Radiomic features define risk and are linked to DNA methylation attributes in primary CNS lymphoma.

Background: The prognostic roles of clinical and laboratory markers have been exploited to model risk in patients with primary CNS lymphoma, but these approaches do not fully explain the observed variation in outcome. To date, neuroimaging or molecular information is not used. The aim of this study was to determine the utility of radiomic features to capture clinically relevant phenotypes, and to link those to molecular profiles for enhanced risk stratification. Methods: In this retrospective study, we investigated 133 patients across 9 sites in Austria (2005-2018) and an external validation site in South Korea (44 patients, 2013-2016). We used T1-weighted contrast-enhanced MRI and an L1-norm regularized Cox proportional hazard model to derive a radiomic risk score. We integrated radiomic features with DNA methylation profiles using machine learning-based prediction, and validated the most relevant biological associations in tissues and cell lines. Results: The radiomic risk score, consisting of 20 mostly textural features, was a strong and independent predictor of survival (multivariate hazard ratio = 6.56 [3.64-11.81]) that remained valid in the external validation cohort. Radiomic features captured gene regulatory differences such as in BCL6 binding activity, which was put forth as testable treatment target for a subset of patients. Conclusions: The radiomic risk score was a robust and complementary predictor of survival and reflected characteristics in underlying DNA methylation patterns. Leveraging imaging phenotypes to assess risk and inform epigenetic treatment targets provides a concept on which to advance prognostic modeling and precision therapy for this aggressive cancer.

Flavin fluorescence lifetime and autofluorescence optical redox ratio for improved visualization and classification of brain tumors.

Purpose: Modern techniques for improved tumor visualization have the aim to maximize the extent of resection during brain tumor surgery and thus improve patient prognosis. Optical imaging of autofluorescence is a powerful and non-invasive tool to monitor metabolic changes and transformation in brain tumors. Cellular redox ratios can be retrieved from fluorescence emitted by the coenzymes reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). Recent studies point out that the influence of flavin mononucleotide (FMN) has been underestimated. Experimental design: Fluorescence lifetime imaging and fluorescence spectroscopy were performed through a modified surgical microscope. We acquired 361 flavin fluorescence lifetime (500-580 nm) and fluorescence spectra (430-740 nm) data points on freshly excised different brain tumors: low-grade gliomas (N=17), high-grade gliomas (N=42), meningiomas (N=23), metastases (N=26) and specimens from the non-tumorous brain (N=3). Results: Protein-bound FMN fluorescence in brain tumors did increase with a shift toward a more glycolytic metabolism (R=-0.87). This increased the average flavin fluorescence lifetime in tumor entities with respect to the non-tumorous brain. Further, these metrics were characteristic for the different tumor entities and showed promise for machine learning based brain tumor classification. Conclusions: Our results shed light on FMN fluorescence in metabolic imaging and outline the potential for supporting the neurosurgeon in visualizing and classifying brain tumor tissue during surgery.

Reconstruction of visible light optical coherence tomography images retrieved from discontinuous spectral data using a conditional generative adversarial network.

Achieving high resolution in optical coherence tomography typically requires the continuous extension of the spectral bandwidth of the light source. This work demonstrates an alternative approach: combining two discrete spectral windows located in the visible spectrum with a trained conditional generative adversarial network (cGAN) to reconstruct a high-resolution image equivalent to that generated using a continuous spectral band. The cGAN was trained using OCT image pairs acquired with the continuous and discontinuous visible range spectra to learn the relation between low- and high-resolution data. The reconstruction performance was tested using 6000 B-scans of a layered phantom, micro-beads and ex-vivo mouse ear tissue. The resultant cGAN-generated images demonstrate an image quality and axial resolution which approaches that of the high-resolution system.

Investigation of the scattering and attenuation properties of cataracts formed in mouse eyes with 1060-nm and 1310-nm swept-source optical coherence tomography.

Cataracts are the leading cause of blindness worldwide. Here we propose optical coherence tomography (OCT) as a quantitative method for investigating cataracts. OCT provides volumetric and non-invasive access to the lens and makes it possible to rapidly observe the formation of opacifications in animal models such as mice. We compared the performance of two different wavelengths - 1060 nm and 1310 nm - for OCT imaging in cataract research. In addition, we present multi-contrast OCT capable of mapping depth-resolved scattering and average anterior cortical attenuation properties of the crystalline lens and quantitatively characterize induced cataract development in the mouse eye. Lastly, we also propose a novel method based on the retinal OCT projection image for quantifying and mapping opacifications in the lens, which showed a good correlation with scattering and attenuation characteristics simultaneously analyzed during the process of cataract formation in the lens.

Fluorescence Lifetime Imaging and Spectroscopic Co-Validation for Protoporphyrin IX-Guided Tumor Visualization in Neurosurgery.

Maximal safe resection is a key strategy for improving patient prognosis in the management of brain tumors. Intraoperative fluorescence guidance has emerged as a standard in the surgery of high-grade gliomas. The administration of 5-aminolevulinic acid prior to surgery induces tumor-specific accumulation of protoporphyrin IX, which emits red fluorescence under blue-light illumination. The technology, however, is substantially limited for low-grade gliomas and weakly tumor-infiltrated brain, where low protoporphyrin IX concentrations are outweighed by tissue autofluorescence. In this context, fluorescence lifetime imaging has shown promise to distinguish spectrally overlapping fluorophores. We integrated frequency-domain fluorescence lifetime imaging in a surgical microscope and combined it with spatially registered fluorescence spectroscopy, which can be considered a research benchmark for sensitive protoporphyrin IX detection. Fluorescence lifetime maps and spectra were acquired for a representative set of fresh ex-vivo brain tumor specimens (low-grade gliomas n = 15, high-grade gliomas n = 80, meningiomas n = 41, and metastases n = 35). Combining the fluorescence lifetime with fluorescence spectra unveiled how weak protoporphyrin IX accumulations increased the lifetime respective to tissue autofluorescence. Infiltration zones (4.1ns ± 1.8ns, p = 0.017) and core tumor areas (4.8ns ± 1.3ns, p = 0.040) of low-grade gliomas were significantly distinguishable from non-pathologic tissue (1.6ns ± 0.5ns). Similarly, fluorescence lifetimes for infiltrated and reactive tissue as well as necrotic and core tumor areas were increased for high-grade gliomas and metastasis. Meningioma tumor specimens showed strongly increased lifetimes (12.2ns ± 2.5ns, p = 0.005). Our results emphasize the potential of fluorescence lifetime imaging to optimize maximal safe resection in brain tumors in future and highlight its potential toward clinical translation.

High-resolution, depth-resolved vascular leakage measurements using contrast-enhanced, correlation-gated optical coherence tomography in mice.

Vascular leakage plays a key role in vision-threatening retinal diseases such as diabetic retinopathy and age-related macular degeneration. Fluorescence angiography is the current gold standard for identification of leaky vasculature in vivo, however it lacks depth resolution, providing only 2D images that complicate precise identification and localization of pathological vessels. Optical coherence tomography (OCT) has been widely adopted for clinical ophthalmology due to its high, micron-scale resolution and rapid volumetric scanning capabilities. Nevertheless, OCT cannot currently identify leaky blood vessels. To address this need, we have developed a new method called exogenous contrast-enhanced leakage OCT (ExCEL-OCT) which identifies the diffusion of tracer particles around leaky vasculature following injection of a contrast agent. We apply this method to a mouse model of retinal neovascularization and demonstrate high-resolution 3D vascular leakage measurements in vivo for the first time.

Evaluation of the Temporal Muscle Thickness as an Independent Prognostic Biomarker in Patients with Primary Central Nervous System Lymphoma.

In this study, we assessed the prognostic relevance of temporal muscle thickness (TMT), likely reflecting patient's frailty, in patients with primary central nervous system lymphoma (PCNSL). In 128 newly diagnosed PCNSL patients TMT was analyzed on cranial magnetic resonance images. Predefined sex-specific TMT cutoff values were used to categorize the patient cohort. Survival analyses, using a log-rank test as well as Cox models adjusted for further prognostic parameters, were performed. The risk of death was significantly increased for PCNSL patients with reduced muscle thickness (hazard ratio of 3.189, 95% CI: 2-097-4.848, p < 0.001). Importantly, the results confirmed that TMT could be used as an independent prognostic marker upon multivariate Cox modeling (hazard ratio of 2.504, 95% CI: 1.608-3.911, p < 0.001) adjusting for sex, age at time of diagnosis, deep brain involvement of the PCNSL lesions, Eastern Cooperative Oncology Group (ECOG) performance status, and methotrexate-based chemotherapy. A TMT value below the sex-related cutoff value at the time of diagnosis is an independent adverse marker in patients with PCNSL. Thus, our results suggest the systematic inclusion of TMT in further translational and clinical studies designed to help validate its role as a prognostic biomarker.

Improved accuracy of quantitative birefringence imaging by polarization sensitive OCT with simple noise correction and its application to neuroimaging.

Polarization-sensitive optical coherence tomography (PS-OCT) enables three-dimensional imaging of biological tissues based on the inherent contrast provided by scattering and polarization properties. In fibrous tissue such as the white matter of the brain, PS-OCT allows quantitative mapping of tissue birefringence. For the popular PS-OCT layout using a single circular input state, birefringence measurements are based on a straight-forward evaluation of phase retardation data. However, the accuracy of these measurements strongly depends on the signal-to-noise ratio (SNR) and is prone to mapping artifacts when the SNR is low. Here we present a simple yet effective approach for improving the accuracy of PS-OCT phase retardation and birefringence measurements. By performing a noise bias correction of the detected OCT signal amplitudes, the impact of the noise floor on retardation measurements can be markedly reduced. We present simulation data to illustrate the influence of the noise bias correction on phase retardation measurements and support our analysis with real-world PS-OCT image data.

Macroscopic fluorescence-lifetime imaging of NADH and protoporphyrin IX improves the detection and grading of 5-aminolevulinic acid-stained brain tumors.

Maximal safe tumor resection remains the key prognostic factor for improved prognosis in brain tumor patients. Despite 5-aminolevulinic acid-based fluorescence guidance the neurosurgeon is, however, not able to visualize most low-grade gliomas (LGG) and infiltration zone of high-grade gliomas (HGG). To overcome the need for a more sensitive visualization, we investigated the potential of macroscopic, wide-field fluorescence lifetime imaging of nicotinamide adenine dinucleotide (NADH) and protoporphyrin IX (PPIX) in selected human brain tumors. For future intraoperative use, the imaging system offered a square field of view of 11 mm at 250 mm free working distance. We performed imaging of tumor tissue ex vivo, including LGG and HGG as well as brain metastases obtained from 21 patients undergoing fluorescence-guided surgery. Half of all samples showed visible fluorescence during surgery, which was associated with significant increase in PPIX fluorescence lifetime. While the PPIX lifetime was significantly different between specific tumor tissue types, the NADH lifetimes did not differ significantly among them. However, mainly necrotic areas exhibited significantly lower NADH lifetimes compared to compact tumor in HGG. Our pilot study indicates that combined fluorescence lifetime imaging of NADH/PPIX represents a sensitive tool to visualize brain tumor tissue not detectable with conventional 5-ALA fluorescence.

Ex-vivo Alzheimer's disease brain tissue investigation: a multiscale approach using 1060-nm swept source optical coherence tomography for a direct correlation to histology.

Significance: Amyloid-beta ( A - ß ) plaques are pathological protein deposits formed in the brain of Alzheimer's disease (AD) patients upon disease progression. Further research is needed to elucidate the complex underlying mechanisms involved in their formation using label-free, tissue preserving, and volumetric techniques. Aim: The aim is to achieve a one-to-one correlation of optical coherence tomography (OCT) data to histological micrographs of brain tissue using 1060-nm swept source OCT. Approach: A - ß plaques were investigated in ex-vivo AD brain tissue using OCT with the capability of switching between two magnifications. For the exact correlation to histology, a 3D-printed tool was designed to generate samples with parallel flat surfaces. Large field-of-view (FoV) and sequentially high-resolution volumes at different locations were acquired. The large FoV served to align the OCT to histology images; the high-resolution images were used to visualize fine details. Results: The instrument and the presented method enabled an accurate correlation of histological micrographs with OCT data. A - ß plaques were identified as hyperscattering features in both FoV OCT modalities. The plaques identified in volumetric OCT data were in good agreement with immunohistochemically derived micrographs. Conclusion: OCT combined with the 3D-printed tool is a promising approach for label-free, nondestructive, volumetric, and fast tissue analysis.

Improved Diagnostic Imaging of Brain Tumors by Multimodal Microscopy and Deep Learning.

Fluorescence-guided surgery is a state-of-the-art approach for intraoperative imaging during neurosurgical removal of tumor tissue. While the visualization of high-grade gliomas is reliable, lower grade glioma often lack visible fluorescence signals. Here, we present a hybrid prototype combining visible light optical coherence microscopy (OCM) and high-resolution fluorescence imaging for assessment of brain tumor samples acquired by 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. OCM provides high-resolution information of the inherent tissue scattering and absorption properties of tissue. We here explore quantitative attenuation coefficients derived from volumetric OCM intensity data and quantitative high-resolution 5-ALA fluorescence as potential biomarkers for tissue malignancy including otherwise difficult-to-assess low-grade glioma. We validate our findings against the gold standard histology and use attenuation and fluorescence intensity measures to differentiate between tumor core, infiltrative zone and adjacent brain tissue. Using large field-of-view scans acquired by a near-infrared swept-source optical coherence tomography setup, we provide initial assessments of tumor heterogeneity. Finally, we use cross-sectional OCM images to train a convolutional neural network that discriminates tumor from non-tumor tissue with an accuracy of 97%. Collectively, the present hybrid approach offers potential to translate into an in vivo imaging setup for substantially improved intraoperative guidance of brain tumor surgeries.

Sex-Specific Differences in Primary CNS Lymphoma.

Sex-specific differences have been increasingly recognized in many human diseases including brain cancer, namely glioblastoma. Primary CNS lymphoma (PCNSL) is an exceedingly rare type of brain cancer that tends to have a higher incidence and worse outcomes in male patients. Yet, relatively little is known about the reasons that contribute to these observed sex-specific differences. Using a population-representative cohort of patients with PCNSL with dense magnetic resonance (MR) imaging and digital pathology annotation (n = 74), we performed sex-specific cluster and survival analyses to explore possible associations. We found three prognostically relevant clusters for females and two for males, characterized by differences in (i) patient demographics, (ii) tumor-associated immune response, and (iii) MR imaging phenotypes. Upon a multivariable analysis, an enhanced FoxP3+ lymphocyte-driven immune response was associated with a shorter overall survival particularly in female patients (HR 1.65, p = 0.035), while an increased extent of contrast enhancement emerged as an adverse predictor of outcomes in male patients (HR 1.05, p < 0.01). In conclusion, we found divergent prognostic constellations between female and male patients with PCNSL that suggest differential roles of tumor-associated immune response and MR imaging phenotypes. Our results further underline the importance of continued sex-specific analyses in the field of brain cancer.

Three-dimensional visualization of opacifications in the murine crystalline lens by in vivo optical coherence tomography.

Diagnostic classification techniques used to diagnose cataracts, the world's leading cause of blindness, are currently based on subjective methods. Here, we present optical coherence tomography as a noninvasive tool for volumetric visualization of lesions formed in the crystalline lens. A custom-made swept-source optical coherence tomography (SS-OCT) system was utilized to investigate the murine crystalline lens. In addition to imaging cataractous lesions in aged wildtype mice, we studied the structure and shape of cataracts in a mouse model of Alzheimer's disease. Hyperscattering opacifications in the crystalline lens were observed in both groups. Post mortem histological analysis were performed to correlate findings in the anterior and posterior part of the lens to 3D OCT in vivo imaging. Our results showcase the capability of OCT to rapidly visualize cataractous lesions in the murine lens and suggest that OCT might be a valuable tool that provides additional insight for preclinical studies of cataract formation.

Towards real-time wide-field fluorescence lifetime imaging of 5-ALA labeled brain tumors with multi-tap CMOS cameras.

Fluorescence guided neurosurgery based on 5-aminolevulinic acid (5-ALA) has significantly increased maximal safe resections. Fluorescence lifetime imaging (FLIM) of 5-ALA could further boost this development by its increased sensitivity. However, neurosurgeons require real-time visual feedback which was so far limited in dual-tap CMOS camera based FLIM. By optimizing the number of phase frames required for reconstruction, we here demonstrate real-time 5-ALA FLIM of human high- and low-grade glioma with up to 12 Hz imaging rate over a wide field of view (11.0 x 11.0 mm). Compared to conventional fluorescence imaging, real-time FLIM offers enhanced contrast of weakly fluorescent tissue.

Surgical microscope with integrated fluorescence lifetime imaging for 5-aminolevulinic acid fluorescence-guided neurosurgery.

SIGNIFICANCE: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed. AIM: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging. APPROACH: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology. RESULTS: Our imaging system enabled macroscopic FLIM of a 6.5 × 6.5 mm2 field of view at spatial resolutions <20 µm. A frame of 512 × 512 pixels with a lifetime accuracy <1 ns was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology. CONCLUSIONS: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.

Retinal analysis of a mouse model of Alzheimer's disease with multicontrast optical coherence tomography.

Significance. Recent Alzheimer's disease (AD) patient studies have focused on retinal analysis, as the retina is the only part of the central nervous system that can be imaged noninvasively by optical methods. However, as this is a relatively new approach, the occurrence and role of retinal pathological features are still debated. Aim. The retina of an APP/PS1 mouse model was investigated using multicontrast optical coherence tomography (OCT) in order to provide a documentation of what was observed in both transgenic and wild-type mice. Approach. Both eyes of 24 APP/PS1 transgenic mice (age: 45 to 104 weeks) and 15 age-matched wild-type littermates were imaged by the custom-built OCT system. At the end of the experiment, retinas and brains were harvested from a subset of the mice (14 transgenic, 7 age-matched control) in order to compare the in vivo results to histological analysis and to quantify the cortical amyloid beta plaque load. Results. The system provided a combination of standard reflectivity data, polarization-sensitive data, and OCT angiograms. Qualitative and quantitative information from the resultant OCT images was extracted on retinal layer thickness and structure, presence of hyper-reflective foci, phase retardation abnormalities, and retinal vasculature. Conclusions. Although multicontrast OCT revealed abnormal structural properties and phase retardation signals in the retina of this APP/PS1 mouse model, the observations were very similar in transgenic and control mice.

Revealing brain pathologies with multimodal visible light optical coherence microscopy and fluorescence imaging.

We present a multimodal visible light optical coherence microscopy (OCM) and fluorescence imaging (FI) setup. Specification and phantom measurements were performed to characterize the system. Two applications in neuroimaging were investigated. First, curcumin-stained brain slices of a mouse model of Alzheimer's disease were examined. Amyloid-beta plaques were identified based on the fluorescence of curcumin, and coregistered morphological images of the brain tissue were provided by the OCM channel. Second, human brain tumor biopsies retrieved intraoperatively were imaged prior to conventional neuropathologic work-up. OCM revealed the three-dimensional structure of the brain parenchyma, and FI added the tumor tissue-specific contrast. Attenuation coefficients computed from the OCM data and the florescence intensity values were analyzed and showed a statistically significant difference for 5-aminolevulinic acid (5-ALA)-positive and -negative brain tissues. OCM findings correlated well with malignant hot spots within brain tumor biopsies upon histopathology. The combination of OCM and FI seems to be a promising optical imaging modality providing complementary contrast for applications in the field of neuroimaging.

Widefield fluorescence lifetime imaging of protoporphyrin IX for fluorescence-guided neurosurgery: An ex vivo feasibility study.

Achieving a maximal safe extent of resection during brain tumor surgery is the goal for improved patient prognosis. Fluorescence-guided neurosurgery using 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX has thereby become a valuable tool enabling a high frequency of complete resections and a prolonged progression-free survival in glioblastoma patients. We present a widefield fluorescence lifetime imaging device with 250 mm working distance, working under similar conditions such as surgical microscopes based on a time-of-flight dual tap CMOS camera. In contrast to intensity-based fluorescence imaging, our method is invariant to light scattering and absorption while being sensitive to the molecular composition of the tissue. We evaluate the feasibility of lifetime imaging of protoporphyrin IX using our system to analyze brain tumor phantoms and fresh 5-ALA-labeled human tissue samples. The results demonstrate the potential of our lifetime sensing device to go beyond the limitation of current intensity-based fluorescence-guided neurosurgery.

Visual and semiquantitative 11C-methionine PET: an independent prognostic factor for survival of newly diagnosed and treatment-naïve gliomas.

Background: Few data exist regarding the prognostic value of L-[S-methyl-11C]methionine (MET) PET for treatment-naïve gliomas. Methods: A total of 160 glioma patients (89 men, 71 women; mean age: 45, range 18-84 y) underwent a MET PET prior to any therapy. The PET scans were evaluated visually and semiquantitatively by tumor-to-background (T/N) ratio thresholds chosen by analysis of receiver operating characteristics. Additionally, isocitrate dehydrogenase 1-R132H (IDH1-R132H) immunohistochemistry was performed. Survival analysis was done using Kaplan-Meier estimates and the Cox proportional hazards model. Results: Significantly shorter mean survival times (7.2 vs 8.6 y; P = 0.024) were seen in patients with amino acid avid gliomas (n = 137) compared with visually negative tumors (n = 33) in MET PET. T/N ratio thresholds of 2.1 and 3.5 were significantly associated with survival (10.3 vs 7 vs 4.3 y; P < 0.001). Mean survival differed significantly using the median T/N ratio of 2.4 as cutoff, independent of histopathology (P < 0.01; mean survival: 10.2 ± 0.8 y vs 5.5 ± 0.6 y). In the subgroup of 142 glioma patients characterized by IDH1-R132H status, METT/N ratio demonstrated a significant prognostic impact in IDH1-R132H wildtype astrocytomas and glioblastoma (P = 0.001). Additionally, multivariate testing revealed semiquantitative MET PET as an independent prognostic parameter for treatment-naïve glioma patients without (P = 0.031) and with IDH1-R132H characterization of gliomas (P = 0.024; odds ratio 1.57). Conclusion: This retrospective analysis demonstrates the value of MET PET as a prognostic parameter on survival in treatment-naïve glioma patients.

Spectroscopic imaging with spectral domain visible light optical coherence microscopy in Alzheimer's disease brain samples.

A visible light spectral domain optical coherence microscopy system was developed. A high axial resolution of 0.88 µm in tissue was achieved using a broad visible light spectrum (425 - 685 nm). Healthy human brain tissue was imaged to quantify the difference between white (WM) and grey matter (GM) in intensity and attenuation. The high axial resolution enables the investigation of amyloid-beta plaques of various sizes in human brain tissue and animal models of Alzheimer's disease (AD). By performing a spectroscopic analysis of the OCM data, differences in the characteristics for WM, GM, and neuritic amyloid-beta plaques were found. To gain additional contrast, Congo red stained AD brain tissue was investigated. A first effort was made to investigate optically cleared mouse brain tissue to increase the penetration depth and visualize hyperscattering structures in deeper cortical regions.