To date only a fraction of the genetic footprint of thyroid function has been clarified. We report a genome-wide association study meta-analysis of thyroid function in up to 271,040 individuals of European ancestry, including reference range thyrotropin (TSH), free thyroxine (FT4), free and total triiodothyronine (T3), proxies for metabolism (T3/FT4 ratio) as well as dichotomized high and low TSH levels. We revealed 259 independent significant associations for TSH (61% novel), 85 for FT4 (67% novel), and 62 novel signals for the T3 related traits. The loci explained 14.1%, 6.0%, 9.5% and 1.1% of the total variation in TSH, FT4, total T3 and free T3 concentrations, respectively. Genetic correlations indicate that TSH associated loci reflect the thyroid function determined by free T3, whereas the FT4 associations represent the thyroid hormone metabolism. Polygenic risk score and Mendelian randomization analyses showed the effects of genetically determined variation in thyroid function on various clinical outcomes, including cardiovascular risk factors and diseases, autoimmune diseases, and cancer. In conclusion, our results improve the understanding of thyroid hormone physiology and highlight the pleiotropic effects of thyroid function on various diseases.

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During oncological surgery, it can be challenging to identify the tumor and establish adequate resection margins. This study proposes a new two-layer approach in which diffuse reflectance spectroscopy (DRS) is used to predict the top layer thickness and classify the layers in two-layered phantom and animal tissue. Using wavelet-based and peak-based DRS spectral features, the proposed method could predict the top layer thickness with an accuracy of up to 0.35 mm. In addition, the tissue types of the first and second layers were classified with an accuracy of 0.95 and 0.99. Distinguishing multiple tissue layers during spectral analyses results in a better understanding of more complex tissue structures encountered in surgical practice.

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Establishing adequate resection margins during colorectal cancer surgery is challenging. Currently, in up to 30% of the cases the tumor is not completely removed, which emphasizes the lack of a real-time tissue discrimination tool that can assess resection margins up to multiple millimeters in depth. Therefore, we propose to combine spectral data from diffuse reflectance spectroscopy (DRS) with spatial information from ultrasound (US) imaging to evaluate multi-layered tissue structures. First, measurements with animal tissue were performed to evaluate the feasibility of the concept. The phantoms consisted of muscle and fat layers, with a varying top layer thickness of 0-10 mm. DRS spectra of 250 locations were obtained and corresponding US images were acquired. DRS features were extracted using the wavelet transform. US features were extracted based on the graph theory and first-order gradient. Using a regression analysis and combined DRS and US features, the top layer thickness was estimated with an error of up to 0.48 mm. The tissue types of the first and second layers were classified with accuracies of 0.95 and 0.99 respectively, using a support vector machine model.

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Background: Although the incidence of positive resection margins in breast-conserving surgery has decreased, both incomplete resection and unnecessary large resections still occur. This is especially the case in the surgical treatment of ductal carcinoma in situ (DCIS). Diffuse reflectance spectroscopy (DRS), an optical technology based on light tissue interactions, can potentially characterize tissue during surgery thereby guiding the surgeon intraoperatively. DRS has shown to be able to discriminate pure healthy breast tissue from pure invasive carcinoma (IC) but limited research has been done on (1) the actual optical characteristics of DCIS and (2) the ability of DRS to characterize measurements that are a mixture of tissue types. Methods: In this study, DRS spectra were acquired from 107 breast specimens from 107 patients with proven IC and/or DCIS (1488 measurement locations). With a generalized estimating equation model, the differences between the DRS spectra of locations with DCIS and IC and only healthy tissue were compared to see if there were significant differences between these spectra. Subsequently, different classification models were developed to be able to predict if the DRS spectrum of a measurement location represented a measurement location with “healthy” or “malignant” tissue. In the development and testing of the models, different definitions for “healthy” and “malignant” were used. This allowed varying the level of homogeneity in the train and test data. Results: It was found that the optical characteristics of IC and DCIS were similar. Regarding the classification of tissue with a mixture of tissue types, it was found that using mixed measurement locations in the development of the classification models did not tremendously improve the accuracy of the classification of other measurement locations with a mixture of tissue types. The evaluated classification models were able to classify measurement locations with > 5% malignant cells with a Matthews correlation coefficient of 0.41 or 0.40. Some models showed better sensitivity whereas others had better specificity. Conclusion: The results suggest that DRS has the potential to detect malignant tissue, including DCIS, in healthy breast tissue and could thus be helpful for surgical guidance.

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Background and Objectives: In patients with rectal cancer who received neoadjuvant (chemo)radiotherapy, fibrosis is induced in and around the tumor area. As tumors and fibrosis have similar visual and tactile feedback, they are hard to distinguish during surgery. To prevent positive resection margins during surgery and spare healthy tissue, it would be of great benefit to have a real-time tissue classification technology that can be used in vivo. Study Design/Materials and Methods: In this study diffuse reflectance spectroscopy (DRS) was evaluated for real-time tissue classification of tumor and fibrosis. DRS spectra of fibrosis and tumor were obtained on excised rectal specimens. After normalization using the area under the curve, a support vector machine was trained using a 10-fold cross-validation. Results: Using spectra of pure tumor tissue and pure fibrosis tissue, we obtained a mean accuracy of 0.88. This decreased to a mean accuracy of 0.61 when tumor measurements were used in which a layer of healthy tissue, mainly fibrosis, was present between the tumor and the measurement surface. Conclusion: It is possible to distinguish pure fibrosis from pure tumor. However, when the measurements on tumor also involve fibrotic tissue, the classification accuracy decreases. Lasers Surg. Med.

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Background: In colorectal cancer surgery there is a delicate balance between complete removal of the tumor and sparing as much healthy tissue as possible. Especially in rectal cancer, intraoperative tissue recognition could be of great benefit in preventing positive resection margins and sparing as much healthy tissue as possible. To better guide the surgeon, we evaluated the accuracy of diffuse reflectance spectroscopy (DRS) for tissue characterization during colorectal cancer surgery and determined the added value of DRS when compared to clinical judgement. Methods: DRS spectra were obtained from fat, healthy colorectal wall and tumor tissue during colorectal cancer surgery and results were compared to histopathology examination of the measurement locations. All spectra were first normalized at 800 nm, thereafter two support vector machines (SVM) were trained using a tenfold cross-validation. With the first SVM fat was separated from healthy colorectal wall and tumor tissue, the second SVM distinguished healthy colorectal wall from tumor tissue. Results: Patients were included based on preoperative imaging, indicating advanced local stage colorectal cancer. Based on the measurement results of 32 patients, the classification resulted in a mean accuracy for fat, healthy colorectal wall and tumor of 0.92, 0.89 and 0.95 respectively. If the classification threshold was adjusted such that no false negatives were allowed, the percentage of false positive measurement locations by DRS was 25% compared to 69% by clinical judgement. Conclusion: This study shows the potential of DRS for the use of tissue classification during colorectal cancer surgery. Especially the low false positive rate obtained for a false negative rate of zero shows the added value for the surgeons. Trail registration This trail was performed under approval from the internal review board committee (Dutch Trail Register NTR5315), registered on 04/13/2015, https://www.trialregister.nl/trial/5175.

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Neoadjuvant radiotherapy, as part of the conventional treatment of rectal cancer, can induce fibrotic tissue formation around the tumor. This complicates the exact determination of the tumor borders during surgery, which might increase the chance of positive resection margins. In a previous ex vivo study, we distinguished tumor tissue from healthy rectal wall and fat with an accuracy of 0.95, using diffuse reflectance spectroscopy (DRS). Since this study did not include fibrosis, the aim of the current ex vivo study was to examine whether differentiation of tumor and fibrosis with DRS is possible. DRS measurements from freshly resected specimen of 16 patients were obtained. In eight patients fibrosis was measured, in the other eight patients tumor was measured. The measurements were performed using a DRS probe with a source-detector distance of 2 mm. The spectra were obtained in the wavelength range of 450-1600 nm. Classification of the measurements was done using a support vector machine (SVM) and a set of features extracted from the spectra. The SVM was evaluated using an eight-fold cross-validation, which was repeated ten times. For all repetitions, the area under the ROC curve was greater than 0.85 (mean = 0.87, STD = 0.02). The mean sensitivity and specificity were 0.85 (STD = 0.03) and 0.88 (STD = 0.01) respectively. It can be concluded that tumor tissue can be distinguished from fibrosis based on spectral features from DRS measurements. The next step will be to conduct an in vivo study, to verify these results during surgery.@en

In 10 to 40% of breast cancer patients treated with breast conserving surgery, ductal carcinoma in situ (DCIS) or invasive carcinoma (IC) is present at the margin of the specimen. Diffuse Reflectance Spectroscopy (DRS) could potentially help the surgeon during surgery in avoiding these tumor positive resection margins. Here we assess the feasibility of DRS for discriminating healthy breast tissue from tumor. For this purpose, DRS measurements (400-1600nm) were obtained in 71 breast specimens. A custom-made grid was placed on top of a slice of the resection specimen to accurately correlate the DRS measurements with histopathology. To assess DRS for the discrimination between healthy and tumor tissue types, a support vector machine based classification algorithm was developed. A total of 1081 locations were included, of which 236 locations were considered homogenous (> 95% fat, connective tissue, IC, or DCIS) and 845 locations consisted of a mixture of tissue types. The spectra of pure DCIS locations were similar to the spectra of pure IC, suggesting that these tissue types could not be separated based on their spectra. In a first analysis it was found that locations of pure fat, pure connective, and pure tumor tissue (both DCIS and IC) could be discriminated with accuracies of 0.95, 0.95, and 0.79 respectively. Classification of mixture locations was more challenging. The accuracy for tumor detection depends on the amount of tumor present in the measurement volume. These results indicate that DRS can potentially be used for the detection of tumor at the resection margin.@en

In vivo data acquisition using fiberoptic diffuse reflectance spectroscopy (DRS) is more complicated and less controlled compared to ex vivo data acquisition. It would be of great benefit if classifiers for in vivo tissue discrimination based on DRS could be trained on data obtained ex vivo. In this study, in vivo and ex vivo DRS measurements are obtained during colorectal cancer surgery. A mixed model statistical analysis is used to examine the differences between the two datasets. Furthermore, classifiers are trained and tested using in vivo and ex vivo data. It is found that with a classifier trained on ex vivo data and tested on in vivo data, similar results are obtained compared to a classifier trained and tested on in vivo data. In conclusion, under the conditions used in this study, classifiers intended for in vivo tissue discrimination can be trained on ex vivo data.@en

Diffuse reflectance spectroscopy (DRS) can discriminate different tissue types based on optical characteristics. Since this technology has the ability to detect tumor tissue, several groups have proposed to use DRS for margin assessment during breast-conserving surgery for breast cancer. Nowadays, an increasing number of patients with breast cancer are being treated by neoadjuvant chemotherapy. Limited research has been published on the influence of neoadjuvant chemotherapy on the optical characteristics of the tissue. Hence, it is unclear whether margin assessment based on DRS is feasible in this specific group of patients. We investigate whether there is an effect of neoadjuvant chemotherapy on optical measurements of breast tissue. To this end, DRS measurements were performed on 92 ex-vivo breast specimens from 92 patients, treated with neoadjuvant chemotherapy and without neoadjuvant chemotherapy. Generalized estimating equation (GEE) models were generated, comparing the measurements of patients with and without neoadjuvant chemotherapy in datasets of different tissue types using a significance level of 5%. As input for the GEE models, either the intensity at a specific wavelength or a fit parameter, derived from the spectrum, was used. In the evaluation of the intensity, no influence of neoadjuvant chemotherapy was found, since none of the wavelengths were significantly different between the measurements with and the measurements without neoadjuvant chemotherapy in any of the datasets. These results were confirmed by the analysis of the fit parameters, which showed a significant difference for the amount of collagen in only one dataset. All other fit parameters were not significant for any of the datasets. These findings may indicate that assessment of the resection margin with DRS is also feasible in the growing population of breast cancer patients who receive neoadjuvant chemotherapy. However, it is possible that we did not detect neoadjuvant chemotherapy effect in the some of the datasets due to the small number of measurements in those datasets.@en

Background and Objectives: There is a clinical need to assess the resection margins of tongue cancer specimens, intraoperatively. In the current ex vivo study, we evaluated the feasibility of hyperspectral diffuse reflectance imaging (HSI) for distinguishing tumor from the healthy tongue tissue. Study Design/Materials and Methods: Fresh surgical specimens (n = 14) of squamous cell carcinoma of the tongue were scanned with two hyperspectral cameras that cover the visible and near-infrared spectrum (400–1,700 nm). Each pixel of the hyperspectral image represents a measure of the diffuse optical reflectance. A neural network was used for tissue-type prediction of the hyperspectral images of the visual and near-infrared data sets separately as well as both data sets combined. Results: HSI was able to distinguish tumor from muscle with a good accuracy. The diagnostic performance of both wavelength ranges (sensitivity/specificity of visual and near-infrared were 84%/80% and 77%/77%, respectively) appears to be comparable and there is no additional benefit of combining the two wavelength ranges (sensitivity and specificity were 83%/76%). Conclusions: HSI has a strong potential for intra-operative assessment of tumor resection margins of squamous cell carcinoma of the tongue. This may optimize surgery, as the entire resection surface can be scanned in a single run and the results can be readily available. Lasers Surg. Med.@en

Precise nerve localization is of major importance in both surgery and regional anesthesia. Optically based techniques can identify tissue through differences in optical properties, like absorption and scattering. The aim of this study was to evaluate the potential of optical spectroscopy (diffuse reflectance spectroscopy) for clinical nerve identification in vivo. Eighteen patients (8 male, 10 female, age 53 ± 13 years) undergoing inguinal lymph node resection or resection or a soft tissue tumor in the groin were included to measure the femoral or sciatic nerve and the surrounding tissues. In vivo optical measurements were performed using Diffuse Reflectance Spectroscopy (400–1600 nm) on nerve, near nerve adipose tissue, muscle, and subcutaneous fat using a needle-shaped probe. Model-based analyses were used to derive verified quantitative parameters as concentrations of optical absorbers and several parameters describing scattering. A total of 628 optical spectra were recorded. Measured spectra reveal noticeable tissue specific characteristics. Optical absorption of water, fat, and oxy- and deoxyhemoglobin was manifested in the measured spectra. The parameters water and fat content showed significant differences (P < 0.005) between nerve and all surrounding tissues. Classification using k-Nearest Neighbor based on the derived parameters revealed a sensitivity of 85% and a specificity of 79%, for identifying nerve from surrounding tissues. Diffuse Reflectance Spectroscopy identifies peripheral nerve bundles. The differences found between tissue groups are assignable to the tissue composition and structure.

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Objective: Identification of peripheral nerve tissue is crucial in both surgery and regional anesthesia. Recently, optical tissue identification methods are presented to facilitate nerve identification in transcutaneous procedures and surgery. Optimization and validation of such techniques require large datasets. The use of alternative models to human in vivo, like human post mortem, or swine may be suitable to test, optimize and validate new optical techniques. However, differences in tissue characteristics and thus optical properties, like oxygen saturation and tissue perfusion are to be expected. This requires a structured comparison between the models. Study Design: Comparative observational study. Methods: Nerve and surrounding tissues in human (in vivo and post mortem) and swine (in vivo and post mortem) were structurally compared macroscopically, histologically, and spectroscopically. Diffuse reflective spectra were acquired (400–1,600 nm) after illumination with a broad band halogen light. An analytical model was used to quantify optical parameters including concentrations of optical absorbers. Results: Several differences were found histologically and in the optical parameters. Histologically nerve and adipose tissue (subcutaneous fat and sliding fat) showed clear similarities between human and swine while human muscle enclosed more adipocytes and endomysial collagen. Optical parameters revealed model dependent differences in concentrations of β-carotene, water, fat, and oxygen saturation. The similarity between optical parameters is, however, sufficient to yield a strong positive correlation after cross model classification. Conclusion: This study shows and discusses similarities and differences in nerve and surrounding tissues between human in vivo and post mortem, and swine in vivo and post mortem; this could support the discussion to use an alternative model to optimize and validate optical techniques for clinical nerve identification. Lasers Surg. Med. 50:253–261, 2018.

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Background: Careful identification of nerves during head and neck surgery is essential to prevent nerve damage. Currently, nerves are identified based on anatomy and appearance, optionally combined with electromyography (EMG). In challenging cases, nerve damage is reported in up to 50%. Recently, optical techniques, like diffuse reflectance spectroscopy (DRS) and fluorescence spectroscopy (FS) show potential to improve nerve identification. Methods: 212 intra-operative DRS/FS measurements were performed. Small nerve branches (1-3 mm), on near-nerve adipose tissue, muscle and subcutaneous fat were measured during 11 surgical procedures. Tissue identification was based on quantified concentrations of optical absorbers and scattering parameters. Results: Clinically comprehensive parameters showed significant differences (<0.05) between the tissues. Classification using k-Nearest Neighbor resulted in 100% sensitivity and a specificity of 83% (accuracy 91%), for the identification of nerve against surrounding tissues. Conclusions: DRS/FS is a potentially useful intraoperative tool for identification of nerves from adjacent tissues. Level of Evidence: Observational proof of principle study.

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This ex-vivo study evaluates the feasibility of diffuse reflectance spectroscopy (DRS) for discriminating tumor from healthy tissue, with the aim to develop a technology that can assess resection margins for the presence of tumor cells during oral cavity cancer surgery. Diffuse reflectance spectra were acquired on fresh surgical specimens from 28 patients with oral cavity squamous cell carcinoma. The spectra (400 to 1600 nm) were detected after illuminating tissue with a source fiber at 0.3-, 0.7-, 1.0-, and 2.0-mm distances from a detection fiber, obtaining spectral information from different sampling depths. The spectra were correlated with histopathology. A total of 76 spectra were obtained from tumor tissue and 110 spectra from healthy muscle tissue. The first- and second-order derivatives of the spectra were calculated and a classification algorithm was developed using fivefold cross validation with a linear support vector machine. The best results were obtained by the reflectance measured with a 1-mm source-detector distance (sensitivity, specificity, and accuracy are 89%, 82%, and 86%, respectively). DRS can accurately discriminate tumor from healthy tissue in an ex-vivo setting using a 1-mm source-detector distance. Accurate validation methods are warranted for larger sampling depths to allow for guidance during oral cavity cancer excision.

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Background: Breast cancer surgeons struggle with differentiating healthy tissue from cancer at the resection margin during surgery. We report on the feasibility of using diffuse reflectance spectroscopy (DRS) for real-time in vivo tissue characterization. Methods: Evaluating feasibility of the technology requires a setting in which measurements, imaging and pathology have the best possible correlation. For this purpose an optical biopsy needle was used that had integrated optical fibers at the tip of the needle. This approach enabled the best possible correlation between optical measurement volume and tissue histology. With this optical biopsy needle we acquired real-time DRS data of normal tissue and tumor tissue in 27 patients that underwent an ultrasound guided breast biopsy procedure. Five additional patients were measured in continuous mode in which we obtained DRS measurements along the entire biopsy needle trajectory. We developed and compared three different support vector machine based classification models to classify the DRS measurements. Results: With DRS malignant tissue could be discriminated from healthy tissue. The classification model that was based on eight selected wavelengths had the highest accuracy and Matthews Correlation Coefficient (MCC) of 0.93 and 0.87, respectively. In three patients that were measured in continuous mode and had malignant tissue in their biopsy specimen, a clear transition was seen in the classified DRS measurements going from healthy tissue to tumor tissue. This transition was not seen in the other two continuously measured patients that had benign tissue in their biopsy specimen. Conclusions: It was concluded that DRS is feasible for integration in a surgical tool that could assist the breast surgeon in detecting positive resection margins during breast surgery. Trail registration NIH US National Library of Medicine-clinicaltrails.gov, NCT01730365. Registered: 10/04/2012 https://clinicaltrials.gov/ct2/show/study/NCT01730365

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Colorectal surgery is the standard treatment for patients with colorectal cancer. To overcome two of the main challenges, the circumferential resection margin and postoperative complications, real-time tissue assessment could be of great benefit during surgery. In this ex vivo study, diffuse reflectance spectroscopy (DRS) was used to differentiate tumor tissue from healthy surrounding tissues in patients with colorectal neoplasia. DRS spectra were obtained from tumor tissue, healthy colon, or rectal wall and fat tissue, for every patient. Data were randomly divided into training (80%) and test (20%) sets. After spectral band selection, the spectra were classified using a quadratic classifier and a linear support vector machine. Of the 38 included patients, 36 had colorectal cancer and 2 had an adenoma. When the classifiers were applied to the test set, colorectal cancer could be discriminated from healthy tissue with an overall accuracy of 0.95 (±0.03). This study demonstrates the possibility to separate colorectal cancer from healthy surrounding tissue by applying DRS. High classification accuracies were obtained both in homogeneous and inhomogeneous tissues. This is a fundamental step toward the development of a tool for real-time in vivo tissue assessment during colorectal surgery.

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Successful breast conserving surgery consists of complete removal of the tumor while sparing healthy surrounding tissue. Despite currently available imaging and margin assessment tools, recognizing tumor tissue at a resection margin during surgery is challenging. Diffuse reflectance spectroscopy (DRS), which uses light for tissue characterization, can potentially guide surgeons to prevent tumor positive margins. However, inter-patient variation and changes in tissue physiology occurring during the resection might hamper this light-based technology. Here we investigate how inter-patient variation and tissue status (in vivo vs ex vivo) affect the performance of the DRS optical parameters. In vivo and ex vivo measurements of 45 breast cancer patients were obtained and quantified with an analytical model to acquire the optical parameters. The optical parameter representing the ratio between fat and water provided the best discrimination between normal and tumor tissue, with an area under the receiver operating characteristic curve of 0.94. There was no substantial influence of other patient factors such as menopausal status on optical measurements. Contrary to expectations, normalization of the optical parameters did not improve the discriminative power. Furthermore, measurements taken in vivo were not significantly different from the measurements taken ex vivo. These findings indicate that DRS is a robust technology for the detection of tumor tissue during breast conserving surgery.

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