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.

Endovascular treatment of epistaxis: indications, management, and outcome.

OBJECTIVE: Epistaxis is a common clinical problem, and the majority of bleedings can be managed conservatively. However, due to extensive and sometimes life-threatening bleeding, further treatment, such as superselective embolization, may be required. We report our experience with endovascular treatment of life-threatening epistaxis. METHODS: All patients presenting with excessive epistaxis, which received endovascular treatment at a German tertiary care facility between January 2001 and December 2009, were retrospectively identified. Demographic data, etiology, origin and clinical relevance of bleeding, interventional approach, therapy-associated complications, and outcome were assessed. RESULTS: A total of 48 patients required 53 embolizations. Depending on the etiology of bleeding, patients were assigned to three groups: 1) idiopathic epistaxis (31/48), 2) traumatic or iatrogenic epistaxis (12/48), and 3) hereditary hemorrhagic telangiectasia (HHT) (5/48). Eleven of 48 patients required blood transfusions, and 9 of these 11 patients (82%) were termed clinically unstable. The sphenopalatine artery was embolized unilaterally in 10 of 53 (18.9%) and bilaterally in 41 of 53 (77.4%) procedures. During the same procedure, additional vessels were embolized in three patients (3/53; 5.7%). In 2 of 53(3.8%) cases, the internal carotid artery (ICA) was occluded. Long-term success rates of embolization were 29 of 31 (93.5%) for group 1 and 11 of 12 (91.7%) for group 2 patients. Embolization of patients with HHT offered at least a temporary relief in three of five (60%) cases. Two major complications (necrosis of nasal tip and transient hemiparesis) occurred after embolization. CONCLUSIONS: Endovascular treatment proves to be effective for prolonged and life-threatening epistaxis. It is easily repeatable if the first procedure is not successful and offers a good risk-benefit profile.

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.

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.

Early detection in head and neck cancer - current state and future perspectives.

Survival and quality of life in head and neck cancer are directly linked to the size of the primary tumor at first detection. In order to achieve substantial gain at these issues, both, primary prevention and secondary prevention, which is early detection of malignant lesions at a small size, have to be improved. So far, there is not only a lack in the necessary infrastructure not only in Germany, but rather worldwide, but additionally the techniques developed so far for early detection have a significance and specificity too low as to warrant safe implementation for screening programs. However, the advancements recently achieved in endoscopy and in quantitative analysis of hypocellular specimens open new perspectives for secondary prevention. Chromoendoscopy and narrow band imaging (NBI) pinpoint suspicious lesions more easily, confocal endomicroscopy and optical coherence tomography obtain optical sections through those lesions, and hyperspectral imaging classifies lesions according to characteristic spectral signatures. These techniques therefore obtain optical biopsies. Once a "bloody" biopsy has been taken, the plethora of parameters that can be quantified objectively has been increased and could be the basis for an objective and quantitative classification of epithelial lesions (multiparametric cytometry, quantitative histology). Finally, cytomics and proteomics approaches, and lab-on-the-chip technology might help to identify patients at high-risk. Sensitivity and specificity of these approaches have to be validated, yet, and some techniques have to be adapted for the specific conditions for early detection of head and neck cancer. On this background it has to be stated that it is still a long way to go until a population based screening for head and neck cancer is available. The recent results of screening for cancer of the prostate and breast highlight the difficulties implemented in such a task.

Prediction of upper aerodigestive tract cancer by slide-based cytometry.

AIM: To evaluate slide-based cytometry in screening for and following up of carcinoma of the upper aerodigestive tract using swabs for a minimal-invasive approach. METHODS: Laser scanning cytometry (LSC) was used for multiparametric analysis of cells stained for cytokeratin and DNA to determine the DNA-index (DI) of the tumor cells. Histograms with 0.95 < DI < 1.05 and 1.9 < DI < 2.1 were defined as DNA euploid and any other DI as DNA aneuploid. After subsequent HE-staining, single cells were relocalized in order to document morphology. Conventional cytology was also performed on a subset of the slides. Routine histopathology of parallel biopsies served as gold standard in all cases. RESULTS: 115 swabs from 109 patients were obtained from the entire upper aerodigestive tract. 16 swabs were classified as insufficient for LSC. In the remaining 99 specimens, 1 benign lesion was misclassified as malignant, while 61 of the 75 malignant lesions were correctly identified. This corresponds to predictive values of 98.4% and 62.2% for the detection of malignant and benign samples by LSC. CONCLUSION: This pilot study demonstrates the validity of LSC screening for the identification of tumor malignancy in the upper aerodigestive tract from swab collected cytological material.

Hyperchromatic cytometry principles for cytomics using slide based cytometry.

BACKGROUND: Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high-content and high-throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object. METHODS AND RESULTS: We demonstrate various approaches for hyperchromatic analysis on a SBC instrument, the Laser Scanning Cytometer. The different components demonstrated here include (1) polychromatic cytometry (staining of the specimen with eight or more different fluorochromes simultaneously), (2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), (3) differential photobleaching (differentiating fluorochromes by their different photostability), (4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and (5) photodestruction (destruction of FRET dyes). Based on the ability to relocate cells that are immobilized on a microscope slide with a precision of approximately 1 microm, identical cells can be reanalyzed on the single cell level after manipulation steps. CONCLUSION: With the intelligent combination of several different techniques, the hyperchromatic cytometry approach allows to quantify and analyze all components of relevance on the single cell level. The information gained per specimen is only limited by the number of available antibodies and sterical hindrance.

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.

Novel aspects of systems biology and clinical cytomics.

The area of Cytomics and Systems Biology became of great impact during the last years. In some fields of the leading cytometric techniques it represents the cutting edge today. Many different applications/variations of multicolor staining were developed for flow- or slide-based cytometric analysis of suspensions and sections to whole animal analysis. Multispectral optical imaging can be used for studying immunological and tumorigenic processes. New methods resulted in the establishment of lipidomics as the systemic research of lipids and their behavior. All of these development push the systemic approach of the analysis of biological specimens to enhance the outcome in the clinic and in drug discovery programs.

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.

Sequential photobleaching of fluorochromes for polychromatic slide-based cytometry.

BACKGROUND: Slide-based cytometry is a key technology for polychromatic cytomic investigations. Here we exploit the relocalization and merge feature of Laser Scanning Cytometry for distinguishing fluorochromes of comparable emission spectra but different photostabilities. METHODS: Blood specimens were stained with the fluorochrome pairs: FITC/ALEXA488, PE/ALEXA532, or APC/ALEXA633. Bleaching was performed by repeated laser excitation. RESULTS: Since ALEXA dyes are photostable as compared to the conventional fluorochromes FITC, PE, and APC, a differentiation within one fluorochrome pair is possible. CONCLUSION: The sequential photobleaching method results in an increased information density on a single cell level and represents an important component to perform polychromatic cytometry.

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.