Desorption electrospray ionization-mass spectrometry (DESI-MS) imaging was used to analyze unmodified human brain tissue sections from 39 subjects sequentially in the positive and negative ionization ...modes. Acquisition of both MS polarities allowed more complete analysis of the human brain tumor lipidome as some phospholipids ionize preferentially in the positive and others in the negative ion mode. Normal brain parenchyma, comprised of grey matter and white matter, was differentiated from glioma using positive and negative ion mode DESI-MS lipid profiles with the aid of principal component analysis along with linear discriminant analysis. Principal component-linear discriminant analyses of the positive mode lipid profiles was able to distinguish grey matter, white matter, and glioma with an average sensitivity of 93.2% and specificity of 96.6%, while the negative mode lipid profiles had an average sensitivity of 94.1% and specificity of 97.4%. The positive and negative mode lipid profiles provided complementary information. Principal component-linear discriminant analysis of the combined positive and negative mode lipid profiles, via data fusion, resulted in approximately the same average sensitivity (94.7%) and specificity (97.6%) of the positive and negative modes when used individually. However, they complemented each other by improving the sensitivity and specificity of all classes (grey matter, white matter, and glioma) beyond 90% when used in combination. Further principal component analysis using the fused data resulted in the subgrouping of glioma into two groups associated with grey and white matter, respectively, a separation not apparent in the principal component analysis scores plots of the separate positive and negative mode data. The interrelationship of tumor cell percentage and the lipid profiles is discussed, and how such a measure could be used to measure residual tumor at surgical margins.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Intraoperative desorption electrospray ionization-mass spectrometry (DESI-MS) is used to characterize tissue smears by comparison with a library of DESI mass spectra of pathologically determined ...tissue types. Measurements are performed in the operating room within 3 min. These mass spectra provide direct information on tumor infiltration into white or gray brain matter based on N-acetylaspartate (NAA) and on membrane-derived complex lipids. The mass spectra also indicate the isocitrate dehydrogenase mutation status of the tumor via detection of 2-hydroxyglutarate, currently assessed postoperatively on biopsied tissue using immunohistochemistry. Intraoperative DESI-MS measurements made at surgeon-defined positions enable assessment of relevant disease state of tissue within the tumor mass and examination of the resection cavity walls for residual tumor. Results for 73 biopsies from 10 surgical resection cases show that DESI-MS allows detection of glioma and estimation of high tumor cell percentage (TCP) at surgical margins with 93% sensitivity and 83% specificity. TCP measurements from NAA are corroborated by indirect measurements based on lipid profiles. Notably, high percentages (>50%) of unresected tumor were found in one-half of the margin biopsy smears, even in cases where postoperative MRI suggested gross total tumor resection. Unresected tumor causes recurrence and malignant progression, as observed within a year in one case examined in this study. These results corroborate the utility of DESI-MS in assessing surgical margins for maximal safe tumor resection. Intraoperative DESI-MS analysis of tissue smears, ex vivo, can be inserted into the current surgical workflow with no alterations. The data underscore the complexity of glioma infiltration.
Examination of tissue sections using desorption electrospray ionization (DESI)-MS revealed phospholipid-derived signals that differ between gray matter, white matter, gliomas, meningiomas, and ...pituitary tumors, allowing their ready discrimination by multivariate statistics. A set of lower mass signals, some corresponding to oncometabolites, including 2-hydroxyglutaric acid and N-acetylaspartic acid, was also observed in the DESI mass spectra, and these data further assisted in discrimination between brain parenchyma and gliomas. The combined information from the lipid and metabolite MS profiles recorded by DESI-MS and explored using multivariate statistics allowed successful differentiation of gray matter (n = 223), white matter (n = 66), gliomas (n = 158), meningiomas (n = 111), and pituitary tumors (n = 154) from 58 patients. A linear discriminant model used to distinguish brain parenchyma and gliomas yielded an overall sensitivity of 97.4% and a specificity of 98.5%. Furthermore, a discriminant model was created for tumor types (i.e., glioma, meningioma, and pituitary), which were discriminated with an overall sensitivity of 99.4% and a specificity of 99.7%. Unsupervised multivariate statistics were used to explore the chemical differences between anatomical regions of brain parenchyma and secondary infiltration. Infiltration of gliomas into normal tissue can be detected by DESI-MS. One hurdle to implementation of DESI-MS intraoperatively is the need for tissue freezing and sectioning, which we address by analyzing smeared biopsy tissue. Tissue smears are shown to give the same chemical information as tissue sections, eliminating the need for sectioning before MS analysis. These results lay the foundation for implementation of intraoperative DESI-MS evaluation of tissue smears for rapid diagnosis.
Zika virus (ZIKV) can infect and cause microcephaly and Zika-associated neurological complications in the developing fetal and adult brains. In terms of pathogenesis, a critical question is how ZIKV ...overcomes the barriers separating the brain from the circulation and gains access to the central nervous system (CNS). Despite the importance of ZIKV pathogenesis, the route ZIKV utilizes to cross CNS barriers remains unclear. Here we show that in mouse models, ZIKV-infected cells initially appeared in the periventricular regions of the brain, including the choroid plexus and the meninges, prior to infection of the cortex. The appearance of ZIKV in cerebrospinal fluid (CSF) preceded infection of the brain parenchyma. Further the brain infection was significantly attenuated by neutralization of the virus in the CSF, indicating that ZIKV in the CSF at the early stage of infection might be responsible for establishing a lethal infection of the brain. We show that cells infected by ZIKV in the choroid plexus were pericytes. Using in vitro systems, we highlight the possibility that ZIKV crosses the blood-CSF barrier by disrupting the choroid plexus epithelial layer. Taken together, our results suggest that ZIKV might exploit the blood-CSF barrier rather than the blood-brain barrier to invade the CNS.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection ...(GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood.
The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation.
The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell-specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI-guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI.
Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions.
Touch spray mass spectrometry using medical swabs is an ambient ionization technique (ionization of unprocessed sample in the open air) that has potential intraoperative application in quickly ...identifying the disease state of tissue and in better characterizing the resection margin. To explore this potential, we studied 29 human brain tumor specimens and obtained evidence that this technique can provide diagnostic molecular information that is relevant to brain cancer. Touch spray using medical swabs involves the physical sampling of tissue using a medical swab on a spatial scale of a few mm
with subsequent ionization occurring directly from the swab tip upon addition of solvent and application of a high voltage. Using a tertiary mixture of acetonitrile, N,N-dimethylformamide, and ethanol, membrane-derived phospholipids and oncometabolites are extracted from the tissue, incorporated into the sprayed microdroplets, vacuumed into the mass spectrometer, and characterized in the resulting mass spectra. The tumor cell load was assessed from the complex phospholipid pattern in the mass spectra and also separately by measurement of N-acetylaspartate. Mutation status of the isocitrate dehydrogenase gene was determined via detection of the oncometabolite 2-hydroxyglutarate. The lack of sample pretreatment makes touch spray mass spectrometry using medical swabs a feasible intraoperative strategy for rapid surgical assessment.
The authors describe a rapid intraoperative ambient ionization mass spectrometry (MS) method for determining isocitrate dehydrogenase (IDH) mutation status from glioma tissue biopsies. This method ...offers new glioma management options and may impact extent of resection goals. Assessment of the IDH mutation is key for accurate glioma diagnosis, particularly for differentiating diffuse glioma from other neoplastic and reactive inflammatory conditions, a challenge for the standard intraoperative diagnostic consultation that relies solely on morphology.
Banked glioma specimens (n = 37) were analyzed by desorption electrospray ionization-MS (DESI-MS) to develop a diagnostic method to detect the known altered oncometabolite in IDH-mutant gliomas, 2-hydroxyglutarate (2HG). The method was used intraoperatively to analyze tissue smears obtained from glioma patients undergoing resection and to rapidly diagnose IDH mutation status (< 5 minutes). Fifty-one tumor core biopsies from 25 patients (14 wild type WT and 11 mutant) were examined and data were analyzed using analysis of variance and receiver operating characteristic curve analysis.
The optimized DESI-MS method discriminated between IDH-WT and IDH-mutant gliomas, with an average sensitivity and specificity of 100%. The average normalized DESI-MS 2HG signal was an order of magnitude higher in IDH-mutant glioma than in IDH-WT glioma. The DESI 2HG signal intensities correlated with independently measured 2HG concentrations (R2 = 0.98). In 1 case, an IDH1 R132H-mutant glioma was misdiagnosed as a demyelinating condition by frozen section histology during the intraoperative consultation, and no resection was performed pending the final pathology report. A second craniotomy and tumor resection was performed after the final pathology provided a diagnosis most consistent with an IDH-mutant glioblastoma. During the second craniotomy, high levels of 2HG in the tumor core biopsies were detected.
This study demonstrates the capability to differentiate rapidly between IDH-mutant gliomas and IDH-WT conditions by DESI-MS during tumor resection. DESI-MS analysis of tissue smears is simple and can be easily integrated into the standard intraoperative pathology consultation. This approach may aid in solving differential diagnosis problems associated with low-grade gliomas and could influence intraoperative decisions regarding extent of resection, ultimately improving patient outcome. Research is ongoing to expand the patient cohort, systematically validate the DESI-MS method, and investigate the relationships between 2HG and tumor heterogeneity.
Intraoperative neurosurgical histopathologic diagnoses rely on evaluation of rapid tissue preparations such as frozen sections and smears with conventional light microscopy. Although useful, these ...techniques are time consuming and therefore cannot provide real-time intraoperative feedback. In vivo molecular imaging techniques are emerging as novel methods for generating real-time diagnostic histopathologic images of tumors and their surrounding tissues. These imaging techniques rely on contrast generated by exogenous fluorescent dyes, autofluorescence of endogenous molecules, fluorescence decay of excited molecules, or light scattering. Large molecular imaging instruments are being miniaturized for clinical in vivo use. This review discusses pertinent imaging systems that have been developed for neurosurgical use and imaging techniques currently under development for neurosurgical molecular imaging.
Interobserver reproducibility in the diagnosis of benign intraductal proliferative lesions has been poor. The aims of the study were to investigate the inter- and intraobserver variability and the ...impact of the addition of an immunostain for high- and low-molecular weight keratins on the variability. Nine pathologists reviewed 81 cases of breast proliferative lesions in three stages and assigned each of the lesions to one of the following three diagnoses: usual ductal hyperplasia, atypical ductal hyperplasia and ductal carcinoma in situ. Hematoxylin and eosin slides and corresponding slides stained with ADH-5 cocktail (cytokeratins (CK) 5, 14. 7, 18 and p63) by immunohistochemistry were evaluated. Concordance was evaluated at each stage of the study. The interobserver agreement among the nine pathologists for diagnosing the 81 proliferative breast lesions was fair (κ-value=0.34). The intraobserver κ-value ranged from 0.56 to 0.88 (moderate to strong). Complete agreement among nine pathologists was achieved in only nine (11%) cases, at least eight agreed in 20 (25%) cases and seven or more agreed in 38 (47%) cases. Following immunohistochemical stain, a significant improvement in the interobserver concordance (overall κ-value=0.50) was observed (P=0.015). There was a significant reduction in the total number of atypical ductal hyperplasia diagnosis made by nine pathologists after the use of ADH-5 immunostain. Atypical ductal hyperplasia still remains a diagnostic dilemma with wide variation in both inter- and intraobserver reproducibility among pathologists. The addition of an immunohistochemical stain led to a significant improvement in the concordance rate. More importantly, there was an 8% decrease in the number of lesions classified as atypical ductal hyperplasia in favor of usual hyperplasia; in clinical practice, this could lead to a decrease in the number of surgeries carried out for intraductal proliferative lesions.
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