought to decrease sensitivity to radiotherapy and some forms of chemotherapy. Presence of hypoxia may be assessed by investigating expression of endogenous markers of hypoxia (EMH) using immunohistochemistry (IHC). In this systematic review we investigated the effect of EMH expression on local control and survival according to treatment modality in head and neck cancer (head and neck squamous cell carcinoma [HNSCC]). A search was performed in MEDLINE and EMBASE. Studies were eligible for inclusion that described EMH expression in relation to outcome in HNSCC patients. Quality was assessed using the Quality in Prognosis Studies (QUIPS) tool. Hazard ratios for locoregional control and survival were extracted. Forty studies of adequate quality were included. HIF-1a, HIF-2a, CA-IX, GLUT-1, and OPN were identified as the best described EMHs. With exception of HIF-2a, all EMHs were significantly related to adverse outcome in multiple studies, especially in studies where patients underwent single-modality treatment. Positive expression was often correlated with adverse clinical characteristics, including disease stage and differentiation grade. In summary, EMH expression was common in HNSCC patients and negatively influenced their prognosis. Future studies should investigate the effect of hypoxia-modified treatment schedules in patients with high In summary, EMH expression. These may include ARCON, treatment with nimorazole, or novel targeted therapies directed at hypoxic tissue. Also, the feasibility of surgical removal of the hypoxic tumor volume prior to radiotherapy should be investigated.

Use of the 2-µm continuous wave thulium laser for the resection of oral squamous cell carcinomas does not impair pathological assessment.

BACKGROUND AND OBJECTIVE: Current resection modalities for oral squamous cell carcinomas (OSCC) vary from cold steel over CO2 laser to monopolar electro-surgery (MO). We compared thulium laser (TL) as a new modality with MO with regards to pathological assessment. STUDY DESIGN/MATERIALS AND METHODS: Forty-two patients who were treated for OSCC by either TL or MO were included. All resected specimens were assessed with special attention to margin interpretation and thermal damage. RESULTS: Depth of thermal damage ranged from 1.0 to 3.5 mm in the TL group compared to 1.0-4.0 mm in the MO group without interfering with the pathological assessment. The percentage of positive margin resections was three times higher in the MO group compared with the TL group. CONCLUSIONS: This study shows resections done by TL do not impair pathological assessment when compared to MO resections.