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%.

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.

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.

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 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.

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.

Clinical applications of slide-based cytometry--an update.

Slide-based cytometric approaches open the possibility to obtain quantitative and objective data from specimens that so far have not been accessible to this kind of analysis. In this review, we will highlight the specific advantages of slide-based cytometry (SBC) and show the applications that have been established for clinical samples. Focuses are cytomic analyses of oncological and hematological samples where the slide-based concept turned out to open new dimensions in understanding underlying cellular networks. We review the recent literature and point out future applications.

Solitary metastasis of lung carcinoma to the internal auditory canal.

We report a case of solitary metastasis of lung carcinoma into the internal auditory canal. On admission, the 60-year-old patient was complaining about a rapidly developing hearing loss with ipsilateral facial nerve paralysis. Magnetic resonance imaging revealed an intracanalicular tumor, which was interpreted as vestibular schwannoma. After tumor resection, the histopathologic examination revealed a metastasis of a squamous cell carcinoma. Computed tomographic scan and bronchoscopy identified an asymptomatic primary pulmonary neoplasm.

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.

Iterative restaining as a pivotal tool for n-color immunophenotyping by slide-based cytometry.

AIM: Slide-based cytometry (SBC) allows to "ask a cell a second time." We used this tool for detailed immunophenotyping of peripheral blood leukocytes (PBLs). METHODS: PBLs primarily stained for CD-markers and DNA were immobilized on a glass slide and analyzed by laser scanning cytometry. Then, iterative restaining was applied for a second and a third analysis. Based on the cells' fixed location, analyses were merged on a single-cell level. RESULTS: We analyzed six virtual immunostainings by "recycling" the PE-channel for four CD-markers. CONCLUSION: Iterative restaining might prove to be a pivotal tool for n-color immunophenotyping exclusive to SBC concepts.

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry.

BACKGROUND: To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. METHODS: The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. RESULTS: With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. CONCLUSIONS: With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.