Radiographic and Clinical Outcomes After Stand-Alone Anterior Lumbar Interbody Fusion for Symptomatic L5-S1 Retrolisthesis.

BACKGROUND AND OBJECTIVES: Degenerative lumbar spondylolisthesis is associated with significant pain and disability. The literature on the treatment options and clinical outcomes for lumbar anterolisthesis is robust, but very few reports specifically evaluate lumbar retrolisthesis. This study investigated surgical outcomes for symptomatic L5-S1 retrolisthesis treated with stand-alone L5-S1 anterior lumbar interbody fusion (ALIF). METHODS: All patients with symptomatic L5-S1 retrolisthesis treated with stand-alone L5-S1 ALIF at a single institution over a 7-year period were identified. Exhaustive nonoperative management had failed for all patients. Patients with previous lumbar fusion were excluded. Preoperative and postoperative radiographic images and patient-reported outcome measures for 20 patients (14 males and 6 females; mean [SD] age, 50.3 [13.7] years) were analyzed. RESULTS: The mean (SD) follow-up was 43.0 (23.7) months (range, 12.1-102.5 months). Patients experienced postoperative improvements in L5-S1 retrolisthesis (P = .048), L5-S1 disk height and angle (P < .001), L5 foraminal height (P < .001), L5-S1 lordosis (P < .001), and lumbar lordosis (P = .01). There were no significant changes in spinopelvic parameters. At the most recent follow-up, minimal clinically important differences in Oswestry Disability Index score, 36-Item Short-Form Survey (SF-36), and numerical rating scale score for leg pain were achieved in 11 of 20 (55%), 7 of 14 (50%), and 7 of 13 (54%) patients, respectively. All patients demonstrated fusion with no graft subsidence at up to 32 months. No patient experienced intraoperative complications, was readmitted, or required a subsequent posterior decompression or fusion because of refractory symptoms. CONCLUSION: In our cohort, stand-alone L5-S1 ALIF was associated with radiographic and clinical improvement in patients with symptomatic L5-S1 retrolisthesis.

Competitive neurosurgery residency programs: Predictors of matching outcome and research productivity.

OBJECTIVE: The aim of this study was to provide a comprehensive assessment of preresidency research and school as predictors of competitive neurosurgery matching and to assess for any correlations between preresidency and intraresidency research productivity. METHODS: Individuals who graduated from US neurosurgery programs from 2018 through 2020 were assessed for medical school, degree (MD, DO, or PhD), preresidency versus intraresidency publications, author order, article type, and neurosurgery matching outcomes. RESULTS: Medical school ranking (top 50) and the number of published papers (≥3) before intern year were predictors for matching to a top-25 residency program after adjusting for other covariates (p < 0.001, p = 0.002, respectively). On average, individuals who published more papers before residency published more papers during residency. For the comprehensive clinical papers category, there was a significant difference between individuals from the top 25 residency programs and others, with a stronger correlation between the number of preresidency publications and intraresidency publications for neurosurgeons who attended a top-25 residency program (r = 0.378 and r = 0.179, respectively; p = 0.02). CONCLUSION: Medical school ranking and research productivity as measured by the number of published papers were independently associated with matching to the top 25 residency programs. In addition, high research productivity in the preresidency years was associated with continued productivity during residency, especially in the category of comprehensive clinical papers.

Academically Inclined: Predictors of Early Career Trajectory and Avenues for Early Intervention Among Neurosurgery Trainees.

BACKGROUND: The relationship of academic activities before and during neurosurgery residency with fellowship or career outcomes has not been studied completely. OBJECTIVE: To assess possible predictors of fellowship and career outcomes among neurosurgery residents. METHODS: US neurosurgery graduates (2018-2020) were assessed retrospectively for peer-reviewed citations of preresidency vs intraresidency publications, author order, and article type. Additional parameters included medical school, residency program, degree (MD vs DO; PhD), postgraduate fellowship, and academic employment. RESULTS: Of 547 neurosurgeons, 334 (61.1%) entered fellowships. Fellowship training was significantly associated with medical school rank and first-author publications. Individuals from medical schools ranked 1 to 50 were 1.6 times more likely to become postgraduate fellows than individuals from medical schools ranked 51 to 92 (odds ratio [OR], 1.63 [95% CI 1.04-2.56]; P = .03). Residents with ≥2 first-author publications were almost twice as likely to complete a fellowship as individuals with <2 first-author publications (OR, 1.91 [95% CI 1.21-3.03]; P = .006). Among 522 graduates with employment data available, academic employment obtained by 257 (49.2%) was significantly associated with fellowship training and all publication-specific variables. Fellowship-trained graduates were twice as likely to pursue academic careers (OR, 1.99 [95% CI 1.34-2.96]; P < .001) as were individuals with ≥3 first-author publications ( P < .001), ≥2 laboratory publications ( P = .04), or ≥9 clinical publications ( P < .001). CONCLUSION: Research productivity, medical school rank, and fellowships are independently associated with academic career outcomes of neurosurgeons. Academically inclined residents may benefit from early access to mentorship, sponsorship, and publishing opportunities.

Publication Speed Across Neurosurgery Journals: A Bibliometric Analysis.

OBJECTIVE: Many factors influence an author's choice for journal submission, including journal impact factor and publication speed. These and other bibliometric data points have not been assessed in journals dedicated to neurosurgery. METHODS: Eight leading neurosurgery journals were analyzed to identify original articles and reviews, collected via randomized, stratified sampling per published issue per year from 2016 to 2020. Bibliometric data on publication speed were gathered for each article. Journal impact factor, article processing fees, and open access availability were determined using Clarivate Journal Citation Reports. Correlation analysis and a linear regression model were used to estimate the effect of impact factor and publication year on publication speed. RESULTS: Across the 8 neurosurgery journals, 1617 published articles were reviewed. The mean (standard deviation) time from submission to acceptance (SA) was 131 (101) days, from acceptance to online publication was 77 (61) days, and from submission to online publication was 207 (123) days. Higher impact factors correlated with longer publication times for all metrics. Later years of publication correlated with longer times from SA and submission to online publication. For each point increase in a journal's impact factor, multivariate regression modeling estimated a 19.2-day increase in time from SA, a 19.7-day increase in time from acceptance to online publication, and a 38.9-day increase in time from submission to online publication (P < 0.001 for all). CONCLUSIONS: Publication speeds vary widely among neurosurgery journals and appear to be associated with the journal impact factor. Time to publication increased over the study period.

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.

Management of basilar fenestration aneurysms: a systematic review with an illustrative case report.

BACKGROUND: Basilar artery (BA) fenestration aneurysm (BAFA) is a rare phenomenon commonly accompanying other aneurysms. Treatment is challenging, and few cases have been reported. This review investigated the management outcomes of BAFAs. METHODS: Publication databases were searched to identify studies evaluating outcomes of endovascular treatment (EVT) and microsurgical treatment of BAFAs from inception through 2021. Outcomes (clinical, angiographic, postoperative complications, and retreatment rates) were collected and analyzed. The authors present their case of a patient treated for a BAFA. RESULTS: Including the authors' case, 184 patients with 209 BAFAs were reported in 68 studies. Most patients (130/175; 74.3%) presented with ruptured aneurysms, most commonly involving the proximal segment of the BA. Most BAFAs were small (52/103, 50.5%) and saccular (119/143, 83.2%). Most patients underwent EVT (143/184, 77.7%); the rest underwent microsurgery. Postoperative complications after EVT occurred in 10 (8.3%) of 120 patients, with 4 of the 10 experiencing strokes. At clinical follow-up, most EVT patients (74/86, 86.0%) showed good outcomes; 3.9% (2/51) had died. Most aneurysms managed with EVT (56/73, 76.7%) showed complete occlusion at follow-up; 7.3% (8/109) were retreated. Postoperative complications occurred in 62.2% (23/37) of microsurgical patients; 5 (21.7%) of the 23 experienced strokes. All patients showed good clinical outcomes at follow-up. Most aneurysms (22/28, 78.6%) treated microsurgically showed complete occlusion at angiographic follow-up, with no retreatment required. CONCLUSION: BAFAs are often symptomatic; thus, treatment is challenging. By the 2000s, treatment had moved from microsurgical to endovascular modalities, with good clinical and angiographic outcomes.

Surgical management outcomes of intracranial arteriovenous malformations after preoperative embolization: a systematic review and meta-analysis.

OBJECTIVE: Preoperative embolization for brain arteriovenous malformations (AVMs) has been shown to mitigate morbidity for high-risk AVMs, chiefly by reducing lesional blood flow before resection. However, associated risks include postembolization AVM rupture, and the effect of preoperative embolization on outcome remains uncertain. We performed a meta-analysis of the literature on preoperative embolization for microsurgically treated AVMs. METHODS: A systematic review and meta-analysis were conducted of all English-language publications reporting clinical outcomes after combined embolization and surgical resection for AVMs. Single- and 2-arm analyses were performed using random-effects modeling. RESULTS: Thirty-six studies with 2108 patients were included in this analysis. Most patients (90.6%) who underwent embolization had at least a 50% obliteration of AVMs on posttreatment preoperative angiography, with a mean rate of obliteration of approximately 80% (range 28.8-100%). Among patients who had combined treatment, 3.4% (95% confidence interval [CI] 2.1-4.6%) experienced embolization-related hemorrhagic complications before surgery. Both treatment groups achieved excellent postsurgical complete resection rates (odds ratio [OR] 1.05; 95% CI 0.60-1.85). Neither the clinical outcome (OR 1.42; 95% CI 0.84-2.40) nor the total number of hemorrhagic complications (OR 1.84; 95% CI 0.88-3.85) was significantly different between the treatment groups. CONCLUSIONS: In this meta-analysis, preoperative embolization appears to have substantially reduced the lesional volume with active AV shunting before AVM resection. Anecdotally, preoperative embolization facilitates safe and efficient resection; however, differences in outcomes were not significant. The decision to pursue preoperative embolization remains a nuanced decision based on individual lesion anatomy and treatment team experience.

Management of injuries on the 16th-century battlefield: Ambroise Paré's contributions to neurosurgery and functional recovery.

During the 1536 siege of Turin in northern Italy, a young French barber-surgeon abandoned the conventional treatment of battle-inflicted wounds, launching a revolution in military medicine and surgical techniques. Ambroise Paré (1510-1590) was born into a working-class Huguenot family in Laval, France, during an era when surgery was not considered a respectable profession. He rose from humble origins as a barber-surgeon, a low-ranked occupation in the French medical hierarchy, to become a royal surgeon (chirurgien ordinaire du Roi) serving 4 consecutive French monarchs. His innovative ideas and surgical practice were a response to the environment created by new military technology on 16th-century European battlefields. Gunpowder weapons caused unfamiliar, complicated injuries that challenged Paré to develop new techniques and surgical instruments. Although Paré's contributions to the treatment of wounds and functional prosthetics are documented, a deeper appreciation of his role in military neurosurgery is needed. This paper examines archives, primary texts, and written accounts by Paré that reveal specific patient cases highlighting his innovative contributions to neurotrauma and neurosurgery during demanding and harrowing circumstances, on and off the battlefield, in 16th-century France. Notably, trepanation indications increased because of battlefield head injuries, and Paré frequently described this technique and improved the design of the trepan tool. His contribution to neurologically related topics is extensive; there are more chapters devoted to the nervous system than to any other organ system in his compendium, Oeuvres. Regarding anatomical knowledge as fundamentally important and admiring the contemporary contributions of Andreas Vesalius, Paré reproduced many images from Vesalius' works at his own great expense. The manner in which Paré's participation in military expeditions enabled collaboration with multidisciplinary artisans on devices, including surgical tools and prosthetics, to restore neurologically associated functionality is also discussed. Deeply religious, in a life filled with adventure, and serving in often horrendous conditions during a time when Galenic dogma still dominated medical practice, Paré developed a reputation for logic, empiricism, technology, and careful treatment. "I have [had] the opportunity to praise God, for what he called me to do in medical operation, which is commonly called surgery, which could not be bought with gold or silver, but by only virtue and great experimentation."

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.

Multiple hippocampal transection for mesial temporal lobe epilepsy: A systematic review.

PURPOSE: Multiple hippocampal transection (MHT) is a surgical technique that offers adequate seizure control with minimal perioperative morbidity. However, there is little evidence available to guide neurosurgeons in selecting this technique for use in appropriate patients. This systematic review analyzes patient-level data associated with MHT for intractable epilepsy, focusing on postoperative seizure control and memory outcomes. METHODS: The systematic review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Relevant articles were identified from 3 databases (PubMed, Medline, Embase) up to August 1, 2021. Inclusion criteria were that the majority of patients had received a diagnosis of intractable epilepsy, the article was written in English, MHT was the primary procedure, and patient-level metadata were included. RESULTS: Fifty-nine unique patients who underwent MHT were identified across 11 studies. Ten (17%) of 59 patients underwent MHT alone. Forty-three (75%) of 57 patients who had a follow-up 12 months or longer were seizure free at last follow-up. With respect to postoperative verbal memory retention, 9 of 38 (24%) patient test scores did not change, 14 (37%) decreased, and 16 (42%) increased. With respect to postoperative nonverbal memory retention, 12 of 38 (34%) patient test scores did not change, 13 (34%) decreased, and 13 (33%) increased. CONCLUSION: There are few reported patients analyzed after MHT. Although the neurocognitive benefits of MHT are unproven, this relatively novel technique has shown promise in the management of seizures in patients with intractable epilepsy. However, structured trials assessing MHT in isolation are warranted.

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.

Redosing of Fluorescein Sodium Improves Image Interpretation During Intraoperative Ex Vivo Confocal Laser Endomicroscopy of Brain Tumors.

BACKGROUND: Fluorescein sodium (FNa) is a fluorescence agent used with a wide-field operating microscope for intraoperative guidance and with confocal laser endomicroscopy (CLE) to evaluate brain tissue. Susceptibility of FNa to degradation over time may affect CLE image quality during prolonged surgeries. This study describes improved characteristics of CLE images after intraoperative redosing with FNa. METHODS: A retrospective analysis was performed using CLE images obtained ex vivo from samples obtained during tumor resections with FNa-based fluorescence guidance with a wide-field operating microscope. The comparison groups included CLE images acquired after FNa redosing (redose imaging group), images from the same patients acquired after the initial FNa dose (initial-dose imaging group), and images from patients in whom redosing was not used (single-dose imaging group). A detailed assessment of image quality and interpretation regarding different FNa dosage and timing of imaging after FNa administration was conducted for all comparison groups. RESULTS: The brightest and most contrasting images were observed in the redose group compared to the initial-dose and single-dose groups (P<0.001). The decay of FNa signal negatively correlated with brightness (rho = -0.52, P<0.001) and contrast (rho = -0.57, P<0.001). Different doses of FNa did not significantly affect the brightness (P=0.15) or contrast (P=0.09) in CLE images. As the mean timing of imaging increased, the percentage of accurately diagnosed images decreased (P=0.03). CONCLUSIONS: The decay of the FNa signal is directly associated with image brightness and contrast. The qualitative interpretation scores of images were highest for the FNa redose imaging group. Redosing with FNa to improve the utility of CLE imaging should be considered a safe and beneficial strategy during prolonged surgeries.

Opportunistic Invasive Fungal Infections Mimicking Progression of Non-Small-Cell Lung Cancer.

BACKGROUND: Many studies have shown that invasive pulmonary aspergillosis, cryptococcosis, and mucormycosis can mimic radiographic and clinical features of primary lung cancer. However, more research surveying the incidence and outcomes of these fungal infections among patients with a history of lung cancer is needed. The aim of this study was to describe the occurrence and clinical outcomes of opportunistic invasive fungal infections that can mimic tumors in non-small-cell lung cancer patients. PATIENTS AND METHODS: Patients seen at Stanford University Medical Center from January 1, 2007, to May 1, 2020, with pulmonary aspergillosis, cryptococcosis, or mucormycosis after non-small-cell lung cancer (NSCLC) diagnosis were reviewed. The European Organization for Research and Treatment of Cancer National Institute of Allergy and Infectious Diseases Mycoses Study Group criteria was used to classify patients with evidence of proven or probable invasive fungal infection within our cohort. RESULTS: A total of 12 patients with proven or probable invasive mold infection (including 8 cases of aspergillosis) and 1 patient with proven cryptococcosis were identified, without any cases of mucormycosis. Of this cohort, 6 patients (46%) showed radiographic findings that were found to be most consistent with lung cancer by radiologists. Eight cases (62%) were suspected of cancer recurrence or progression by the treatment team on the basis of additional considerations of medical history and clinical symptoms. Most patients had active NSCLC or had a history of recurrence without active NSCLC at the time of fungal discovery (11 patients; 85%). Most patients died without full recovery (7 patients; 54%). CONCLUSIONS: Invasive pulmonary aspergillosis and cryptococcosis can often be mistaken as cancer recurrence or progression in patients with a history of NSCLC because of mimicking radiographic and clinical characteristics.

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression.

Sialylation, a post-translational modification that impacts the structure, activity, and longevity of glycoproteins has been thought to be controlled primarily by the expression of sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-Bu3ManNAc, a "high flux" metabolic precursor for the sialic acid biosynthetic pathway. We then analyzed N-glycan sialylation using solid phase extraction of glycopeptides (SPEG) mass spectrometry-based proteomics under conditions that selectively captured sialic acid-containing glycopeptides, referred to as "sialoglycosites." Gene ontology (GO) analysis showed that flux-based changes to sialylation were broadly distributed across classes of proteins in 1,3,4-O-Bu3ManNAc-treated cells. Only three categories of proteins, however, were "highly responsive" to flux (defined as two or more sialylation changes of 10-fold or greater). Two of these categories were cell signaling and cell adhesion, which reflect well-known roles of sialic acid in oncogenesis. A third category-protein folding chaperones-was unexpected because little precedent exists for the role of glycosylation in the activity of these proteins. The highly flux-responsive proteins were all linked to cancer but sometimes as tumor suppressors, other times as proto-oncogenes, or sometimes both depending on sialylation status. A notable aspect of our analysis of metabolically glycoengineered breast cells was decreased sialylation of a subset of glycosites, which was unexpected because of the increased intracellular levels of sialometabolite "building blocks" in the 1,3,4-O-Bu3ManNAc-treated cells. Sites of decreased sialylation were minor in the MCF10A (<25% of all glycosites) and T-47D (<15%) cells but dominated in the MDA-MB-231 line (~60%) suggesting that excess sialic acid could be detrimental in advanced cancer and cancer cells can evolve mechanisms to guard against hypersialylation. In summary, flux-driven changes to sialylation offer an intriguing and novel mechanism to switch between context-dependent pro- or anti-cancer activities of the several oncoproteins identified in this study. These findings illustrate how metabolic glycoengineering can uncover novel roles of sialic acid in oncogenesis.

Integration of genetic and metabolic features related to sialic acid metabolism distinguishes human breast cell subtypes.

In this report we use 'high-flux' tributanoyl-modified N-acetylmannosamine (ManNAc) analogs with natural N-acetyl as well as non-natural azido- and alkyne N-acyl groups (specifically, 1,3,4-O-Bu3ManNAc, 1,3,4-O-Bu3ManNAz, and 1,3,4-O-Bu3ManNAl respectively) to probe intracellular sialic acid metabolism in the near-normal MCF10A human breast cell line in comparison with earlier stage T-47D and more advanced stage MDA-MB-231 breast cancer lines. An integrated view of sialic acid metabolism was gained by measuring intracellular sialic acid production in tandem with transcriptional profiling of genes linked to sialic acid metabolism. The transcriptional profiling showed several differences between the three lines in the absence of ManNAc analog supplementation that helps explain the different sialoglycan profiles naturally associated with cancer. Only minor changes in mRNA transcript levels occurred upon exposure to the compounds confirming that metabolic flux alone can be a key determinant of sialoglycoconjugate display in breast cancer cells; this result complements the well-established role of genetic control (e.g., the transcription of STs) of sialylation abnormalities ubiquitously associated with cancer. A notable result was that the different cell lines produced significantly different levels of sialic acid upon exogenous ManNAc supplementation, indicating that feedback inhibition of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE)-generally regarded as the 'gatekeeper' enzyme for titering flux into sialic acid biosynthesis-is not the only regulatory mechanism that limits production of this sugar. A notable aspect of our metabolic glycoengineering approach is its ability to discriminate cell subtype based on intracellular metabolism by illuminating otherwise hidden cell type-specific features. We believe that this strategy combined with multi-dimensional analysis of sialic acid metabolism will ultimately provide novel insights into breast cancer subtypes and provide a foundation for new methods of diagnosis.