Hyperspectral Imaging Using Flexible Endoscopy for Laryngeal Cancer Detection.

Hyperspectral imaging (HSI) is increasingly gaining acceptance in the medical field. Up until now, HSI has been used in conjunction with rigid endoscopy to detect cancer in vivo. The logical next step is to pair HSI with flexible endoscopy, since it improves access to hard-to-reach areas. While the flexible endoscope's fiber optic cables provide the advantage of flexibility, they also introduce an interfering honeycomb-like pattern onto images. Due to the substantial impact this pattern has on locating cancerous tissue, it must be removed before the HS data can be further processed. Thereby, the loss of information is to minimize avoiding the suppression of small-area variations of pixel values. We have developed a system that uses flexible endoscopy to record HS cubes of the larynx and designed a special filtering technique to remove the honeycomb-like pattern with minimal loss of information. We have confirmed its feasibility by comparing it to conventional filtering techniques using an objective metric and by applying unsupervised and supervised classifications to raw and pre-processed HS cubes. Compared to conventional techniques, our method successfully removes the honeycomb-like pattern and considerably improves classification performance, while preserving image details.

Photosensitizing effects of hypericin on head neck squamous cell carcinoma in vitro.

Clinical outcome of patients suffering from head neck squamous cell carcinomas is still poor due to recurrent disease and surgical limitations. There is still a demand for multimodality approaches and new therapeutic options. Hypericin is a promising phototoxic drug which was investigated for its effects on head neck squamous cell carcinoma cells in vitro. FaDu cells incubated with or without hypericin were illuminated (450-700 nm, 50,000 lx) for different time periods. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide- and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay were used to score metabolic and apoptotic activity. Even after the shortest illumination FaDu cells incubated with hypericin showed massive reduction of metabolism and excessive apoptosis. This was present even with the lowest hypericin concentration. Cells without hypericin or without illumination were not affected. These photosensitizing effects of hypericin could be suitable for clinical application and could lead to the development of an intraoperative photodynamic therapy of head neck squamous cell carcinomas.

Hyperspectral hybrid method classification for detecting altered mucosa of the human larynx.

BACKGROUND: In the field of earth observation, hyperspectral detector systems allow precise target detections of surface components from remote sensing platforms. This enables specific land covers to be identified without the need to physically travel to the areas examined. In the medical field, efforts are underway to develop optical technologies that detect altering tissue surfaces without the necessity to perform an excisional biopsy. With the establishment of expedient classification procedures, hyperspectral imaging may provide a non-invasive diagnostic method that allows determination of pathological tissue with high reliability. In this study, we examined the performance of a hyperspectral hybrid method classification for the automatic detection of altered mucosa of the human larynx. MATERIALS AND METHODS: Hyperspectral Imaging was performed in vivo and 30 bands from 390 to 680 nm for 5 cases of laryngeal disorders (2x hemorrhagic polyp, 3x leukoplakia) were obtained. Image stacks were processed with unsupervised clustering (linear spectral unmixing), spectral signatures were extracted from unlabeled cluster maps and subsequently applied as end-members for supervised classification (spectral angle mapper) of further medical cases with identical diagnosis. RESULTS: Linear spectral unmixing clearly highlighted altered mucosa as single spectral clusters in all cases. Matching classes were identified, and extracted spectral signatures could readily be applied for supervised classifications. Automatic target detection performed well, as the considered classes showed notable correspondence with pathological tissue locations. CONCLUSIONS: Using hyperspectral classification procedures derived from remote sensing applications for diagnostic purposes can create concrete benefits for the medical field. The approach shows that it would be rewarding to collect spectral signatures from histologically different lesions of laryngeal disorders in order to build up a spectral library and to prospectively allow non-invasive optical biopsies.

Transarterial endovascular treatment in the management of life-threatening intra- and postoperative haemorrhages after otorhinolaryngological surgery.

Management of life-threatening postsurgical bleeding is complex. If conservative or surgical therapy is demanding, an endovascular treatment can be considered. The goal of this study was to evaluate the outcome of endovascular approaches in the diagnosis and therapy of otherwise intractable postoperative haemorrhages with a study design of outcomes research. Charts of all patients with postsurgical bleedings receiving endovascular treatment were reviewed for clinical outcome, complications, and demographic data. 15 patients were identified. They had rhinosurgery (12/15), tonsillectomy (2/15) or transoral tumour debulking (1/15) prior to the endovascular procedure. In more than 70%, the source of bleeding was directly located angiographically and subsequently superselectively embolized. The remaining patients suffered from post-rhinosurgical epistaxis and underwent a bilateral embolization of the sphenopalatine artery. All bleedings were successfully controlled and no procedure-related complication was noted. In conclusion, endovascular treatment of life-threatening postsurgical haemorrhages should be considered if the source of bleeding is unknown or if surgery is difficult and may result in devastating postoperative complications.

In vivo detection of head and neck tumors by hyperspectral imaging combined with deep learning methods.

Currently, there are no fast and accurate screening methods available for head and neck cancer, the eighth most common tumor entity. For this study, we used hyperspectral imaging, an imaging technique for quantitative and objective surface analysis, combined with deep learning methods for automated tissue classification. As part of a prospective clinical observational study, hyperspectral datasets of laryngeal, hypopharyngeal and oropharyngeal mucosa were recorded in 98 patients before surgery in vivo. We established an automated data interpretation pathway that can classify the tissue into healthy and tumorous using convolutional neural networks with 2D spatial or 3D spatio-spectral convolutions combined with a state-of-the-art Densenet architecture. Using 24 patients for testing, our 3D spatio-spectral Densenet classification method achieves an average accuracy of 81%, a sensitivity of 83% and a specificity of 79%.

Peptide microarrays coupled to machine learning reveal individual epitopes from human antibody responses with neutralizing capabilities against SARS-CoV-2.

The coronavirus SARS-CoV-2 is the causative agent for the disease COVID-19. To capture the IgA, IgG, and IgM antibody response of patients infected with SARS-CoV-2 at individual epitope resolution, we constructed planar microarrays of 648 overlapping peptides that cover the four major structural proteins S(pike), N(ucleocapsid), M(embrane), and E(nvelope). The arrays were incubated with sera of 67 SARS-CoV-2 positive and 22 negative control samples. Specific responses to SARS-CoV-2 were detectable, and nine peptides were associated with a more severe course of the disease. A random forest model disclosed that antibody binding to 21 peptides, mostly localized in the S protein, was associated with higher neutralization values in cellular anti-SARS-CoV-2 assays. For antibodies addressing the N-terminus of M, or peptides close to the fusion region of S, protective effects were proven by antibody depletion and neutralization assays. The study pinpoints unusual viral binding epitopes that might be suited as vaccine candidates.

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.

Approaching clinical proteomics: current state and future fields of application in cellular proteomics.

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter- and intra-assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Cone-Beam Computed Tomography (CBCT) dacryocystography for imaging of the nasolacrimal duct system.

PURPOSE: To evaluate the usefulness and safety of cone-beam computed tomography (CBCT) dacryocystography in detecting lesions, identifying coexisting soft-tissue changes and determining treatment options in patients with epiphora. PATIENTS AND METHODS: Unilateral digital subtraction dacryocystography and CBCT dacryocystography were carried out on 45 patients. Stenoses and occlusions were identified and coexisting changes such as septal deviation and dacryoliths were noted. The diameter of the bony lacrimal duct of affected and unaffected side was measured and related to the clinically evident epiphora. An attempt was made to base the subsequent therapeutic planning on the CBCT dacryocystographic findings. Additionally, the radiation dose levels for CBCT dacryocystography in comparison to those of multislice computed tomography (MSCT) were evaluated in a standardized head-neck Rando-Alderson phantom. RESULTS: Nasolacrimal duct obstructions were present in 37/45 patients, 18 with a stenosis and 19 with an occlusion in parts of the lacrimal outflow system. The minimal bony diameter of the side with epiphora was significantly decreased compared to the unaffected side. Coexisting soft-tissue changes did not correlate significantly with the clinical sign of epiphora. Eight patients showed no underlying reason for the epiphora and were treated conservatively. A total of eleven patients received interventional therapy for their stenosis and 23 patients had to be treated surgically. A further three patients received medical treatment for infection, before surgery and interventional therapy, respectively, were carried out. Dose levels for CBCT imaging remained far below those of MSCT. CONCLUSION: CBCT dacryocystography is a safe and time-efficient modality for assessing the nasolacrimal duct system in patients with epiphora. CBCT dacryocystography provides detailed images of the nasolacrimal drainage system, surrounding soft tissue, and bony structures in one diagnostic tour. It allows clear measurement of the bony nasolacrimal duct and displays information beyond that of the drainage lumen, improving the planning of therapeutic interventional and surgical procedures.

DNA methylation of indoleamine 2,3-dioxygenase 1 (IDO1) in head and neck squamous cell carcinomas correlates with IDO1 expression, HPV status, patients' survival, immune cell infiltrates, mutational load, and interferon γ signature.

BACKGROUND: The immune checkpoint, indoleamine 2,3-dioxygenase 1, is under investigation as target of novel immunotherapies for cancers, including head and neck squamous cell carcinomas (HNSCC). The aim of our study was to analyze DNA methylation of the encoding gene (IDO1) in HNSCC. METHODS: Methylation of three CpG sites within the promoter, promoter flank, and gene body was investigated and correlated with mRNA expression, immune cell infiltration, mutational burden, human papillomavirus (HPV)-status, and overall survival in a cohort of N = 528 HNSCC patients obtained from The Cancer Genome Atlas. In addition, IDO1 immunohistochemistry and DNA methylation analysis was performed in an independent cohort of N = 138 HNSCC samples. FINDINGS: Significant inverse correlations of IDO1 methylation and IDO1 mRNA expression were found in the promoter and promoter flank region (Spearman's ρ = -0.163 and ρ = -0.377, respectively) while a positive correlation was present in the gene body (ρ = 0.502; all P < 0.001). IDO1 DNA methylation significantly correlated with IDO1 protein expressing immune cells as well as tumor cells. IDO1 promoter flank hypermethylation was significantly associated with poor overall survival (P < 0.001). In addition, we discovered significant correlations between IDO1 methylation and expression with RNA signatures of immune cell infiltrates and with HPV-status, mutational load (methylation only), and interferon γ signature. INTERPRETATION: Our results suggest IDO1 expression levels are epigenetically regulated by DNA methylation. This study provides rationale to test IDO1 methylation as potential biomarker for prediction of response to IDO1 immune checkpoint inhibitors in HNSCC.

Comparison of immunophenotyping by slide-based cytometry and by flow cytometry.

BACKGROUND: Flow cytometry (FCM) is the gold standard for immunophenotyping of peripheral blood leukocytes (PBLs). Slide-based cytometry (SBC) systems, for example the laser scanning cytometer (LSC(R), CompuCyte), can give additional information (repeated staining and scanning, morphology). In order to adequately judge the clinical usefulness of LSC for immunophenotyping it is obligatory to compare it with FCM. AIM: The aim of this study was to systematically compare immunophenotyping by both FCM and LSC methods and to test the correlation of the results. METHODS: PBLs were stained with directly labeled monoclonal antibodies with the whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in parallel by a FACScan (BD-Biosciences) using standard protocols and by LSC with different triggers (forward scatter, CD45 FITC, or 7-AAD). For 7-AAD measurements by LSC, slides were additionally fixed with acetone before 7-AAD staining. RESULTS: Calculating the percentage distribution of PBLs obtained by LSC and by FCM showed very good correlation with regression coefficients close to 1.0 for the major populations and the lymphocyte sub-populations (neutrophils, monocytes, and lymphocytes; T-helper-, T-cytotoxic-, B-, NK-cells). The best trigger for LSC was 7-AAD. CONCLUSION: LSC can be recommended for immunophenotyping of PBLs especially in cases where only limited sample volumes are available or where additional analysis of the cells' morphology is important. The detection of rare leukocytes or weak antigens is limited; in these cases appropriate amplification steps for immunofluorescence should be engaged.

Development of an image pre-processor for operational hyperspectral laryngeal cancer detection.

Hyperspectral imaging (HSI) is a technology with high potential in the field of non-invasive detection of cancer. However, in complex imaging situations like HSI of the larynx with a rigid endoscope, various image interferences can disable a proper classification of cancerous tissue. We identified three main problems: i) misregistration of single images in a HS cube due to patient heartbeat ii) image noise and iii) specular reflections (SR). Consequently, an image pre-processor is developed in the current paper to overcome these image interferences. It encompasses i) image registration ii) noise removal by minimum noise fraction (MNF) transformation and iii) a novel SR detection method. The results reveal that the pre-processor improves classification performance, while the newly developed SR detection method outperforms global thresholding technique hitherto used by 46%. The novel pre-processor will be used for future studies towards the development of an operational scheme for HS-based larynx cancer detection. RGB image of the larynx derived from the hyperspectral cube and corresponding specular reflections (a) manually segmented and (b) detected by a novel specular reflection detection method.

Preoperative detection of laryngeal cancer in mucosal swabs by slide-based cytometry.

The aim of our study was to evaluate slide-based cytometry in screening for laryngeal cancer using swabs a minimally invasive approach. Laser scanning cytometry (LSC) was used for the multiparametric analysis of cells stained for cytokeratin and DNA to determine the DNA-index (DI) of the tumour cells. Histograms with DI < 0.95, 1.05 < DI < 1.9, and 2.1 < DI were defined as DNA aneuploid. After subsequent haemotoxylin-eosin (HE)-staining, single cells were re-localised and an analysis by conventional cytology was performed. Additionally, routine histopathology of parallel biopsies was obtained in all cases. Fifty one swabs from 49 lesions were analyzed. Seven and 17 swabs, were classified as insufficient for LSC and cytology, respectively. One and two benign lesions, were misclassified as malignant, respectively. Out of 34 malignant lesions, LSC detected 25 and cytology 14. LSC was superior to cytology in all of the statistical parameters tested. This pilot study demonstrates the validity of LSC for the preoperative detection of malignancy in laryngeal tumours using swabs.

Expression profiles of p53, p63, and p73 in benign salivary gland tumors.

The tumor-suppressor protein p53 has recently been shown to belong to a family that includes two structurally related proteins, p63 and p73. In contrast to p53, p63 and p73 play an essential role in epithelial development, stem cell identity and cellular differentiation. Salivary gland tumors carry a wide spectrum of histopathological forms, which may share a common single-cell origin from the epithelial progenitor basal duct cells and have a different tendency of malignant progression. This study was performed to examine the expression of p53, p63, and p73 in benign salivary gland tumors. Expression and mutation of p53, p73, and p63 were examined by direct DNA sequencing, reverse transcription PCR using isoform-specific primers, and by immunohistochemistry in normal parotid tissue ( n=10), and various tumors of the salivary gland (42 pleomorphic adenomas, 12 myoepitheliomas, 8 basal cell adenomas, 5 oncocytomas, 5 canalicular adenomas, and 20 adenolymphomas). In normal parotid tissue the expression of p63 and p73 was restricted to few basal and myoepithelial cells. Ductal luminal and acinus cells were completely negative for the expression of all three family members. In contrast, in salivary gland tumors, strong nuclear staining for p63 and p73 was observed. Myoepithelial and basaloid cells and the basal epithelial layer of adenolyphomas and oncocytomas were positive for p63 and also, to a lesser extent, to p73. Mutations of p53 were detected in 4 of 42 (10%) pleomorphic adenomas, in 3 of 12 (25%) myoepitheliomas, and in 1 of 8 (13%) basal cell adenomas but not in other tumors. We failed to detect specific mutations of p63 and p73. Using isoform-specific PCR, we found that all isoforms of p63 were expressed in normal parotid tissue whereas the pleomorphic adenomas, myoepitehliomas, and basal cell adenomas dominantly expressed the transactivation-incompetent truncated isoforms. Our data indicate that p63 and p73 are upregulated in salivary gland tumors and may serve as a marker of epithelial and myoepithelial progenitor cells in salivary glands. The prevalence of p53 mutations and the observation of the expression of DeltaNp63 isoforms only in pleomorphic adenomas, myoepitheliomas, and also basal cell adenomas may reflect their possible malignant potential.

Slide-based cytometry for predicting malignancy in solid salivary gland tumors by fine needle aspirate biopsies.

BACKGROUND: To minimize hospitalization and morbidity for a patient with a solid tumor of a salivary gland, malignancy must be confirmed or excluded as soon as possible. This information cannot be obtained preoperatively by existing standard procedures. Minimal-invasive approaches with adequate diagnostic analysis represent a promising precondition for optimized therapy. METHODS: For fine needle aspirate biopsies (FNABs), laser scanning cytometry (LSC) offers a semi-automated slide-based technology for objective and quantitative analysis. We have established an assay for FNABs from salivary gland tumors. FNAB cells were stained for cytokeratin and DNA followed by LSC analysis. The cells were subsequently HE-stained and were relocalized on the slide. The LSC analysis quantitatively determines the DNA index (DI) of the tumor cells taking leukocytes as internal DNA diploid standard. Histograms with 0.95 < DI < 1.05 and 1.9 2.5 (i.e., 5c exceeding rate, 5cER) was calculated. Samples with DNA aneuploid peaks or with 5cER > 5% were classified as malignant. Routine histopathology was performed as a control. RESULTS: FNABs from 51 solid salivary gland tumors (41 parotid gland, six submandibular, four parapharyngeal) were analyzed with this assay. Eleven of 14 malignant tumors were DNA aneuploid by LSC analysis. All benign tumors showed diploid DNA content. The positive predictive value for malignancy was 1.0, the negative predictive value was 0.93, the correlation with routine histopathology was highly significant (p = 7.6 x 10(-9), Fisher's exact test). The calculated specificity of LSC analysis was 1.0 and the sensitivity was 0.79. CONCLUSIONS: This pilot study demonstrates the validity of slide-based cytometry for the preoperative prediction of malignancy in solid tumors being inaccessible for incision biopsy but suitable for FNABs such as those of the parotid gland.

Comparative functional cell biological analysis of mesenchymal stem cells of the head and neck region: potential impact on wound healing, trauma, and infection.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent mesenchymal progenitor cells, originally identified in bone-marrow. Little is known about MSCs of the head and neck region. We investigated cell biological properties with a potential impact on wound healing of 2 different tissue-resident MSC populations. METHODS: MSCs were isolated from human nasal mucosa (nmMSCs) and parotid gland (pgMSCs). Clonogenic potential, cell surface markers, cytokine secretion, chemokine receptor expression, mobility, and adhesion to extracellular matrix were examined in unstimulated and stimulated MSCs. RESULTS: NmMSCs had the higher clonogenic potential. PgMSCs showed a broader panel of chemokine receptor expression and displayed higher mobility, especially after challenge with bacterial lipopolysaccharide (LPS). NmMSCs were less mobile and showed increased LPS-induced secretion of the inflammatory cytokine interleukin-8 (IL-8) compared with pgMSCs. CONCLUSION: These data highlight functional differences between tissue-resident MSCs of the head and neck region, which may impact functional properties of these cells in response to trauma or infection.

Histologic diagnoses in persistently swollen cervical lymph nodes.

BACKGROUND: Biopsy and histological examination of persistently enlarged cervical lymph nodes represent a major health care issue and have high impact on further clinical therapy. Tertiary health centers are faced with an increased demand for diagnostic workup to rule out malignancy. We performed a retrospective study from January 2000 to June 2008 to identify patients referred to us for diagnostic biopsy and to document the histopathological result. METHODS: Patients with a diagnostic biopsy, but neither clinical signs of head and neck cancer nor other malignancies, were identified within the records. Patient characteristics and histopathological diagnosis were retrieved. RESULTS: Three hundred twenty-six patients were identified (146 women, and 180 men). One hundred twenty-three patients (38%; 44 women, and 79 men) had a malignancy: 61 with metastatic disease and 62 with malignant lymphoma; the youngest was 15 years old and the oldest was 92 years old. CONCLUSION: Persistently swollen cervical lymph nodes should trigger a thorough clinical examination and prompt biopsy for histopathological workup.

Hyperspectral imaging of mucosal surfaces in patients.

The aim of this study was to proof applicability of hyperspectral imaging for the analysis and classification of human mucosal surfaces in vivo. The larynx as a prototypical anatomically well-defined surgical test area was analyzed by microlaryngoscopy with a polychromatic lightsource and a synchronous triggered monochromatic CCD-camera. Image stacks (5 benign, 7 malignant tumors) were analyzed by established software (principal component analysis PCA, hyperspectral classification, spectral profiles). Hyperspectral image datacubes were analyzed and classified by conventional software. In PCA, images at 590-680 nm loaded most onto the first PC which typically contained 95% of the total information. Hyperspectral classification clustered the data highlighting altered mucosa. The spectral profiles clearly differed between the different groups. Hyperspectral imaging can be applied to mucosal surfaces. This approach opens the way to analyze spectral characteristics of histologically different lesions in order to build up a spectral library and to allow non-touch optical biopsy.