Autofluorescence Imaging Research Articles

Autofluorescence Excitation Imaging of Nonmelanoma Skin Cancer for Margin Assessment Before Mohs Micrographic Surgery: A Pilot Study.

Autofluorescence photography can detect specific light-tissue interactions and record important pathophysiological changes associated with nonmelanoma skin cancer (NMSC), which has been ascribed to the fluorescence of an aromatic amino acid, tryptophan. To assess the impact of a novel, autofluorescence imaging (AFI) device on margin control for NMSCs before Mohs micrographic surgery (MMS) in an effort to decrease overall operating time. Before the initial stage of MMS, NMSCs were measured with a 2-mm margin as standard of care (normal margin). The tumor was then imaged with the AFI device. A 2-mm margin was drawn around the fluorescent area captured by the AFI device and was referred to as the camera margin. The tumor was excised based on the normal margin and evaluated on frozen histological section. Imaging based on the AFI device resulted in appropriate recommendations for margin control in 8 of 11 tumors. Four of these tumors did not fluoresce and demonstrated a lack of tumor residuum on stage I specimen, as anticipated. There were no side effects from the AFI device. This is an initial pilot study that supports the use of a novel, noninvasive imaging device to help with margin assessment before MMS. On optimization, this device has potential to extend applicability to surgical excisions for tumors that do not fulfill criteria for MMS.

Abstract 4169: A deep learning approach to guide acquisition region selection for imaging mass cytometry

Abstract The growth in cancer immunotherapy agents requires an understanding of the immune contexture of the tumor microenvironment. This can be aided by high-plex imaging and analysis to obtain phenotypes of specific cells and study their biodistribution and interactions. Imaging Mass Cytometry™ (IMC) is the method of choice for single-step staining and high-plex imaging of tissues, avoiding the complications of autofluorescence and cyclic imaging. IMC has expanded its capabilities with three distinct imaging modes: Preview, Cell, and Tissue. The Preview Mode is a rapid scanning system that captures a comprehensive overview of the stained tissue, mapping out the distribution of over 40 markers and revealing tissue heterogeneity. This enables researchers to make informed decisions about which areas warrant closer examination on the same. Building on this, Cell Mode offers high-resolution imaging for detailed analysis of the Regions of Interest (ROI) identified during Preview, all using the same slide. Tissue Mode complements these by providing a fast acquisition of the entire tissue at a lower resolution, which is optimal for quantitative pixel-based analysis of tissue biology. These modes support automated, continuous imaging of more than 40 large tissue samples (400 mm2) weekly. Following Preview Mode, the selection of ROIs for high-resolution imaging is a critical step, enhanced by automated AI algorithms to ensure it is informed by biomarker expression. Tissue sections of colon adenocarcinoma were stained with a 30-marker IMC panel of structural, tumor, stroma, immune cell, and immune activation markers. Images were acquired on Hyperion XTi™ (Standard BioTools), first in Preview Mode, then in Cell Mode with automatic selection of ROIs using Phenoplex™ (Visiopharm). ROIs were automatically selected based on two criteria: 1) actively proliferating and non-proliferating tumor regions; 2) cold and hot tumor regions as identified by hotspots of lymphoid and myeloid immune markers within stromal or epithelial tumor regions. Single cell analysis of the high-resolution images obtained in Cell Mode, was performed as a multi-step workflow using Phenoplex. Tissue segmentation was used to divide the tissue into epithelial tumor (E-Cadherin/panCK), stromal, and tumor margin regions; cell segmentation was based on Iridium DNA channels; and phenotyping was performed using the guided highplex workflow. These data were used to compare the immune contexture through a series of t-SNE plots partitioned by spatial region and clinical variables. This work demonstrates that the multi-modal features of Hyperion XTi can greatly accelerate the ability of IMC users to gain useful insights from complex biological samples. Phenoplex enables a comprehensive workflow for the analysis of this complex data, providing automated ROI selection, and phenotyping and spatial analyses of high-resolution IMC images for biological assessment. Citation Format: Fabian Schneider, Smriti Kala, Sam Lim, Clinton Hupple, Nina Lane, James Robert Mansfield. A deep learning approach to guide acquisition region selection for imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4169.

Autofluorescence is a biomarker of neural stem cell activation state

Neural stem cells (NSCs) must exit quiescence to produce neurons; however, our understanding of this process remains constrained by the technical limitations of current technologies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Using this non-destructive, live-cell, and label-free strategy, we found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) have unique autofluorescence profiles. Specifically, qNSCs display an enrichment of autofluorescence localizing to a subset of lysosomes, which can be used as a graded marker of NSC quiescence to predict cell behavior at single-cell resolution. Coupling autofluorescence imaging with single-cell RNA sequencing, we provide resources revealing transcriptional features linked to deep quiescence and rapid NSC activation. Together, we describe an approach for tracking mouse NSC activation state and expand our understanding of adult neurogenesis.

Adaptive Optics Retinal Imaging in RDH12-Associated Early Onset Severe Retinal Dystrophy.

RDH12 is among the most common genes found in individuals with early-onset severe retinal (EOSRD). Adaptive optics scanning light ophthalmoscopy (AOSLO) enables resolution of individual rod and cone photoreceptors in the retina. This study presents the first AOSLO imaging of individuals with RDH12-associated EOSRD. Case series of patients who attended Moorfields Eye Hospital (London, UK). Spectral-domain optical coherence tomography, near-infrared reflectance (NIR), and blue autofluorescence imaging were analyzed. En face image sequences of photoreceptors were recorded using either of two AOSLO modalities. Cross-sectional analysis was undertaken for seven patients and longitudinal analysis for one patient. Nine eyes from eight patients are presented in this case series. The mean age at the time of the assessment was 11.2 ± 6.5years of age (range 7-29). A subfoveal continuous ellipsoid zone (EZ) line was present in eight eyes. Posterior pole AOSLO revealed patches of cone mosaics. Average cone densities at regions of interest 0.5° to the fovea ranged from 12,620 to 23,660 cells/mm2, whereas intercell spacing ranged from 7.0 to 9.7µm. This study demonstrates that AOSLO can provide useful high-quality images in patients with EOSRD, even during childhood, with nystagmus, and early macular atrophy. Cones at the posterior pole can appear as scattered islands or, possibly later in life, as a single subfoveal conglomerate. Detailed image analysis suggests that retinal pigment epithelial stress and dysfunction may be the initial step toward degeneration, with NIR being a useful tool to assess retinal well-being in RDH12-associated EOSRD.

Abstract 914: Combination analysis of tumor-associated collagen frameworks and tumor immune phenotype of lung carcinomas using virtual staining

Abstract An emerging predictive parameter of immunotherapy response is the patient’s tumor immune status, generally classified as “inflamed”, "immune excluded” and “desert”. Historically, classification was performed semi-quantitatively with somewhat subjective parameters as evidenced by the recent Delphi Workshop consensus. There are a variety of approaches to classifying immune status, most based upon analysis of H&E images (counting tumor infiltrating lymphocytes, TILs), or counting subpopulations of immune cells using immunologic staining. However, use of multiplexed or multiple stains has recently been explored to better quantify the counts and types of immune cells in individual tissue compartments. While the immune microenvironment represents an important parameter in predicting patient response, other regions such as the extracellular matrix (ECM) may yield complementary information, as collagen-rich ECMs may present a barrier to drug diffusion and the orientation of fibers may direct the migration of malignant cells. Here we present a proof of concept virtual staining enhanced image analysis pipeline, which converts autofluorescence signals from a single tissue section into virtual H&E and Masson’s Trichrome along with virtual detection of pan cytokeratin (AE1/AE3/PCK26) and CD45 (LCA) positive cells. We apply image analysis to the panel of virtual stains to study the spatial and case-by-case heterogeneity of tumor collagen frameworks and immune phenotype. Using a standard slide scanner (Axio Scan Z1, Zeiss), multiple autofluorescence images were captured from unstained sections (4-6um thick) of lung tissue. The virtual staining was performed by four deep neural networks trained in a supervised learning fashion. Select chemical stains were performed on previously scanned tissues and reviewed by pathologists side-by-side with virtual stains to ensure consistency and quality control. Four perfectly registered WSI virtual stain images were generated from each tissue section. The multi-stain results were rendered from a variety of lung cancers, including tissue microarray slides. Image analysis was performed using HALO (Indica Labs) and custom python scripts. We identified unique areas of tumor and adjacent stroma with heterogeneous immune phenotype and collagen characteristics, suggesting that an interplay might exist that could be utilized to improve patient selection or prognosis when deploying advanced AI tools. Future work includes applying this virtual stain technique retrospectively to cases with known immunotherapy treatment responses to determine the prognostic significance of the combined immune/ECM phenotype. Citation Format: Serge Alexanian, Yair Rivenson, Ning Xuan, Brian Cone, Zihang Fang, Sean Meyering, Luis A. Carvalho, David Palacios, Raymond Kozikowski. Combination analysis of tumor-associated collagen frameworks and tumor immune phenotype of lung carcinomas using virtual staining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 914.

Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei.

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

Genetic and Clinical Features of ABCA4-Associated Retinopathy in a Japanese Nationwide Cohort

PurposeTo clarify the genetic and clinical features of Japanese patients with ABCA4-associated retinopathy. DesignRetrospective, multicenter cohort study MethodsPatients with retinal degeneration and biallelic ABCA4 variants were recruited from 13 different hospitals. Whole exome sequencing analysis was used for genetic testing. Comprehensive ophthalmic examinations were performed on matched patients. The primary outcome measure was identifying multimodal retinal imaging findings associated with disease progression. ResultsThis study included 63 patients: 19 with missense/missense, 23 with missense/truncation, and 21 with truncation/truncation genotypes. In total, 62 variants were identified, including 29 novel variants. Six patients had a mild phenotype characterized by foveal-sparing or preserved foveal structure, including four with missense/missense and two with missense/truncation genotypes. The p.Arg212His variant was the most frequent in patients with mild phenotypes (4/12 alleles). Clinical findings showed a disease duration-dependent worsening of the phenotypic stage. Patients with the truncation/truncation genotype exhibited rapid retinal degeneration within a few years and definite fundus autofluorescence imaging patterns, including hyper autofluorescence at the macula and few or no flecks. ConclusionsOur results indicate that missense/missense or missense/truncation genotypes, including the p.Arg212His variant, are associated with a relatively mild phenotype. In contrast, the truncation/truncation genotype causes rapid and severe retinal degeneration in Japanese patients with ABCA4-associated retinopathy. These data are vital in predicting patient prognosis, guiding genetic counseling, and stratifying patients for future clinical trials.

Intraoperative Techniques That Define the Mucosal Margins of Oral Cancer In-Vivo: A Systematic Review.

This systematic review investigates techniques for determining adequate mucosal margins during the resection of oral squamous cell carcinoma (SCC). The primary treatment involves surgical removal with ≥5 mm margins, highlighting the importance of accurate differentiation between SCC and dysplasia during surgery. A comprehensive Embase and PubMed literature search was performed. Studies underwent quality assessment using QUADAS-2. After the full-text screening and exclusion of studies exhibiting high bias, eight studies were included, focusing on three margin visualization techniques: autofluorescence, iodine staining, and narrow-band imaging (NBI). Negative predictive value (NPV) was calculable across the studies, though reference standards varied. Results indicated NPVs for autofluorescence, iodine, and NBI ranging from 61% to 100%, 92% to 99%, and 86% to 100%, respectively. Autofluorescence did not significantly enhance margins compared to white light-guided surgery, while iodine staining demonstrated improvement for mild or moderate dysplasia. NBI lacked comparison with a white light-guided surgery cohort. We recommend studying and comparing the diagnostic accuracy of iodine staining and NBI in larger cohorts of patients with oral SCC, focusing on discriminating between SCC and (severe) dysplasia. Furthermore, we advise reporting the diagnostic accuracy alongside the treatment effects to improve the assessment of these techniques.

Subthreshold laser treatment for non-center involved diabetic macular edema via non-damaging retinal laser therapy (NRT).

To evaluate the efficacy of subthreshold laser treatment via non-damaging retinal laser therapy (NRT) in patients with non-center involved diabetic macular edema (non-CI DME). In this prospective controlled study, NRT with 577nm wavelength was performed to the edematous inner subfields as needed at 3 monthly intervals, while the control group received no treatment. If CI-DME developed in either group, intravitreal anti-VEGF was performed and the eye was excluded from subsequent analysis. A total of 75 eyes (36 study eyes, 39 controls) were evaluated. The change in superior, nasal and temporal inner subfield thicknesses over time and between groups was found significant (P = 0.004, P < 0.001, P = 0.04 respectively). Best corrected visual acuity (BCVA) change was not significant over time and between groups (P = 0.69). Rates of CI-DME development requiring intravitreal anti-VEGF treatment were not different during the first and second years (P = 0.171, 0.908). No laser scar was detected in any eye in fundus autofluorescence imaging. NRT performed as needed at 3 monthly intervals is effective after 21months of follow up in the treatment of non-CI DME and it was safe. With this method, it may be foreseen that BCVA will be better preserved in the long term by avoiding the possible side effects of conventional laser.

The impact of short-term postoperative face-up position on unintentional retinal displacement after pars plana vitrectomy for rhegmatogenous retinal detachment.

To assess the effectiveness and safety of a rigorous short-term supine position in preventing inadvertent retinal displacement after pars plana vitrectomy (PPV) with gas tamponade for rhegmatogenous retinal detachment (RRD). We conducted a retrospective observational analysis of a case series at two ophthalmological surgical centers. We included eyes diagnosed with macula-off RRD that maintained a strict face-up position for three hours immediately after PPV with intraoperative perfluorocarbon liquid (PFCL) and 20% sulfur hexafluoride (SF6) tamponade. Fundus autofluorescence (FAF) imaging was performed at one month post-operatively to identify unintentional retinal displacement through the detection of retinal vessel prints (RVPs) on FAF imaging using an ultrawide-field (UWF) imaging system. A total of 29 eyes with macula-off RRD were included in the study. The average age of the participants was 59.62 years. RRD involved one quadrant in two eyes, two quadrants in fourteen eyes, three quadrants in seven eyes, and four quadrants in six eyes. UWF-FAF imaging at one month follow-up after complete reattachment of the retina revealed RVPs in seven out of the 29 eyes (24.13%), with a mean displacement of 0.22 mm. In every case the displacement occurred downward. Our results suggest that adhering to a strict face-up position for three hours after PPV with PFCL and gas tamponade for macula-off RRD may lead to a low frequency and severity of inadvertent post-operative retinal displacement.

Research progresses of near-infrared autofluorescence imaging used in surgery of parathyroid glands

Research progresses of near-infrared autofluorescence imaging used in surgery of parathyroid glands

Metabolic response of auditory brainstem neurons to their broad physiological activity range.

Neurons exhibit a high energetic need, and the question arises as how they metabolically adapt to changing activity states. This is relevant for interpreting functional neuroimaging in different brain areas. Particularly, neurons with a broad firing range might exhibit metabolic adaptations. Therefore, we studied MNTB (medial nucleus of the trapezoid body) principal neurons, which generate action potentials (APs) at frequencies up to several hundred hertz. We performed the experiments in acute brainstem slices of the Mongolian gerbil (Meriones unguiculatus) at 22.5-24.5°C. Upon electrical stimulation of afferent MNTB fibres with 400 stimuli at varying frequencies, we monitored autofluorescence levels of NAD(P)H and FAD and determined the extremum amplitudes of their biphasic response. Additionally, we compared these data with alterations in O2 concentrations measured with an electrochemical sensor. These O2 changes are prominent since MNTB neurons rely on oxidative phosphorylation as shown by our pharmacological experiments. We calculated the O2 consumption rate as change in O2 concentration divided by stimulus durations, because these periods varied inversely with stimulus frequency as a result of the constant number of 400 stimuli applied. The O2 consumption rate increased with stimulation frequency up to a constant value at 600 Hz; that is, energy demand depends on temporal characteristics of activity despite the same number of stimuli. The rates showed no correlation with peak amplitudes of NAD(P)H or FAD, whilst the values of the two molecules were linearly correlated. This points at the complexity of analysing autofluorescence imaging for quantitative metabolic studies, because these values report only relative net changes of many superimposed oxidative and reductive processes. Monitoring O2 concentration rates is, thus, an important tool to improve the interpretation of NAD(P)H/FAD autofluorescence data, as they do not under all conditions and in all systems appropriately reflect the metabolic activity or energy demand.

Shwachman-Diamond syndrome associated with rod-cone dystrophy.

To report a patient with Shwachman-Diamond syndrome and concomitant rod-cone dystrophy who underwent bone marrow transplantation. Retrospective single case report. A female patient with Shwachman-Diamond syndrome was referred to a tertiary hospital to investigate possible pigmentary retinopathy at the age of 16. She described poor night vision and was found to have reduced visual acuity (6/20 right, 6/38 left). Over the ten-year follow-up period, her visual acuity remained relatively stable with no new visual symptoms. Optical coherence tomography revealed progressive, diffuse outer retinal thinning with disruption of the ellipsoid zone, which initially was relatively preserved subfoveally. Fundus autofluorescence images revealed generalised areas of hypoautofluorescence beyond the vascular arcades and a perimacular ring of increased autofluorescence. The flash electroretinogram was in keeping with a severe rod-cone dystrophy. The pattern visual evoked potential was abnormal but detectable indicating macular pathway dysfunction, suggesting encroachment into central macular regions but with some functional preservation. We report a patient with Shwachman-Diamond syndrome with severe early-onset rod-cone dystrophy noted at the age of 16. Slow anatomical progression has been observed over the subsequent ten years, with relative functional macular preservation to support a visual acuity of 6/36 in both eyes.

ATXN7-Related Cone-Rod Dystrophy

Reliable biomarkers with diagnostic and prognostic values are needed for upcoming gene therapy trials for spinocerebellar ataxias. To identify ophthalmological biomarkers in a sample of spinocerebellar ataxia type 7 (SCA7) carriers. This article presents baseline data from a cross-sectional natural history study conducted in Paris, France, reference centers for rare diseases from May 2020 to April 2021. Data were analyzed from September to December 2022. Fifteen adult ATXN7 pathogenic expansion carriers (9 with preataxia and 6 with ataxia) were included, all with a Scale for the Assessment and Rating of Ataxia (SARA) score of 15 of 40 or lower. Patients were recruited at the Paris Brain Institute, and all contacted patients accepted to participate in the study. Three visits (baseline, 6 months, and 12 months) were planned, including neurological examination (SARA and Composite Cerebellar Functional Severity Score), ophthalmological examination (best-corrected visual acuity, microperimetry, full-field electroretinogram, optical coherence tomography, and fundus autofluorescence imaging), and neurofilament light chain (NfL) measurements. Here we report the baseline ophthalmic data from the cohort and determine whether there is a correlation between disease scores and ophthalmic results. Among the 15 included SCA7 carriers (median [range] age, 38 [18-60] years; 8 women and 7 men), 12 displayed cone or cone-rod dystrophy, with the number of CAG repeats correlating with disease severity (ρ, 0.73, 95% CI, 0.34 to 0.90; P < .001). Two patients with cone-rod dystrophy exhibited higher repeat numbers and greater ataxia scores (median [range] SARA score, 9 [7-15]) compared to those with only cone dystrophy (median [range] SARA score, 2 [0-5]). A correlation emerged for outer nuclear layer thickness with SARA score (ρ, -0.88; 95% CI, -0.96 to -0.59; P < .001) and NfL levels (ρ, -0.87; 95% CI, -0.86 to 0.96; P < .001). Moreover, ataxia severity was correlated with visual acuity (ρ: 0.89; 95% CI, 0.68 to 0.96; P < .001) and retinal sensitivity (ρ, -0.88; 95% CI, -0.96 to 0.59; P < .001). In this cross-sectional study, retinal abnormalities were found at preataxic stages of the disease. Most of the carriers presented with cone dystrophy and preserved rod function. The outer nuclear layer thickness correlated with SARA score and plasma NfL levels suggesting nuclear layer thickness to be a biomarker of disease severity. These findings contribute to understanding the dynamics of SCA7-related retinal dystrophy and may help lay the groundwork for future therapeutic intervention monitoring and clinical trials. ClinicalTrials.gov Identifier: NCT04288128.

Virtual histological staining of unlabeled autopsy tissue

Traditional histochemical staining of post-mortem samples often confronts inferior staining quality due to autolysis caused by delayed fixation of cadaver tissue, and such chemical staining procedures covering large tissue areas demand substantial labor, cost and time. Here, we demonstrate virtual staining of autopsy tissue using a trained neural network to rapidly transform autofluorescence images of label-free autopsy tissue sections into brightfield equivalent images, matching hematoxylin and eosin (H&E) stained versions of the same samples. The trained model can effectively accentuate nuclear, cytoplasmic and extracellular features in new autopsy tissue samples that experienced severe autolysis, such as COVID-19 samples never seen before, where the traditional histochemical staining fails to provide consistent staining quality. This virtual autopsy staining technique provides a rapid and resource-efficient solution to generate artifact-free H&E stains despite severe autolysis and cell death, also reducing labor, cost and infrastructure requirements associated with the standard histochemical staining.

Autofluorescence imaging of exuviae as a tool for studying slide preparations of micro-arthropods, exemplified by a museum collection of the enigmatic crustacean “y-larvae” (Pancrustacea: Facetotecta)

In recent years, fluorescence microscopy has revitalized the study of invertebrate comparative morphology. Here we explore the usefulness of combining confocal laser scanning microscopy (CLSM) and cuticular autofluorescence to examine the taxonomically challenging marine planktonic “y-larvae” (Pancrustacea: Facetotecta). To gauge the effectiveness of CLSM with autofluorescence in producing taxonomically useful images, we applied it to seven distinct y-naupliar species or morphospecies that had previously undergone scrutiny by other techniques. The specimens were part of a museum collection of glycerin-jelly slides of exuviae of last-stage y-nauplii, a key instar for studying the taxonomy of y-larvae. For Hansenocaris demodex, the level of detail obtained from a single specimen was comparable to that previously obtained by scanning electron microscopy (SEM). For Hansenocaris aquila, revisiting the original holotype specimen resulted in a dramatic increase in our understanding of the species’ morphology, including taxonomically pivotal information about its spinose labrum and a digitally rotated lateral view. CLSM analyses of the other five specimens, which represented a broad spectrum of y-naupliar morphology, efficiently generated more such information. Novel observations were made concerning putative external rudiments of both the first and second maxillae in late nauplii as well as the extreme dorso-ventral flattening of some naupliar types. CLSM observation of museum slides of naupliar exuviae using cuticular autofluorescence thus shows great promise of becoming an excellent tool for studying the morphology and taxonomy of y-larvae, and we suggest that this technique might also profitably be applied to other forms of larval exuviae.

Genetics, Clinical Characteristics, and Natural History of PDE6B-Associated Retinal Dystrophy

To analyze the clinical characteristics, natural history, and genetics of PDE6B-associated retinal dystrophy. Retrospective, observational cohort study. Review of medical records and retinal imaging, including fundus autofluorescence (FAF) imaging and spectral-domain optical coherence tomography (SD-OCT) of patients with molecularly confirmed PDE6B-associated retinal dystrophy in a single tertiary referral center. Genetic results were reviewed, and the detected variants were assessed. Forty patients (80 eyes) were identified and evaluated longitudinally. The mean age (±SD, range) was 42.1 years (± 19.0, 10-86) at baseline, with a mean follow-up time of 5.2 years. Twenty-nine (72.5%) and 27 (67.5%) patients had no or mild visual acuity impairment at baseline and last visit, respectively. Best-corrected visual acuity (BCVA) was 0.56 ± 0.72 LogMAR (range -0.12 to 2.80) at baseline and 0.63 ± 0.73 LogMAR (range 0.0-2.80) at the last visit. BCVA was symmetrical in 87.5% of patients. A hyperautofluorescent ring was observed on FAF in 48 and 46 eyes at baseline and follow-up visit, respectively, with a mean area of 7.11 ± 4.13 mm2 at baseline and mean of 6.13 ± 3.62 mm2 at the follow-up visit. Mean horizontal ellipsoid zone width at baseline was 1946.1 ± 917.2 µm, which decreased to 1763.9 ± 827.9 µm at follow-up. Forty-four eyes had cystoid macular edema at baseline (55%), and 41 eyes (51.3%) at follow-up. There were statistically significant changes during the follow-up period in terms of BCVA and the ellipsoid zone width. Genetic analysis identified 43 variants in the PDE6B gene, including 16 novel variants. This study details the natural history of PDE6B-retinopathy in the largest cohort to date. Most patients had mild to no BCVA loss, with slowly progressive disease, based on FAF and OCT metrics. There is a high degree of disease symmetry and a wide window for intervention.

Deep learning-based correction of cataract-induced influence on macular pigment optical density measurement by autofluorescence spectroscopy.

Measurements of macular pigment optical density (MPOD) using the autofluorescence spectroscopy yield underestimations of actual values in eyes with cataracts. Previously, we proposed a correction method for this error using deep learning (DL); however, the correction performance was validated through internal cross-validation. This cross-sectional study aimed to validate this approach using an external validation dataset. MPODs at 0.25°, 0.5°, 1°, and 2° eccentricities and macular pigment optical volume (MPOV) within 9° eccentricity were measured using SPECTRALIS (Heidelberg Engineering, Heidelberg, Germany) in 197 (training dataset inherited from our previous study) and 157 eyes (validating dataset) before and after cataract surgery. A DL model was trained to predict the corrected value from the pre-operative value using the training dataset, and we measured the discrepancy between the corrected value and the actual postoperative value. Subsequently, the prediction performance was validated using a validation dataset. Using the validation dataset, the mean absolute values of errors for MPOD and MPOV corrected using DL ranged from 8.2 to 12.4%, which were lower than values with no correction (P < 0.001, linear mixed model with Tukey's test). The error depended on the autofluorescence image quality used to calculate MPOD. The mean errors in high and moderate quality images ranged from 6.0 to 11.4%, which were lower than those of poor quality images. The usefulness of the DL correction method was validated. Deep learning reduced the error for a relatively good autofluorescence image quality. Poor-quality images were not corrected.

Evaluation of Usage of Different Diagnostic Aids for Oral Cancer by Oral and Maxillofacial Surgeons: An Original Research

ABSTRACT Objective: The purpose of this study was to assess how oral and maxillofacial surgeons used various diagnostic tools for oral cancer. Materials and Methods: A cross-sectional methodology was used, and a standardized questionnaire was given to oral and maxillofacial surgeons randomly chosen sample. The questionnaire gathered information on demographics and the use of diagnostic tools. Data analysis methods included Chi-square testing and descriptive statistics. Results: The study included 200 oral and maxillofacial surgeons in total. The most often used diagnostic tool (95%) was visual inspection, followed by toluidine blue staining (48%) and brush biopsy (32%). Less frequently used were newer methods like optical coherence tomography (12.5%) and autofluorescence imaging (15%). No significant correlations between demographic factors and patterns of use of diagnostic tools were found by Chi-square tests. Conclusion: The results show that oral and maxillofacial surgeons frequently use brush biopsy, toluidine blue staining, and ocular evaluation. However, there is a need for more widespread adoption of cutting-edge technologies. By removing obstacles and offering training opportunities, one can increase the use of diagnostic tools, improving patient outcomes and the diagnosis of oral cancer.

Abstract B013: Development of oropharyngeal tumor organoids for disease modeling and high throughput drug screening

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with approximately 550,000 new cases diagnosed each year. The oropharynx is one of the most common sites of occurrence, and disease in this region most often presents in the palatine tonsil, followed by the base of tongue and soft palate. Human papilloma virus (HPV) is a common cause of HNSCC, giving rise to a distinct subset of tumors with a different clinical presentation, disease course, and response to treatment. In order to further characterize HPV+ HNSCC tumors and their therapy response, we have leveraged our high throughput 3D tumor organoid screening platform. Our approach entails seeding tumor cells in extracellular matrix gels deposited around the perimeter of tissue culture wells in ring-like structures, with screening results available within a week from surgery (Phan et al, 2019; Al Shihabi et al, 2022; Nguyen et al, 2022; Al Shihabi et al 2023). We particularly focused on tonsil tumors, and develop organoids from both HNSCC and contralateral normal tonsil tissue. Thus far, we have procured tonsil samples from 31 patients to develop organoids, perform high throughput drug screening, and immunohistochemical characterization including H&amp;E staining, Ki67, p16 and p53 immunohistochemical staining. We have leveraged dynamic optical contrast imaging (DOCI), a label-free tissue autofluorescence imaging method that is being investigated clinically (Kim et al, 2017), to assist in distinguishing tumor organoids from normal tissue organoids. Lastly, given the importance of radiation as a treatment modality for HPV+ HNSCC, we have also optimized a chemoradiation pipeline to test possible combinatorial effects. Taken together, our results demonstrate that 3D organoids of HNSCC generated from patient samples can successfully preserve native tissue characteristics and can be used for high throughput drug screening and radiation de-escalation testing, affording critical insight into the management of HNSCC in patients. Citation Format: Krishna K. Bommakanti, Luda Lin, Yazeed Alhiyari, Andreanne Sannajust, Brandon Mo, Lauran Evans, Peyton Tebon, Maie St. John, Alice Soragni. Development of oropharyngeal tumor organoids for disease modeling and high throughput drug screening [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B013.