Subclonal evolution and expansion of spatially distinct THY1-positive cells is associated with recurrence in glioblastoma.

PURPOSE: Glioblastoma(GBM) is a lethal disease characterized by inevitable recurrence. Here we investigate the molecular pathways mediating resistance, with the goal of identifying novel therapeutic opportunities. EXPERIMENTAL DESIGN: We developed a longitudinal in vivo recurrence model utilizing patient-derived explants to produce paired specimens(pre- and post-recurrence) following temozolomide(TMZ) and radiation(IR). These specimens were evaluated for treatment response and to identify gene expression pathways driving treatment resistance. Findings were clinically validated using spatial transcriptomics of human GBMs. RESULTS: These studies reveal in replicate cohorts, a gene expression profile characterized by upregulation of mesenchymal and stem-like genes at recurrence. Analyses of clinical databases revealed significant association of this transcriptional profile with worse overall survival and upregulation at recurrence. Notably, gene expression analyses identified upregulation of TGFß signaling, and more than one-hundred-fold increase in THY1 levels at recurrence. Furthermore, THY1-positive cells represented <10% of cells in treatment-naïve tumors, compared to 75-96% in recurrent tumors. We then isolated THY1-positive cells from treatment-naïve patient samples and determined that they were inherently resistant to chemoradiation in orthotopic models. Additionally, using image-guided biopsies from treatment-naïve human GBM, we conducted spatial transcriptomic analyses. This revealed rare THY1+ regions characterized by mesenchymal/stem-like gene expression, analogous to our recurrent mouse model, which co-localized with macrophages within the perivascular niche. We then inhibited TGFBRI activity in vivo which decreased mesenchymal/stem-like protein levels, including THY1, and restored sensitivity to TMZ/IR in recurrent tumors. CONCLUSIONS: These findings reveal that GBM recurrence may result from tumor repopulation by pre-existing, therapy-resistant, THY1-positive, mesenchymal cells within the perivascular niche.

Association of Autologous Tumor Lysate-Loaded Dendritic Cell Vaccination With Extension of Survival Among Patients With Newly Diagnosed and Recurrent Glioblastoma: A Phase 3 Prospective Externally Controlled Cohort Trial.

Importance: Glioblastoma is the most lethal primary brain cancer. Clinical outcomes for glioblastoma remain poor, and new treatments are needed. Objective: To investigate whether adding autologous tumor lysate-loaded dendritic cell vaccine (DCVax-L) to standard of care (SOC) extends survival among patients with glioblastoma. Design, Setting, and Participants: This phase 3, prospective, externally controlled nonrandomized trial compared overall survival (OS) in patients with newly diagnosed glioblastoma (nGBM) and recurrent glioblastoma (rGBM) treated with DCVax-L plus SOC vs contemporaneous matched external control patients treated with SOC. This international, multicenter trial was conducted at 94 sites in 4 countries from August 2007 to November 2015. Data analysis was conducted from October 2020 to September 2021. Interventions: The active treatment was DCVax-L plus SOC temozolomide. The nGBM external control patients received SOC temozolomide and placebo; the rGBM external controls received approved rGBM therapies. Main Outcomes and Measures: The primary and secondary end points compared overall survival (OS) in nGBM and rGBM, respectively, with contemporaneous matched external control populations from the control groups of other formal randomized clinical trials. Results: A total of 331 patients were enrolled in the trial, with 232 randomized to the DCVax-L group and 99 to the placebo group. Median OS (mOS) for the 232 patients with nGBM receiving DCVax-L was 19.3 (95% CI, 17.5-21.3) months from randomization (22.4 months from surgery) vs 16.5 (95% CI, 16.0-17.5) months from randomization in control patients (HR = 0.80; 98% CI, 0.00-0.94; P = .002). Survival at 48 months from randomization was 15.7% vs 9.9%, and at 60 months, it was 13.0% vs 5.7%. For 64 patients with rGBM receiving DCVax-L, mOS was 13.2 (95% CI, 9.7-16.8) months from relapse vs 7.8 (95% CI, 7.2-8.2) months among control patients (HR, 0.58; 98% CI, 0.00-0.76; P < .001). Survival at 24 and 30 months after recurrence was 20.7% vs 9.6% and 11.1% vs 5.1%, respectively. Survival was improved in patients with nGBM with methylated MGMT receiving DCVax-L compared with external control patients (HR, 0.74; 98% CI, 0.55-1.00; P = .03). Conclusions and Relevance: In this study, adding DCVax-L to SOC resulted in clinically meaningful and statistically significant extension of survival for patients with both nGBM and rGBM compared with contemporaneous, matched external controls who received SOC alone. Trial Registration: ClinicalTrials.gov Identifier: NCT00045968.

Rapid Automated Analysis of Skull Base Tumor Specimens Using Intraoperative Optical Imaging and Artificial Intelligence.

BACKGROUND: Accurate specimen analysis of skull base tumors is essential for providing personalized surgical treatment strategies. Intraoperative specimen interpretation can be challenging because of the wide range of skull base pathologies and lack of intraoperative pathology resources. OBJECTIVE: To develop an independent and parallel intraoperative workflow that can provide rapid and accurate skull base tumor specimen analysis using label-free optical imaging and artificial intelligence. METHODS: We used a fiber laser-based, label-free, nonconsumptive, high-resolution microscopy method (<60 seconds per 1 × 1 mm2), called stimulated Raman histology (SRH), to image a consecutive, multicenter cohort of patients with skull base tumor. SRH images were then used to train a convolutional neural network model using 3 representation learning strategies: cross-entropy, self-supervised contrastive learning, and supervised contrastive learning. Our trained convolutional neural network models were tested on a held-out, multicenter SRH data set. RESULTS: SRH was able to image the diagnostic features of both benign and malignant skull base tumors. Of the 3 representation learning strategies, supervised contrastive learning most effectively learned the distinctive and diagnostic SRH image features for each of the skull base tumor types. In our multicenter testing set, cross-entropy achieved an overall diagnostic accuracy of 91.5%, self-supervised contrastive learning 83.9%, and supervised contrastive learning 96.6%. Our trained model was able to segment tumor-normal margins and detect regions of microscopic tumor infiltration in meningioma SRH images. CONCLUSION: SRH with trained artificial intelligence models can provide rapid and accurate intraoperative analysis of skull base tumor specimens to inform surgical decision-making.

Correlation between brain tissue oxygen tension and regional cerebral oximetry in uninjured human brain under conditions of changing ventilation strategy.

Controversy surrounds regional cerebral oximetry (rSO2) because extracranial contamination and unmeasured changes in cerebral arterial:venous ratio confound readings. Correlation of rSO2 with brain tissue oxygen (PbrO2), a "gold standard" for cerebral oxygenation, could help resolve this controversy but PbrO2 measurement is highly invasive. This was a prospective cohort study. The primary aim was to evaluate correlation between PbrO2 and rSO2 and the secondary aim was to investigate the relationship between changing ventilation regimens and measurement of PbrO2 and rSO2. Patients scheduled for elective removal of cerebral metastases were anesthetized with propofol and remifentanil, targeted to a BIS range 40-60. rSO2 was measured using the INVOS 5100B monitor and PbrO2 using the Licox brain monitoring system. The Licox probe was placed into an area of normal brain within the tumor excision corridor. FiO2 and minute ventilation were sequentially adjusted to achieve two set points: (1) FiO2 0.3 and paCO2 30 mmHg, (2) FiO2 1.0 and paCO2 40 mmHg. PbrO2 and rSO2 were recorded at each. Nine participants were included in the final analysis, which showed a positive Spearman's correlation (r = 0.50, p = 0.036) between PbrO2 and rSO2. From set point 1 to set point 2, PbrO2 increased from median 6.0, IQR 4.0-11.3 to median 22.5, IQR 9.8-43.6, p = 0.015; rSO2 increased from median 68.0, IQR 62.5-80.5 to median 83.0, IQR 74.0-90.0, p = 0.047. Correlation between PbrO2 and rSO2 is evident. Increasing FiO2 and PaCO2 results in significant increases in cerebral oxygenation measured by both monitors.

A customized 3D implant to target laser interstitial thermal therapy ablation of a posterior fossa mass.

Laser interstitial thermal therapy (LITT) is a minimally invasive neurosurgical technique that has been demonstrated to successfully ablate intracranial tumors. While LITT for supratentorial lesions can often be straightforward, ablation of infratentorial lesions can be difficult with current targeting technologies and instrumentation. The anatomical difficulty of targeting posterior fossa masses can be further complicated in patients who have had a prior craniectomy or other procedure that removed the bone that is required to set the surgical trajectory. This article describes use of a three-dimensional (3D)-printed customized surgical implant to improve and enable targeting of posterior fossa lesions using LITT, particularly in the setting of prior craniectomy. A 3D-printed implant was customized for a patient with a history of metastatic lung cancer and prior posterior fossa craniectomy who presented for treatment of a progressively enlarging contrast-enhancing lesion in the right cerebellar hemisphere. The device included a built-in bolt trajectory for LITT ablation. The temporary implant was successfully fabricated for use with laser ablation of a right cerebellar mass. Three potential trajectories for the LITT bolt were incorporated into the temporary implant, but only the primary trajectory was utilized. Laser ablation was performed with the implant and a SideFire laser probe. Customized 3D-printed implants can enable the use of LITT for patients who would not otherwise be candidates.

Rapid, label-free detection of diffuse glioma recurrence using intraoperative stimulated Raman histology and deep neural networks.

BACKGROUND: Detection of glioma recurrence remains a challenge in modern neuro-oncology. Noninvasive radiographic imaging is unable to definitively differentiate true recurrence versus pseudoprogression. Even in biopsied tissue, it can be challenging to differentiate recurrent tumor and treatment effect. We hypothesized that intraoperative stimulated Raman histology (SRH) and deep neural networks can be used to improve the intraoperative detection of glioma recurrence. METHODS: We used fiber laser-based SRH, a label-free, nonconsumptive, high-resolution microscopy method (<60 sec per 1 × 1 mm2) to image a cohort of patients (n = 35) with suspected recurrent gliomas who underwent biopsy or resection. The SRH images were then used to train a convolutional neural network (CNN) and develop an inference algorithm to detect viable recurrent glioma. Following network training, the performance of the CNN was tested for diagnostic accuracy in a retrospective cohort (n = 48). RESULTS: Using patch-level CNN predictions, the inference algorithm returns a single Bernoulli distribution for the probability of tumor recurrence for each surgical specimen or patient. The external SRH validation dataset consisted of 48 patients (recurrent, 30; pseudoprogression, 18), and we achieved a diagnostic accuracy of 95.8%. CONCLUSION: SRH with CNN-based diagnosis can be used to improve the intraoperative detection of glioma recurrence in near-real time. Our results provide insight into how optical imaging and computer vision can be combined to augment conventional diagnostic methods and improve the quality of specimen sampling at glioma recurrence.

Near real-time intraoperative brain tumor diagnosis using stimulated Raman histology and deep neural networks.

Intraoperative diagnosis is essential for providing safe and effective care during cancer surgery1. The existing workflow for intraoperative diagnosis based on hematoxylin and eosin staining of processed tissue is time, resource and labor intensive2,3. Moreover, interpretation of intraoperative histologic images is dependent on a contracting, unevenly distributed, pathology workforce4. In the present study, we report a parallel workflow that combines stimulated Raman histology (SRH)5-7, a label-free optical imaging method and deep convolutional neural networks (CNNs) to predict diagnosis at the bedside in near real-time in an automated fashion. Specifically, our CNNs, trained on over 2.5 million SRH images, predict brain tumor diagnosis in the operating room in under 150 s, an order of magnitude faster than conventional techniques (for example, 20-30 min)2. In a multicenter, prospective clinical trial (n = 278), we demonstrated that CNN-based diagnosis of SRH images was noninferior to pathologist-based interpretation of conventional histologic images (overall accuracy, 94.6% versus 93.9%). Our CNNs learned a hierarchy of recognizable histologic feature representations to classify the major histopathologic classes of brain tumors. In addition, we implemented a semantic segmentation method to identify tumor-infiltrated diagnostic regions within SRH images. These results demonstrate how intraoperative cancer diagnosis can be streamlined, creating a complementary pathway for tissue diagnosis that is independent of a traditional pathology laboratory.

Thirty-Day Hospital Readmission and Surgical Complication Rates for Shunting in Normal Pressure Hydrocephalus: A Large National Database Analysis.

BACKGROUND: Research on age-related complications secondary to shunts in normal pressure hydrocephalus (NPH) is primarily limited to single-center studies and small cohorts. OBJECTIVE: To determine the rates of hospital readmission and surgical complications, and factors that predict them, following shunt surgery for NPH in a large healthcare network. METHODS: Surgical procedures, complications, and readmissions for adults undergoing ventricular shunting for NPH were determined using de-identified claims from a privately insured United States healthcare network in years 2007-2014. Univariate and multivariate statistics were used to determine factors that predict poor surgical outcomes. The primary outcome variable was surgical complications or readmissions (composite variable for any major perioperative complication or 30-d readmission). RESULTS: The 30-d readmission rate for 974 patients with NPH who underwent ventricular shunting was 7.29%; the most common reasons for readmission were shunt-related complications, infection, hemorrhage, altered mental status, and cardiopulmonary and musculoskeletal problems. The perioperative complication rate was 21.15%, including intraparenchymal hemorrhage (5.85%) and extra-axial (subdural or epidural) hematoma (5.54%). The overall rate of having a surgical complication or 30-d readmission was 25.15%. Age did not predict surgical complication or 30-d readmission. Preoperative comorbidities independently associated with poor outcome were myocardial infarction within 1 yr (OR = 3.984, 95% CI = 1.105-14.368); existing cerebrovascular disease (odds ratio [OR] = 2.206, 95% CI = 1.544-3.152); and moderate/severe renal disease (OR = 2.000, 95% CI = 1.155-3.464). CONCLUSION: The rate of complications or readmission within 30 d of ventricular shunting for NPH is 25.15%. Preoperative comorbidities of myocardial infarction within 1 yr, cerebrovascular disease, and moderate/severe renal disease are independent risk factors for poor outcome.

Correction to: First results on survival from a large Phase 3 clinical trial of an autologous dendritic cell vaccine in newly diagnosed glioblastoma.

Following publication of the original article [1], the authors reported an error in the spelling of one of the author names. In this Correction the incorrect and correct author names are indicated and the author name has been updated in the original publication. The authors also reported an error in the Methods section of the original article. In this Correction the incorrect and correct versions of the affected sentence are indicated. The original article has not been updated with regards to the error in the Methods section.

Serious adverse events following Normal Pressure Hydrocephalus surgery.

OBJECTIVE: Recent Normal Pressure Hydrocephalus (NPH) practice guidelines describe a serious adverse event (SAE) rate following surgery of 11%. PATIENTS & METHODS: We conducted a retrospective review of 162 consecutive patients who have undergone work-up at our center's multidisciplinary NPH clinic over a 47 month time period (2/2014-12/2017). Of these, 22 ultimately underwent neurosurgical ventricular shunt surgery as treatment for NPH. Clinical records were reviewed for SAEs categorized as possibly/probably/definitely related to NPH surgery. RESULTS: In 10/22 (45.5%) operated subjects, there were 11 qualifying SAEs over this 3-year period: 1 central nervous system infections, 4 subdural hematomas, 2 seizures resulting in hospitalization, 1 catheter malfunction, 2 perioperative AEs, and 1 death of uncertain cause. Eight SAEs were coded as probably/definitely related. Six occurred >3 months from the time of surgery. CONCLUSIONS: SAEs following NPH surgery are common. Additional studies are needed to determine the long-term safety of NPH surgery in older adults.

First results on survival from a large Phase 3 clinical trial of an autologous dendritic cell vaccine in newly diagnosed glioblastoma.

BACKGROUND: Standard therapy for glioblastoma includes surgery, radiotherapy, and temozolomide. This Phase 3 trial evaluates the addition of an autologous tumor lysate-pulsed dendritic cell vaccine (DCVax®-L) to standard therapy for newly diagnosed glioblastoma. METHODS: After surgery and chemoradiotherapy, patients were randomized (2:1) to receive temozolomide plus DCVax-L (n = 232) or temozolomide and placebo (n = 99). Following recurrence, all patients were allowed to receive DCVax-L, without unblinding. The primary endpoint was progression free survival (PFS); the secondary endpoint was overall survival (OS). RESULTS: For the intent-to-treat (ITT) population (n = 331), median OS (mOS) was 23.1 months from surgery. Because of the cross-over trial design, nearly 90% of the ITT population received DCVax-L. For patients with methylated MGMT (n = 131), mOS was 34.7 months from surgery, with a 3-year survival of 46.4%. As of this analysis, 223 patients are ≥ 30 months past their surgery date; 67 of these (30.0%) have lived ≥ 30 months and have a Kaplan-Meier (KM)-derived mOS of 46.5 months. 182 patients are ≥ 36 months past surgery; 44 of these (24.2%) have lived ≥ 36 months and have a KM-derived mOS of 88.2 months. A population of extended survivors (n = 100) with mOS of 40.5 months, not explained by known prognostic factors, will be analyzed further. Only 2.1% of ITT patients (n = 7) had a grade 3 or 4 adverse event that was deemed at least possibly related to the vaccine. Overall adverse events with DCVax were comparable to standard therapy alone. CONCLUSIONS: Addition of DCVax-L to standard therapy is feasible and safe in glioblastoma patients, and may extend survival. Trial registration Funded by Northwest Biotherapeutics; Clinicaltrials.gov number: NCT00045968; https://clinicaltrials.gov/ct2/show/NCT00045968?term=NCT00045968&rank=1 ; initially registered 19 September 2002.

Rapid intraoperative histology of unprocessed surgical specimens via fibre-laser-based stimulated Raman scattering microscopy.

Conventional methods for intraoperative histopathologic diagnosis are labour- and time-intensive, and may delay decision-making during brain-tumour surgery. Stimulated Raman scattering (SRS) microscopy, a label-free optical process, has been shown to rapidly detect brain-tumour infiltration in fresh, unprocessed human tissues. Here, we demonstrate the first application of SRS microscopy in the operating room by using a portable fibre-laser-based microscope and unprocessed specimens from 101 neurosurgical patients. We also introduce an image-processing method - stimulated Raman histology (SRH) - which leverages SRS images to create virtual haematoxylin-and-eosin-stained slides, revealing essential diagnostic features. In a simulation of intraoperative pathologic consultation in 30 patients, we found a remarkable concordance of SRH and conventional histology for predicting diagnosis (Cohen's kappa, κ > 0.89), with accuracy exceeding 92%. We also built and validated a multilayer perceptron based on quantified SRH image attributes that predicts brain-tumour subtype with 90% accuracy. Our findings provide insight into how SRH can now be used to improve the surgical care of brain tumour patients.

Comparison of Diffusion Tensor Imaging and Magnetic Resonance Perfusion Imaging in Differentiating Recurrent Brain Neoplasm From Radiation Necrosis.

RATIONALE AND OBJECTIVES: To compare differences in diffusion tensor imaging (DTI) and dynamic susceptibility-weighted contrast-enhanced (DSC) magnetic resonance (MR) perfusion imaging characteristics of recurrent neoplasm and radiation necrosis in patients with brain tumors previously treated with radiotherapy with or without surgery and chemotherapy. MATERIALS AND METHODS: Patients with a history of brain neoplasm previously treated with radiotherapy with or without chemotherapy and surgery who developed a new enhancing lesion on posttreatment surveillance MRI were enrolled. DSC perfusion MRI and DTI were performed. Region of interest cursors were manually drawn in the contrast-enhancing lesions, in the perilesional white matter edema, and in the contralateral normal-appearing frontal lobe white matter. DTI and DSC perfusion MR indices were compared in recurrent tumor versus radiation necrosis. RESULTS: Twenty-two patients with 24 lesions were included. Sixteen (67%) lesions were placed into the recurrent neoplasm group and eight (33%) lesions were placed into the radiation necrosis group using biopsy results as the gold standard in all but three patients. Mean apparent diffusion coefficient values, mean parallel eigenvalues, and mean perpendicular eigenvalues in the contrast-enhancing lesion were significantly lower, and relative cerebral blood volume was significantly higher for the recurrent neoplasm group compared to the radiation necrosis group (P < 0.01, P = 0.03, P < 0.01, and P < 0.01, respectively). CONCLUSIONS: The combined assessment of DTI and DSC MR perfusion properties of new contrast-enhancing lesions is helpful in distinguishing recurrent neoplasm from radiation necrosis in patients with a history of brain neoplasm previously treated with radiotherapy with or without surgery and chemotherapy.

Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy.

Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a nondestructive, label-free optical method, to reveal glioma infiltration in animal models. We show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ = 0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density, and protein/lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density, and protein/lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery.

Lateral inhibition of Notch signaling in neoplastic cells.

During normal development, heterogeneous expression of Notch ligands can result in pathway suppression in the signal-sending cell, a process known as lateral inhibition. It is unclear if an analogous phenomenon occurs in malignant cells. We observed significant induction of Notch ligands in glioblastoma neurospheres and pancreatic carcinoma cells cultured in low oxygen, suggesting that this phenomenon could occur around hypoxic regions. To model lateral inhibition in these tumors, the ligand Jagged1 was overexpressed in glioblastoma and pancreatic carcinoma cells, resulting in overall induction of pathway targets. However, when ligand high and ligand low cells from a single line were co-cultured and then separated, we noted suppression of Notch pathway targets in the former and induction in the latter, suggesting that neoplastic lateral inhibition can occur. We also found that repression of Notch pathway targets in signal-sending cells may occur through the activity of a Notch ligand intracellular domain, which translocates into the nucleus. Understanding how this neoplastic lateral inhibition process functions in cancer cells may be important in targeting ligand driven Notch signaling in solid tumors.

Tenascin-C: a novel candidate marker for cancer stem cells in glioblastoma identified by tissue microarrays.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, with dismal survival outcomes. Recently, cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here, we have investigated the expression of TNC in tissue microarrays including 17 GBMs, 18 WHO grade III astrocytomas, 15 WHO grade II astrocytomas, 4 WHO grade I astrocytomas, and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas, whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133(+)/TNC(+) and CD133(-)/TNC(+) cell populations is much higher than that of the CD133(+)/TNC(-) and CD133(-)/TNC(-) populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs, where the TNC(+) population is identified as a CSC population overlapping with part of the CD133(-) cell population.

Silencing BMI1 eliminates tumor formation of pediatric glioma CD133+ cells not by affecting known targets but by down-regulating a novel set of core genes.

Clinical outcome of children with malignant glioma remains dismal. Here, we examined the role of over-expressed BMI1, a regulator of stem cell self-renewal, in sustaining tumor formation in pediatric glioma stem cells. Our investigation revealed BMI1 over-expression in 29 of 54 (53.7%) pediatric gliomas, 8 of 8 (100%) patient derived orthotopic xenograft (PDOX) mouse models, and in both CD133+ and CD133- glioma cells. We demonstrated that lentiviral-shRNA mediated silencing of suppressed cell proliferation in vitro in cells derived from 3 independent PDOX models and eliminated tumor-forming capacity of CD133+ and CD133- cells derived from 2 PDOX models in mouse brains. Gene expression profiling showed that most of the molecular targets of BMI1 ablation in CD133+ cells were different from that in CD133- cells. Importantly, we found that silencing BMI1 in CD133+ cells derived from 3 PDOX models did not affect most of the known genes previously associated with the activated BMI1, but modulated a novel set of core genes, including RPS6KA2, ALDH3A2, FMFB, DTL, API5, EIF4G2, KIF5c, LOC650152, C20ORF121, LOC203547, LOC653308, and LOC642489, to mediate the elimination of tumor formation. In summary, we identified the over-expressed BMI1 as a promising therapeutic target for glioma stem cells, and suggest that the signaling pathways associated with activated BMI1 in promoting tumor growth may be different from those induced by silencing BMI1 in blocking tumor formation. These findings highlighted the importance of careful re-analysis of the affected genes following the inhibition of abnormally activated oncogenic pathways to identify determinants that can potentially predict therapeutic efficacy.

Immunohistochemical staining, laser capture microdissection, and filter-aided sample preparation-assisted proteomic analysis of target cell populations within tissue samples.

An important problem involves isolating subpopulations of cells defined by protein markers in clinical tissue samples for proteomic studies. We describe a method termed Immunohistochemical staining, laser capture microdissection (LCM) and filter-aided sample preparation (FASP)-Assisted Proteomic analysis of Target cell populations within tissue samples (ILFAPT). The principle of ILFAPT is that a target cell population expressing a protein of interest can be lit up by immunohistochemical staining and isolated from tissue sections using LCM for FASP and proteomic analysis. Using this method, we isolated a small population of CD90(+) stem-like cells from glioblastoma multiforme tissue sections and identified 674 high-confidence (false discovery rate < 0.01) proteins from 32 nL of CD90(+) cells by LC-MS/MS using an Orbitrap Elite mass spectrometer. We further quantified the relative abundance of proteins identified from equal volumes of LCM-captured CD90(+) and CD90(-) cells, where 109 differentially expressed proteins were identified. The major group of these differentially expressed proteins was relevant to cell adhesion and cellular movement. This ILFAPT method has demonstrated the ability to provide in-depth proteome analysis of a very small specific cell population within tissues. It can be broadly applied to the study of target cell populations within clinical specimens.

Restoration of the orbital aesthetic subunit with the thoracodorsal artery system of flaps in patients undergoing radiation therapy.

Objectives To demonstrate the advantages of the thoracodorsal artery scapular tip autogenous transplant (Tdast) for patients requiring restoration of the orbital aesthetic subunit. Design Prospective case series. Setting Tertiary center. Participants Ten patients (M:F,6:4) with a mean age of 56 years (range, 21 to 78 years) underwent restoration of the orbital aesthetic subunit and radiation therapy between 2001 and 2008. Main Outcome Measures The two reconstructive advantages of the thoracodorsal artery system of flaps for orbital reconstruction are a long pedicle and the suitability of the scapula tip to meet the three-dimensional requirements of the orbit. Patients were assessed 1 year or more after treatment for cosmetic outcome, work status, and socialization. Results Eight of 10 patients benefited from the three-dimensional nature of the scapula tip bone and 7 of 10 avoided vein grafting. Four of five evaluable patients reported "frequently" socializing outside their home. Four of five evaluable patients working before undergoing their treatment were able to return to work posttreatment. Seven of nine patients with postoperative photographs had minimal or no facial contour deformity. Conclusions The Tdast can restore orbital contour without osteotomy, and the thoracodorsal artery system of flaps has a long vascular pedicle that reduces vein grafting. Patients have an acceptable cosmetic result and return to preoperative work status and socialization.