Intraoperative delineation of p16+ oropharyngeal carcinoma of unknown primary origin with fluorescence lifetime imaging: Preliminary report.

BACKGROUND: This study evaluated whether fluorescence lifetime imaging (FLIm), coupled with standard diagnostic workups, could enhance primary lesion detection in patients with p16+ head and neck squamous cell carcinoma of the unknown primary (HNSCCUP). METHODS: FLIm was integrated into transoral robotic surgery to acquire optical data on six HNSCCUP patients' oropharyngeal tissues. An additional 55-patient FLIm dataset, comprising conventional primary tumors, trained a machine learning classifier; the output predicted the presence and location of HNSCCUP for the six patients. Validation was performed using histopathology. RESULTS: Among the six HNSCCUP patients, p16+ occult primary was surgically identified in three patients, whereas three patients ultimately had no identifiable primary site in the oropharynx. FLIm correctly detected HNSCCUP in all three patients (ROC-AUC: 0.90 ± 0.06), and correctly predicted benign oropharyngeal tissue for the remaining three patients. The mean sensitivity was 95% ± 3.5%, and specificity 89% ± 12.7%. CONCLUSIONS: FLIm may be a useful diagnostic adjunct for detecting HNSCCUP.

Intraoperative Mapping of Parathyroid Glands Using Fluorescence Lifetime Imaging.

BACKGROUND: Hypoparathyroidism is a common complication following thyroidectomy. There is a need for technology to aid surgeons in identifying the parathyroid glands. In contrast to near infrared technologies, fluorescence lifetime imaging (FLIm) is not affected by ambient light and may be valuable in identifying parathyroid tissue, but has never been evaluated in this capacity. METHODS: We used FLIm to measure the UV induced (355 nm) time-resolved autofluorescence signatures (average lifetimes in 3 spectral emission channels) of thyroid, parathyroid, lymphoid and adipose tissue in 21 patients undergoing thyroid and parathyroid surgery. The Mann-Whitney U test was used to assess the ability of FLIm to discriminate normocellular parathyroid from each of the other tissues. Various machine learning classifiers (random forests, neural network, support vector machine) were then evaluated to recognize parathyroid through a leave-one-out cross-validation. RESULTS: Statistically significant differences in average lifetime were observed between parathyroid and each of the other tissue types in spectral channels 2 and 3 respectively. The largest change was observed between adipose tissue and parathyroid (P < 0.001), while less pronounced but still significant changes were observed when comparing parathyroid with lymphoid tissue (P < 0.05) and thyroid (P < 0.01). A random forest classifier trained on average lifetimes was found to detect parathyroid tissue with 100% sensitivity and 93% specificity at the acquisition run level. CONCLUSION: We found that FLIm derived parameters can distinguish the parathyroid glands and other adjacent tissue types and has promise in scanning the surgical field to identify parathyroid tissue in real-time.

Mesoscopic fluorescence lifetime imaging: Fundamental principles, clinical applications and future directions.

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.

Intraoperative Margin Assessment in Oral and Oropharyngeal Cancer Using Label-Free Fluorescence Lifetime Imaging and Machine Learning.

OBJECTIVE: To demonstrate the diagnostic ability of label-free, point-scanning, fiber-based Fluorescence Lifetime Imaging (FLIm) as a means of intraoperative guidance during oral and oropharyngeal cancer removal surgery. METHODS: FLIm point-measurements acquired from 53 patients (n = 67893 pre-resection in vivo, n = 89695 post-resection ex vivo) undergoing oral or oropharyngeal cancer removal surgery were used for analysis. Discrimination of healthy tissue and cancer was investigated using various FLIm-derived parameter sets and classifiers (Support Vector Machine, Random Forests, CNN). Classifier output for the acquired set of point-measurements was visualized through an interpolation-based approach to generate a probabilistic heatmap of cancer within the surgical field. Classifier output for dysplasia at the resection margins was also investigated. RESULTS: Statistically significant change (P 0.01) between healthy and cancer was observed in vivo for the acquired FLIm signal parameters (e.g., average lifetime) linked with metabolic activity. Superior classification was achieved at the tissue region level using the Random Forests method (ROC-AUC: 0.88). Classifier output for dysplasia (% probability of cancer) was observed to lie between that of cancer and healthy tissue, highlighting FLIm's ability to distinguish various conditions. CONCLUSION: The developed approach demonstrates the potential of FLIm for fast, reliable intraoperative margin assessment without the need for contrast agents. SIGNIFICANCE: Fiber-based FLIm has the potential to be used as a diagnostic tool during cancer resection surgery, including Transoral Robotic Surgery (TORS), helping ensure complete resections and improve the survival rate of oral and oropharyngeal cancer patients.

FLImBrush: dynamic visualization of intraoperative free-hand fiber-based fluorescence lifetime imaging.

A free-hand scanning approach to medical imaging allows for flexible, lightweight probes to image intricate anatomies for modalities such as fluorescence lifetime imaging (FLIm), optical coherence tomography (OCT) and ultrasound. While very promising, this approach faces several key challenges including tissue motion during imaging, varying lighting conditions in the surgical field, and sparse sampling of the tissue surface. These challenges limit the coregistration accuracy and interpretability of the acquired imaging data. Here we report FLImBrush as a robust method for the localization and visualization of intraoperative free-hand fiber optic fluorescence lifetime imaging (FLIm). FLImBrush builds upon an existing method while employing deep learning-based image segmentation, block-matching based motion correction, and interpolation-based visualization to address the aforementioned challenges. Current results demonstrate that FLImBrush can provide accurate localization of FLIm point-measurements while producing interpretable and complete visualizations of FLIm data acquired from a tissue surface. Each of the main processing steps was shown to be capable of real-time processing (> 30 frames per second), highlighting the feasibility of FLImBrush for intraoperative imaging and surgical guidance. Current findings show the feasibility of integrating FLImBrush into a range of surgical applications including cancer margins assessment during head and neck surgery.

Fluorescence lifetime imaging for intraoperative cancer delineation in transoral robotic surgery.

This study evaluates the potential for fluorescence lifetime imaging (FLIm) to enhance intraoperative decisionmaking during robotic-assisted surgery of oropharyngeal cancer. Using a custom built FLIm instrument integrated with the da Vinci robotic surgical platform, we first demonstrate that cancer in epithelial tissue diagnosed by histopathology can be differentiated from surrounding healthy epithelial tissue imaged in vivo prior to cancer resection and ex vivo on the excised specimen. Second, we study the fluorescence properties of tissue imaged in vivo at surgical resection margins (tumor bed). Fluorescence lifetimes and spectral intensity ratios were calculated for three spectral channels, producing a set of six FLIm parameters. Current results from 10 patients undergoing TORS procedures demonstrate that healthy epithelium can be resolved from cancer (P < .001) for at least one FLIm parameter. We also showed that a multiparameter linear discriminant analysis approach provides superior discrimination to individual FLIm parameters for tissue imaged both in vivo and ex vivo. Overall, this study highlights the potential for FLIm to be developed into a diagnostic tool for clinical cancer applications of the oropharynx. This technique could help to circumvent the issues posed by the lack of tactile feedback associated with robotic surgical platforms to better enable cancer delineation.

Allosteric modulation of beta1 integrin function induces lung tissue repair.

The cellular cytoskeleton, adhesion receptors, extracellular matrix composition, and their spatial distribution are together fundamental in a cell's balanced mechanical sensing of its environment. We show that, in lung injury, extracellular matrix-integrin interactions are altered and this leads to signalling alteration and mechanical missensing. The missensing, secondary to matrix alteration and cell surface receptor alterations, leads to increased cellular stiffness, injury, and death. We have identified a monoclonal antibody against ß1 integrin which caused matrix remodelling and enhancement of cell survival. The antibody acts as an allosteric dual agonist/antagonist modulator of ß1 integrin. Intriguingly, this antibody reversed both functional and structural tissue injury in an animal model of degenerative disease in lung.

Assessment of valvular calcification and inflammation by positron emission tomography in patients with aortic stenosis.

BACKGROUND: The pathophysiology of aortic stenosis is incompletely understood, and the relative contributions of valvular calcification and inflammation to disease progression are unknown. METHODS AND RESULTS: Patients with aortic sclerosis and mild, moderate, and severe stenosis were compared prospectively with age- and sex-matched control subjects. Aortic valve severity was determined by echocardiography. Calcification and inflammation in the aortic valve were assessed by 18F-sodium fluoride (18F-NaF) and 18F-fluorodeoxyglucose (18F-FDG) uptake with the use of positron emission tomography. One hundred twenty-one subjects (20 controls; 20 aortic sclerosis; 25 mild, 33 moderate, and 23 severe aortic stenosis) were administered both 18F-NaF and 18F-FDG. Quantification of tracer uptake within the valve demonstrated excellent interobserver repeatability with no fixed or proportional biases and limits of agreement of ±0.21 (18F-NaF) and ±0.13 (18F-FDG) for maximum tissue-to-background ratios. Activity of both tracers was higher in patients with aortic stenosis than in control subjects (18F-NaF: 2.87±0.82 versus 1.55±0.17; 18F-FDG: 1.58±0.21 versus 1.30±0.13; both P<0.001). 18F-NaF uptake displayed a progressive rise with valve severity (r(2)=0.540, P<0.001), with a more modest increase observed for 18F-FDG (r(2)=0.218, P<0.001). Among patients with aortic stenosis, 91% had increased 18F-NaF uptake (>1.97), and 35% had increased 18F-FDG uptake (>1.63). A weak correlation between the activities of these tracers was observed (r(2)=0.174, P<0.001). CONCLUSIONS: Positron emission tomography is a novel, feasible, and repeatable approach to the evaluation of valvular calcification and inflammation in patients with aortic stenosis. The frequency and magnitude of increased tracer activity correlate with disease severity and are strongest for 18F-NaF. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT01358513.

Organization and expression of the human serpin gene cluster at 14q32.1.

The serpins are a superfamily of gene sequences that have been conserved through evolution. These genes encode protein products that perform a variety of functions in vivo, and their regulation differs among different cell types. About one-third of the serpin genes in the human genome are located at 14q32.1, and the serpin genes in this ~370 kb region are organized into discrete proximal, central, and distal subclusters of four, three, and four genes each. In this report we discuss the genomic organization of the 14q32.1 serpin gene cluster, and we summarize what is known about the regulation of each serpin gene in this region. An approach for studying locus-wide regulation of chromosomal serpin genes in situ is also described. Using this approach, specific mutations in the proximal serpin subcluster were prepared by homologous recombination. These mutant alleles define a serpin locus control region that regulates gene activity and chromatin structure of the entire proximal subcluster. Prospects for further analyses of this complex genomic domain are discussed.

Sequence organization and matrix attachment regions of the human serine protease inhibitor gene cluster at 14q32.1.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system for studying the regulation of gene activity and chromatin structure. We demonstrated previously that the six known serpin genes in this region were organized into two subclusters of three genes each that occupied approximately 370 kb of DNA. To more fully understand the genomic organization of this region, we annotated a 1-Mb sequence contig from data from the Genoscope sequencing consortium (http://www.genoscope.cns.fr/ ). We report that 11 different serpin genes reside within the 14q32.1 cluster, including two novel alpha1-antiproteinase-like gene sequences, a kallistatin-like sequence, and two recently identified serpins that had not been mapped previously to 14q32.1. The genomic regions proximal and distal to the serpin cluster contain a variety of unrelated gene sequences of diverse function. To gain insight into the chromatin organization of the region, sequences with putative nuclear matrix-binding potential were identified by using the MAR-Wiz algorithm, and these MAR-Wiz candidate sequences were tested for nuclear matrix-binding activity in vitro. Several differences between the MAR-Wiz predictions and the results of biochemical tests were observed. The genomic organization of the serpin gene cluster is discussed.

Chromosomal elements regulate gene activity and chromatin structure of the human serpin gene cluster at 14q32.1.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 contains a number of genes that are specifically expressed in hepatic cells. Cell-specific enhancers have been identified in several of these genes, but elements involved in locus-wide gene and chromatin control have yet to be defined. To identify regulatory elements in this region, we prepared a series of mutant chromosomal alleles by homologous recombination and transferred the specifically modified human chromosomes to hepatic cells for functional tests. We report that deletion of an 8-kb DNA segment upstream of the human alpha1-antitrypsin gene yields a mutant serpin allele that fails to be activated in hepatic cells. Within this region, a 2.3-kb DNA segment between kb -8.1 and -5.8 contains a previously unrecognized control region that is required not only for serpin gene activation but also for chromatin remodeling of the entire locus.