Intraoperative in vivo confocal laser endomicroscopy imaging at glioma margins: can we detect tumor infiltration?

OBJECTIVE: Confocal laser endomicroscopy (CLE) is a US Food and Drug Administration-cleared intraoperative real-time fluorescence-based cellular resolution imaging technology that has been shown to image brain tumor histoarchitecture rapidly in vivo during neuro-oncological surgical procedures. An important goal for successful intraoperative implementation is in vivo use at the margins of infiltrating gliomas. However, CLE use at glioma margins has not been well studied. METHODS: Matching in vivo CLE images and tissue biopsies acquired at glioma margin regions of interest (ROIs) were collected from 2 institutions. All images were reviewed by 4 neuropathologists experienced in CLE. A scoring system based on the pathological features was implemented to score CLE and H&E images from each ROI on a scale from 0 to 5. Based on the H&E scores, all ROIs were divided into a low tumor probability (LTP) group (scores 0-2) and a high tumor probability (HTP) group (scores 3-5). The concordance between CLE and H&E scores regarding tumor probability was determined. The intraclass correlation coefficient (ICC) and diagnostic performance were calculated. RESULTS: Fifty-six glioma margin ROIs were included for analysis. Interrater reliability of the scoring system was excellent when used for H&E images (ICC [95% CI] 0.91 [0.86-0.94]) and moderate when used for CLE images (ICC [95% CI] 0.69 [0.40-0.83]). The ICCs (95% CIs) of the LTP group (0.68 [0.40-0.83]) and HTP group (0.68 [0.39-0.83]) did not differ significantly. The concordance between CLE and H&E scores was 61.6%. The sensitivity and specificity values of the scoring system were 79% and 37%. The positive predictive value (PPV) and negative predictive value were 65% and 53%, respectively. Concordance, sensitivity, and PPV were greater in the HTP group than in the LTP group. Specificity was higher in the newly diagnosed group than in the recurrent group. CONCLUSIONS: CLE may detect tumor infiltration at glioma margins. However, it is not currently dependable, especially in scenarios where low probability of tumor infiltration is expected. The proposed scoring system has excellent intrinsic interrater reliability, but its interrater reliability is only moderate when used with CLE images. These results suggest that this technology requires further exploration as a method for consistent actionable intraoperative guidance with high dependability across the range of tumor margin scenarios. Specific-binding and/or tumor-specific fluorophores, a CLE image atlas, and a consensus guideline for image interpretation may help with the translational utility of CLE.

Integrated molecular and multiparametric MRI mapping of high-grade glioma identifies regional biologic signatures.

Sampling restrictions have hindered the comprehensive study of invasive non-enhancing (NE) high-grade glioma (HGG) cell populations driving tumor progression. Here, we present an integrated multi-omic analysis of spatially matched molecular and multi-parametric magnetic resonance imaging (MRI) profiling across 313 multi-regional tumor biopsies, including 111 from the NE, across 68 HGG patients. Whole exome and RNA sequencing uncover unique genomic alterations to unresectable invasive NE tumor, including subclonal events, which inform genomic models predictive of geographic evolution. Infiltrative NE tumor is alternatively enriched with tumor cells exhibiting neuronal or glycolytic/plurimetabolic cellular states, two principal transcriptomic pathway-based glioma subtypes, which respectively demonstrate abundant private mutations or enrichment in immune cell signatures. These NE phenotypes are non-invasively identified through normalized K2 imaging signatures, which discern cell size heterogeneity on dynamic susceptibility contrast (DSC)-MRI. NE tumor populations predicted to display increased cellular proliferation by mean diffusivity (MD) MRI metrics are uniquely associated with EGFR amplification and CDKN2A homozygous deletion. The biophysical mapping of infiltrative HGG potentially enables the clinical recognition of tumor subpopulations with aggressive molecular signatures driving tumor progression, thereby informing precision medicine targeting.

Intraoperative confocal laser endomicroscopy for interpretation of a sellar hemangioblastoma: illustrative case.

BACKGROUND: Intraoperative frozen sections play a critical role in surgical strategy because of their ability to provide rapid histopathological information. In cases in which intraoperative biopsy carries a significant risk of bleeding, intraoperative confocal laser endomicroscopy (CLE) can assist in decision-making. OBSERVATIONS: The authors present a rare case of a large sellar hemangioblastoma. Preoperative radiographic imaging and normal pituitary function suggested a differential diagnosis that included hemangioblastoma. The patient underwent partial preoperative embolization and a right-sided pterional craniotomy for resection of the lesion. Gross intraoperative examination revealed a highly vascular sellar lesion requiring circumferential dissection to minimize blood loss. The serious vascularity precluded intraoperative frozen section analysis, and CLE imaging was performed. CLE imaging provided excellent visualization of the remarkable vascular structure and characteristic histoarchitecture with microvasculature, intracytoplasmic vacuoles, and atypical cells consistent with hemangioblastoma. Resection and decompression of the chiasm was accomplished, and the patient was discharged with improved vision. The final histopathological diagnosis was hemangioblastoma. LESSONS: When the benefits of obtaining intraoperative frozen sections greatly outweigh the associated risks, CLE imaging can aid in decision-making. CLE imaging offers real-time, on-the-fly evaluation of intraoperative tissue without the need to biopsy a vascular lesion.

Intraoperative confocal laser endomicroscopy: prospective in vivo feasibility study of a clinical-grade system for brain tumors.

OBJECTIVE: The authors evaluated the feasibility of using the first clinical-grade confocal laser endomicroscopy (CLE) system using fluorescein sodium for intraoperative in vivo imaging of brain tumors. METHODS: A CLE system cleared by the FDA was used in 30 prospectively enrolled patients with 31 brain tumors (13 gliomas, 5 meningiomas, 6 other primary tumors, 3 metastases, and 4 reactive brain tissue). A neuropathologist classified CLE images as interpretable or noninterpretable. Images were compared with corresponding frozen and permanent histology sections, with image correlation to biopsy location using neuronavigation. The specificities and sensitivities of CLE images and frozen sections were calculated using permanent histological sections as the standard for comparison. A recently developed surgical telepathology software platform was used in 11 cases to provide real-time intraoperative consultation with a neuropathologist. RESULTS: Overall, 10,713 CLE images from 335 regions of interest were acquired. The mean duration of the use of the CLE system was 7 minutes (range 3-18 minutes). Interpretable CLE images were obtained in all cases. The first interpretable image was acquired within a mean of 6 (SD 10) images and within the first 5 (SD 13) seconds of imaging; 4896 images (46%) were interpretable. Interpretable image acquisition was positively correlated with study progression, number of cases per surgeon, cumulative length of CLE time, and CLE time per case (p ≤ 0.01). The diagnostic accuracy, sensitivity, and specificity of CLE compared with frozen sections were 94%, 94%, and 100%, respectively, and the diagnostic accuracy, sensitivity, and specificity of CLE compared with permanent histological sections were 92%, 90%, and 94%, respectively. No difference was observed between lesion types for the time to first interpretable image (p = 0.35). Deeply located lesions were associated with a higher percentage of interpretable images than superficial lesions (p = 0.02). The study met the primary end points, confirming the safety and feasibility and acquisition of noninvasive digital biopsies in all cases. The study met the secondary end points for the duration of CLE use necessary to obtain interpretable images. A neuropathologist could interpret the CLE images in 29 (97%) of 30 cases. CONCLUSIONS: The clinical-grade CLE system allows in vivo, intraoperative, high-resolution cellular visualization of tissue microstructure and identification of lesional tissue patterns in real time, without the need for tissue preparation.

Characterization of ex vivo and in vivo intraoperative neurosurgical confocal laser endomicroscopy imaging.

Background: The new US Food and Drug Administration-cleared fluorescein sodium (FNa)-based confocal laser endomicroscopy (CLE) imaging system allows for intraoperative on-the-fly cellular level imaging. Two feasibility studies have been completed with intraoperative use of this CLE system in ex vivo and in vivo modalities. This study quantitatively compares the image quality and diagnostic performance of ex vivo and in vivo CLE imaging. Methods: Images acquired from two prospective CLE clinical studies, one ex vivo and one in vivo, were analyzed quantitatively. Two image quality parameters - brightness and contrast - were measured using Fiji software and compared between ex vivo and in vivo images for imaging timing from FNa dose and in glioma, meningioma, and intracranial metastatic tumor cases. The diagnostic performance of the two studies was compared. Results: Overall, the in vivo images have higher brightness and contrast than the ex vivo images (p < 0.001). A weak negative correlation exists between image quality and timing of imaging after FNa dose for the ex vivo images, but not the in vivo images. In vivo images have higher image quality than ex vivo images (p < 0.001) in glioma, meningioma, and intracranial metastatic tumor cases. In vivo imaging yielded higher sensitivity and negative predictive value than ex vivo imaging. Conclusions: In our setting, in vivo CLE optical biopsy outperforms ex vivo CLE by producing higher quality images and less image deterioration, leading to better diagnostic performance. These results support the in vivo modality as the modality of choice for intraoperative CLE imaging.

Real-time intraoperative surgical telepathology using confocal laser endomicroscopy.

OBJECTIVE: Communication between neurosurgeons and pathologists is mandatory for intraoperative decision-making and optimization of resection, especially for invasive masses. Handheld confocal laser endomicroscopy (CLE) technology provides in vivo intraoperative visualization of tissue histoarchitecture at cellular resolution. The authors evaluated the feasibility of using an innovative surgical telepathology software platform (TSP) to establish real-time, on-the-fly remote communication between the neurosurgeon using CLE and the pathologist. METHODS: CLE and a TSP were integrated into the surgical workflow for 11 patients with brain masses (6 patients with gliomas, 3 with other primary tumors, 1 with metastasis, and 1 with reactive brain tissue). Neurosurgeons used CLE to generate video-flow images of the operative field that were displayed on monitors in the operating room. The pathologist simultaneously viewed video-flow CLE imaging using a digital tablet and communicated with the surgeon while physically located outside the operating room (1 pathologist was in another state, 4 were at home, and 6 were elsewhere in the hospital). Interpretations of the still CLE images and video-flow CLE imaging were compared with the findings on the corresponding frozen and permanent H&E histology sections. RESULTS: Overall, 24 optical biopsies were acquired with mean ± SD 2 ± 1 optical biopsies per case. The mean duration of CLE system use was 1 ± 0.3 minutes/case and 0.25 ± 0.23 seconds/optical biopsy. The first image with identifiable histopathological features was acquired within 6 ± 0.1 seconds. Frozen sections were processed within 23 ± 2.8 minutes, which was significantly longer than CLE usage (p < 0.001). Video-flow CLE was used to correctly interpret tissue histoarchitecture in 96% of optical biopsies, which was substantially higher than the accuracy of using still CLE images (63%) (p = 0.005). CONCLUSIONS: When CLE is employed in tandem with a TSP, neurosurgeons and pathologists can view and interpret CLE images remotely and in real time without the need to biopsy tissue. A TSP allowed neurosurgeons to receive real-time feedback on the optically interrogated tissue microstructure, thereby improving cross-functional communication and intraoperative decision-making and resulting in significant workflow advantages over the use of frozen section analysis.

Introduction of In Vivo Confocal Laser Endomicroscopy and Real-Time Telepathology for Remote Intraoperative Neurosurgery-Pathology Consultation.

BACKGROUND: Precise communication between neurosurgeons and pathologists is crucial for optimizing patient care, especially for intraoperative diagnoses. Confocal laser endomicroscopy (CLE) combined with a telepathology software platform (TSP) provides a novel venue for neurosurgeons and pathologists to review CLE images and converse intraoperatively in real-time. OBJECTIVE: To describe the feasibility of integrating CLE and a TSP in the surgical workflow for real-time review of in vivo digital fluorescence tissue imaging in 3 patients with intracranial tumors. METHODS: Although the neurosurgeon used the CLE probe to generate fluorescence images of histoarchitecture within the operative field that were displayed on monitors in the operating room, the pathologist simultaneously remotely viewed the CLE images. The neurosurgeon and pathologist discussed in real-time the histological structures of intraoperative imaging locations. RESULTS: The neurosurgeon placed the CLE probe at various locations on and around the tumor, in the surgical resection bed, and on surrounding brain tissue with communication through the TSP. The neurosurgeon oriented the pathologist to the location of the CLE, and the pathologist and neurosurgeon discussed the CLE images in real-time. The TSP and CLE were integrated successfully and rapidly in the operating room in all 3 cases. No patient had perioperative complications. CONCLUSION: Two novel digital neurosurgical cellular imaging technologies were combined with intraoperative neurosurgeon-pathologist communication to guide the identification of abnormal histoarchitectural tissue features in real-time. CLE with the TSP may allow rapid decision-making during tumor resection that may hold significant advantages over the frozen section process and surgical workflow in general.

Intracranial mesenchymal tumors with FET-CREB fusion are composed of at least two epigenetic subgroups distinct from meningioma and extracranial sarcomas.

'Intracranial mesenchymal tumor, FET-CREB fusion-positive' occurs primarily in children and young adults and has previously been termed intracranial angiomatoid fibrous histiocytoma (AFH) or intracranial myxoid mesenchymal tumor (IMMT). Here we performed genome-wide DNA methylation array profiling of 20 primary intracranial mesenchymal tumors with FET-CREB fusion to further study their ontology. These tumors resolved into two distinct epigenetic subgroups that were both divergent from all other analyzed intracranial neoplasms and soft tissue sarcomas, including meningioma, clear cell sarcoma of soft tissue (CCS), and AFH of extracranial soft tissue. The first subgroup (Group A, 16 tumors) clustered nearest to but independent of solitary fibrous tumor and AFH of extracranial soft tissue, whereas the second epigenetic subgroup (Group B, 4 tumors) clustered nearest to but independent of CCS and also lacked expression of melanocytic markers (HMB45, Melan A, or MITF) characteristic of CCS. Group A tumors most often occurred in adolescence or early adulthood, arose throughout the neuroaxis, and contained mostly EWSR1-ATF1 and EWSR1-CREB1 fusions. Group B tumors arose most often in early childhood, were located along the cerebral convexities or spinal cord, and demonstrated an enrichment for tumors with CREM as the fusion partner (either EWSR1-CREM or FUS-CREM). Group A tumors more often demonstrated stellate/spindle cell morphology and hemangioma-like vasculature, whereas Group B tumors more often demonstrated round cell or epithelioid/rhabdoid morphology without hemangioma-like vasculature, although robust comparison of these clinical and histologic features requires future study. Patients with Group B tumors had inferior progression-free survival relative to Group A tumors (median 4.5 vs. 49 months, p = 0.001). Together, these findings confirm that intracranial AFH-like neoplasms and IMMT represent histologic variants of a single tumor type ('intracranial mesenchymal tumor, FET-CREB fusion-positive') that is distinct from meningioma and extracranial sarcomas. Additionally, epigenomic evaluation may provide important prognostic subtyping for this unique tumor entity.

High-Dose Fluorescein Reveals Unusual Confocal Endomicroscope Imaging of Low-Grade Glioma.

Background: Fluorescence-guided brain tumor surgery using fluorescein sodium (FNa) for contrast is effective in high-grade gliomas. However, the effectiveness of this technique for visualizing noncontrast-enhancing and low-grade gliomas is unknown. This report is the first documented case of the concurrent use of wide-field fluorescence-guided surgery and confocal laser endomicroscopy (CLE) with high-dose FNa (40 mg/kg) for intraoperative visualization of tumor tissue cellularity in a nonenhancing glioma. Case Description: A patient underwent fluorescence-guided surgery for a left frontal lobe mass without contrast enhancement on magnetic resonance imaging. The patient received 40 mg/kg FNa intravenously at the induction of anesthesia. Surgery was performed under visualization with a Yellow 560 filter and white-light wide-field imaging. Intraoperative CLE produced high-quality images of the lesion 1.5 h after FNa injection. Frozen-section analysis demonstrated findings comparable to those of intraoperative CLE visualization and consistent with World Health Organization (WHO) glioma grades II-III. The patient recovered without complications. Analysis of the permanent histologic sections identified the tumor as an anaplastic oligodendroglioma, IDH-mutant, 1p/19q co-deleted, consistent with WHO grade III because of discrete foci of hypercellularity and increased mitotic figures, but large regions of the lesion were low grade. Conclusions: The use of high-dose FNa in this patient with a nonenhancing borderline low-grade/high-grade glioma produced actionable wide-field fluorescence imaging using the operating microscope and improved CLE visualization of tumor cellularity. Higher doses of FNa for intraoperative CLE imaging and possible simultaneous wide-field fluorescence surgical guidance in nonenhancing gliomas merit further investigation.

Molecular Imaging of Glucose Metabolism for Intraoperative Fluorescence Guidance During Glioma Surgery.

PURPOSE: This study evaluated the use of molecular imaging of fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as a discriminatory marker for intraoperative tumor border identification in a murine glioma model. PROCEDURES: 2-NBDG was assessed in GL261 and U251 orthotopic tumor-bearing mice. Intraoperative fluorescence of topical and intravenous 2-NBDG in normal and tumor regions was assessed with an operating microscope, handheld confocal laser scanning endomicroscope (CLE), and benchtop confocal laser scanning microscope (LSM). Additionally, 2-NBDG fluorescence in tumors was compared with 5-aminolevulinic acid-induced protoporphyrin IX fluorescence. RESULTS: Intravenously administered 2-NBDG was detectable in brain tumor and absent in contralateral normal brain parenchyma on wide-field operating microscope imaging. Intraoperative and benchtop CLE showed preferential 2-NBDG accumulation in the cytoplasm of glioma cells (mean [SD] tumor-to-background ratio of 2.76 [0.43]). Topically administered 2-NBDG did not create sufficient tumor-background contrast for wide-field operating microscope imaging or under benchtop LSM (mean [SD] tumor-to-background ratio 1.42 [0.72]). However, topical 2-NBDG did create sufficient contrast to evaluate cellular tissue architecture and differentiate tumor cells from normal brain parenchyma. Protoporphyrin IX imaging resulted in a more specific delineation of gross tumor margins than intravenous or topical 2-NBDG and a significantly higher tumor-to-normal-brain fluorescence intensity ratio. CONCLUSION: After intravenous administration, 2-NBDG selectively accumulated in the experimental brain tumors and provided bright contrast under wide-field fluorescence imaging with a clinical-grade operating microscope. Topical 2-NBDG was able to create a sufficient contrast to differentiate tumor from normal brain cells on the basis of visualization of cellular architecture with CLE. 5-Aminolevulinic acid demonstrated superior specificity in outlining tumor margins and significantly higher tumor background contrast. Given the nontoxicity of 2-NBDG, its use as a topical molecular marker for noninvasive in vivo intraoperative microscopy is encouraging and warrants further clinical evaluation.

Uncertainty quantification in the radiogenomics modeling of EGFR amplification in glioblastoma.

Radiogenomics uses machine-learning (ML) to directly connect the morphologic and physiological appearance of tumors on clinical imaging with underlying genomic features. Despite extensive growth in the area of radiogenomics across many cancers, and its potential role in advancing clinical decision making, no published studies have directly addressed uncertainty in these model predictions. We developed a radiogenomics ML model to quantify uncertainty using transductive Gaussian Processes (GP) and a unique dataset of 95 image-localized biopsies with spatially matched MRI from 25 untreated Glioblastoma (GBM) patients. The model generated predictions for regional EGFR amplification status (a common and important target in GBM) to resolve the intratumoral genetic heterogeneity across each individual tumor-a key factor for future personalized therapeutic paradigms. The model used probability distributions for each sample prediction to quantify uncertainty, and used transductive learning to reduce the overall uncertainty. We compared predictive accuracy and uncertainty of the transductive learning GP model against a standard GP model using leave-one-patient-out cross validation. Additionally, we used a separate dataset containing 24 image-localized biopsies from 7 high-grade glioma patients to validate the model. Predictive uncertainty informed the likelihood of achieving an accurate sample prediction. When stratifying predictions based on uncertainty, we observed substantially higher performance in the group cohort (75% accuracy, n = 95) and amongst sample predictions with the lowest uncertainty (83% accuracy, n = 72) compared to predictions with higher uncertainty (48% accuracy, n = 23), due largely to data interpolation (rather than extrapolation). On the separate validation set, our model achieved 78% accuracy amongst the sample predictions with lowest uncertainty. We present a novel approach to quantify radiogenomics uncertainty to enhance model performance and clinical interpretability. This should help integrate more reliable radiogenomics models for improved medical decision-making.

Intracranial mesenchymal tumor with FET-CREB fusion-A unifying diagnosis for the spectrum of intracranial myxoid mesenchymal tumors and angiomatoid fibrous histiocytoma-like neoplasms.

Intracranial mesenchymal tumors with FET-CREB fusions are a recently described group of neoplasms in children and young adults characterized by fusion of a FET family gene (usually EWSR1, but rarely FUS) to a CREB family transcription factor (ATF1, CREB1, or CREM), and have been variously termed intracranial angiomatoid fibrous histiocytoma or intracranial myxoid mesenchymal tumor. The clinical outcomes, histologic features, and genomic landscape are not well defined. Here, we studied 20 patients with intracranial mesenchymal tumors proven to harbor FET-CREB fusion by next-generation sequencing (NGS). The 16 female and four male patients had a median age of 14 years (range 4-70). Tumors were uniformly extra-axial or intraventricular and located at the cerebral convexities (n = 7), falx (2), lateral ventricles (4), tentorium (2), cerebellopontine angle (4), and spinal cord (1). NGS demonstrated that eight tumors harbored EWSR1-ATF1 fusion, seven had EWSR1-CREB1, four had EWSR1-CREM, and one had FUS-CREM. Tumors were uniformly well circumscribed and typically contrast enhancing with solid and cystic growth. Tumors with EWSR1-CREB1 fusions more often featured stellate/spindle cell morphology, mucin-rich stroma, and hemangioma-like vasculature compared to tumors with EWSR1-ATF1 fusions that most often featured sheets of epithelioid cells with mucin-poor collagenous stroma. These tumors demonstrated polyphenotypic immunoprofiles with frequent positivity for desmin, EMA, CD99, MUC4, and synaptophysin, but absence of SSTR2A, myogenin, and HMB45 expression. There was a propensity for local recurrence with a median progression-free survival of 12 months and a median overall survival of greater than 60 months, with three patients succumbing to disease (all with EWSR1-ATF1 fusions). In combination with prior case series, this study provides further insight into intracranial mesenchymal tumors with FET-CREB fusion, which represent a distinct group of CNS tumors encompassing both intracranial myxoid mesenchymal tumor and angiomatoid fibrous histiocytoma-like neoplasms.

Intraoperative Confocal Laser Endomicroscopy Ex Vivo Examination of Tissue Microstructure During Fluorescence-Guided Brain Tumor Surgery.

BACKGROUND: Noninvasive intraoperative optical biopsy that provides real-time imaging of histoarchitectural (cell resolution) features of brain tumors, especially at the margin of invasive tumors, would be of great value. To assess clinical-grade confocal laser endomicroscopy (CLE) and to prepare for its use intraoperatively in vivo, we performed an assessment of CLE ex vivo imaging in brain lesions. METHODS: Tissue samples from patients who underwent intracranial surgeries with fluorescein sodium (FNa)-based wide-field fluorescence guidance were acquired for immediate intraoperative ex vivo optical biopsies with CLE. Hematoxylin-eosin-stained frozen section analysis of the same specimens served as the gold standard for blinded neuropathology comparison. FNa 2 to 5 mg/kg was administered upon induction of anesthesia, and FNa 5 mg/kg was injected for CLE contrast improvement. Histologic features were identified, and the diagnostic accuracy of CLE was assessed. RESULTS: Of 77 eligible patients, 47 patients with 122 biopsies were enrolled, including 32 patients with gliomas and 15 patients with other intracranial lesions. The positive predictive value of CLE optical biopsies was 97% for all specimens and 98% for gliomas. The specificity of CLE was 90% for all specimens and 94% for gliomas. The second FNa injection in seven patients, a mean of 2.6 h after the first injection, improved image quality and increased the percentage of accurately diagnosed images from 67% to 93%. Diagnostic CLE features of lesional glioma biopsies and normal brain were identified. Seventeen histologic features were identified. CONCLUSIONS: Results demonstrated high specificity and positive predictive value of ex vivo intraoperative CLE optical biopsies and justify an in vivo intraoperative trial. This new portable, noninvasive intraoperative imaging technique provides diagnostic features to discriminate lesional tissue with high specificity and is feasible for incorporation into the fluorescence-guided surgery workflow, particularly for patients with invasive brain tumors.

Single-Cell RNA Sequencing of a Postmenopausal Normal Breast Tissue Identifies Multiple Cell Types That Contribute to Breast Cancer.

The human breast is composed of diverse cell types. Studies have delineated mammary epithelial cells, but the other cell types in the breast have scarcely been characterized. In order to gain insight into the cellular composition of the tissue, we performed droplet-mediated RNA sequencing of 3193 single cells isolated from a postmenopausal breast tissue without enriching for epithelial cells. Unbiased clustering analysis identified 10 distinct cell clusters, seven of which were nonepithelial devoid of cytokeratin expression. The remaining three cell clusters expressed cytokeratins (CKs), representing breast epithelial cells; Cluster 2 and Cluster 7 cells expressed luminal and basal CKs, respectively, whereas Cluster 9 cells expressed both luminal and basal CKs, as well as other CKs of unknown specificity. To assess which cell type(s) potentially contributes to breast cancer, we used the differential gene expression signature of each cell cluster to derive gene set variation analysis (GSVA) scores and classified breast tumors in The Cancer Gene Atlas (TGGA) dataset (n = 1100) by assigning the highest GSVA scoring cell cluster number for each tumor. The results showed that five clusters (Clusters 2, 3, 7, 8, and 9) could categorize >85% of breast tumors collectively. Notably, Cluster 2 (luminal epithelial) and Cluster 3 (fibroblast) tumors were equally prevalent in the luminal breast cancer subtypes, whereas Cluster 7 (basal epithelial) and Cluster 9 (other epithelial) tumors were present primarily in the triple-negative breast cancer (TNBC) subtype. Cluster 8 (immune) tumors were present in all subtypes, indicating that immune cells may contribute to breast cancer regardless of the subtypes. Cluster 9 tumors were significantly associated with poor patient survival in TNBC, suggesting that this epithelial cell type may give rise to an aggressive TNBC subset.

Identifying the spatial and temporal dynamics of molecularly-distinct glioblastoma sub-populations.

Glioblastomas (GBMs) are the most aggressive primary brain tumours and have no known cure. Each individual tumour comprises multiple sub-populations of genetically-distinct cells that may respond differently to targeted therapies and may contribute to disappointing clinical trial results. Image-localized biopsy techniques allow multiple biopsies to be taken during surgery and provide information that identifies regions where particular sub-populations occur within an individual GBM, thus providing insight into their regional genetic variability. These sub-populations may also interact with one another in a competitive or cooperative manner; it is important to ascertain the nature of these interactions, as they may have implications for responses to targeted therapies. We combine genetic information from biopsies with a mechanistic model of interacting GBM sub-populations to characterise the nature of interactions between two commonly occurring GBM sub-populations, those with EGFR and PDGFRA genes amplified. We study population levels found across image-localized biopsy data from a cohort of 25 patients and compare this to model outputs under competitive, cooperative and neutral interaction assumptions. We explore other factors affecting the observed simulated sub-populations, such as selection advantages and phylogenetic ordering of mutations, which may also contribute to the levels of EGFR and PDGFRA amplified populations observed in biopsy data.

Confocal Laser Endomicroscopy Assessment of Pituitary Tumor Microstructure: A Feasibility Study.

This is the first study to assess confocal laser endomicroscopy (CLE) use within the transsphenoidal approach and show the feasibility of obtaining digital diagnostic biopsies of pituitary tumor tissue after intravenous fluorescein injection. We confirmed that the CLE probe reaches the tuberculum sellae through the transnasal transsphenoidal corridor in cadaveric heads. Next, we confirmed that CLE provides images with identifiable histological features of pituitary adenoma. Biopsies from nine patients who underwent pituitary adenoma surgery were imaged ex vivo at various times after fluorescein injection and were assessed by a blinded board-certified neuropathologist. With frozen sections used as the standard, pituitary adenoma was diagnosed as "definitively" for 13 and as "favoring" in 3 of 16 specimens. CLE digital biopsies were diagnostic for pituitary adenoma in 10 of 16 specimens. The reasons for nondiagnostic CLE images were biopsy acquisition <1 min or >10 min after fluorescein injection (n = 5) and blood artifacts (n = 1). In conclusion, fluorescein provided sufficient contrast for CLE at a dose of 2 mg/kg, optimally 1-10 min after injection. These results provide a basis for further in vivo studies using CLE in transsphenoidal surgery.

Using the Post-Descendens Hypoglossal Nerve in Hypoglossal-Facial Anastomosis: An Anatomic and Histologic Feasibility Study.

BACKGROUND: Hypoglossal-facial anastomosis (HFA) is a popular facial reanimation technique. Mobilizing the intratemporal segment of the facial nerve and using the post-descendens hypoglossal nerve (ie, the segment distal to the take-off of descendens hypoglossi) have been proposed to improve results. However, no anatomic study has verified the feasibility of this technique. OBJECTIVE: To assess the anatomic feasibility of HFA and the structural compatibility between the 2 nerves when the intratemporal facial and post-descendens hypoglossal nerves are used. METHODS: The facial and hypoglossal nerves were exposed bilaterally in 10 sides of 5 cadaveric heads. The feasibility of a side-to-end (ie, partial end-to-end) HFA with partial sectioning of the post-descendens hypoglossal nerve and the mobilized intratemporal facial nerve was assessed. The axonal count and cross-sectional area of the facial and hypoglossal nerves at the point of anastomosis were assessed. RESULTS: The HFA was feasible in all specimens with a mean (standard deviation) 9.3 (5.5) mm of extra length on the facial nerve. The axonal counts and cross-sectional areas of the hypoglossal and facial nerves matched well. Considering the reduction in the facial nerve cross-sectional area after paralysis, the post-descendens hypoglossal nerve can provide adequate axonal count and area to accommodate the facial nerve stump. CONCLUSION: Using the post-descendens hypoglossal nerve for side-to-end anastomosis with the mobilized intratemporal facial nerve is anatomically feasible and provides adequate axonal count for facial reanimation. When compared with use of the pre-descendens hypoglossal nerve, this technique preserves C1 fibers and has a potential to reduce glottic complications.

Temporal Horn Choroid Plexus Papilloma Presenting with Seizures in Adulthood: Clinical Case Report and Review of the Literature.

BACKGROUND: Choroid plexus papillomas (CPPs) are benign World Health Organization grade I tumors that comprise 2%-4% of all brain tumors among children and less than 1% of brain tumors in adults. Most adult cases occur in the fourth ventricle, with only 1 previous report describing an adult patient with a temporal horn CPP. CASE DESCRIPTION: We report a rare case of a temporal horn CPP presenting in an adult with seizures. We performed a minimally invasive subtemporal approach for gross total resection of the lesion. CONCLUSIONS: CPP presenting in the temporal horn is rare among adults. We discuss the surgical nuances of the subtemporal approach for resection and review the literature regarding adult presentation of CPP and the treatment strategies for adult CPP.

Clinical, pathologic, and imaging characteristics of pituitary null cell adenomas as defined according to the 2017 World Health Organization criteria: a case series from two pituitary centers.

PURPOSE: The 2017 World Health Organization classification of pituitary tumors redefined pituitary null cell adenomas (NCAs) by restricting this diagnostic category to pituitary tumors that are negative for pituitary transcription factors and adenohypophyseal hormones. The clinical behavior of this redefined entity has not been widely studied, and this is a major shortcoming of the classification. This study evaluated the imaging and clinical features of NCAs from two pituitary centers and compared them with those of gonadotroph adenomas (GAs). METHODS: Imaging, pathologic, and clinical characteristics of NCAs and GAs were retrospectively reviewed. Tumor immunohistochemistry was performed to confirm absence of adenohypophyseal hormones and pituitary transcription factor expression. RESULTS: Thirty-one NCAs were compared with 38 GAs. NCAs were more likely to invade the cavernous sinus (15/31 [48%] vs. 5/38 [13%], P = .003) and had a higher proliferative index (i.e., MIB-1 > 3%, 11/31 [35%] vs. 5/38 [13%], P = .04). Gross total resection was less likely in the NCA group (19/31 [61%] vs. 33/38 [87], P = .02). Progression-free survival was worse in the NCA cohort (5-year progression-free survival, 0.70 vs. 1.00; P = .011, by log-rank test). CONCLUSIONS: Compared with GAs, NCAs are more invasive at the time of presentation and have a more aggressive clinical course. This study provides evidence that NCAs represent a distinct clinicopathologic entity with behavior that differs adversely from that of GAs. This may inform clinical decision-making, including frequency of postoperative tumor surveillance and timing of adjunctive treatments.

Progress in Confocal Laser Endomicroscopy for Neurosurgery and Technical Nuances for Brain Tumor Imaging With Fluorescein.

Background: Previous studies showed that confocal laser endomicroscopy (CLE) images of brain tumors acquired by a first-generation (Gen1) CLE system using fluorescein sodium (FNa) contrast yielded a diagnostic accuracy similar to frozen surgical sections and histologic analysis. We investigated performance improvements of a second-generation (Gen2) CLE system designed specifically for neurosurgical use. Methods: Rodent glioma models were used for in vivo and rapid ex vivo CLE imaging. FNa and 5-aminolevulinic acid were used as contrast agents. Gen1 and Gen2 CLE images were compared to distinguish cytoarchitectural features of tumor mass and margin and surrounding and normal brain regions. We assessed imaging parameters (gain, laser power, brightness, scanning speed, imaging depth, and Z-stack [3D image acquisition]) and evaluated optimal values for better neurosurgical imaging performance with Gen2. Results: Efficacy of Gen1 and Gen2 was similar in identifying normal brain tissue, vasculature, and tumor cells in masses or at margins. Gen2 had smaller field of view, but higher image resolution, and sharper, clearer images. Other advantages of the Gen2 were auto-brightness correction, user interface, image metadata handling, and image transfer. CLE imaging with FNa allowed identification of nuclear and cytoplasmic contours in tumor cells. Injection of higher dosages of FNa (20 and 40 mg/kg vs. 0.1-8 mg/kg) resulted in better image clarity and structural identification. When used with 5-aminolevulinic acid, CLE was not able to detect individual glioma cells labeled with protoporphyrin IX, but overall fluorescence intensity was higher (p < 0.01) than in the normal hemisphere. Gen2 Z-stack imaging allowed a unique 3D image volume presentation through the focal depth. Conclusion: Compared with Gen1, advantages of Gen2 CLE included a more responsive and intuitive user interface, collection of metadata with each image, automatic Z-stack imaging, sharper images, and a sterile sheath. Shortcomings of Gen2 were a slightly slower maximal imaging speed and smaller field of view. Optimal Gen2 imaging parameters to visualize brain tumor cytoarchitecture with FNa as a fluorescent contrast were defined to aid further neurosurgical clinical in vivo and rapid ex vivo use. Further validation of the Gen2 CLE for microscopic visualization and diagnosis of brain tumors is ongoing.