Gordon Li

New tools for studying microglia in the mouse and human CNS

Significance Microglia are the tissue resident macrophages of the brain and spinal cord, implicated in important developmental, homeostatic, and disease processes, although our understanding of their roles is complicated by an inability to distinguish microglia from related cell types. Although they share many features with other macrophages, microglia have distinct developmental origins and functions. Here we validate a stable and robustly expressed microglial marker for both mouse and human, transmembrane protein 119 (Tmem119). We use custom-made antibodies against Tmem119 to perform deep RNA sequencing of developing microglia, and demonstrate that microglia mature by the second postnatal week in mice. The antibodies, cell isolation methods, and RNAseq profiles presented here will greatly facilitate our understanding of microglial function in health and disease. The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma

Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.

Effects of age and comorbidities on complication rates and adverse outcomes after lumbar laminectomy in elderly patients.

Study Design. This is a retrospective cohort study using the National Inpatient Sample database. Objective. The objective is to report mortality and complications after lumbar laminectomy in the elderly. Summary of Background Data.— As the population continues to age in the United States, it is important to consider the surgical complications and outcomes in the elderly. A review of the literature reveals controversy over the safety of lumbar laminectomy in the elderly and disagreement over estimates of risks in this population. Methods. Outcome measures were abstracted from the National Inpatient Sample. Multivariate analysis was performed to analyze the effect of patient and hospital characteristics on outcome measures. Results. A total of 471,215 patients underwent lumbar laminectomy without fusion for lumbar stenosis from 1993 to 2002. The in-hospital mortality rate was 0.17%, and the complication rate was 12.17%. Postoperative hemorrhage or hematoma (5.2%) and nonspecific renal complications (2.8%) were the most common complications. Complication and mortality rates increased with age and comorbidities with an 18.9% complication rate and 1.4% mortality rate in patients over the age of 85 with 3 or more comorbidities, 14.7% complication rate and 0.22% mortality rate in patients over 85 with no comorbidities, and only a 6% complication rate and 0.05% mortality rate in patient between 18 and 44 with no comorbidities. Multivariate analysis revealed increased odds of mortality with increasing number of comorbidities and complications in the greater than 85 year age group. Increasing age, number of comorbidities, complication rate, and female sex also increased the odds of discharge to institution other than home. Conclusion. Elderly patients with comorbidities are at a higher risk for complications and adverse outcome after lumbar spine surgery. The effects of age and comorbidities on patient outcomes have been quantified. This information is critical in counseling elderly patients about the risk of surgery in their age group.

Risk of Leptomeningeal Disease in Patients Treated With Stereotactic Radiosurgery Targeting the Postoperative Resection Cavity for Brain Metastases

PURPOSE We sought to determine the risk of leptomeningeal disease (LMD) in patients treated with stereotactic radiosurgery (SRS) targeting the postsurgical resection cavity of a brain metastasis, deferring whole-brain radiation therapy (WBRT) in all patients. METHODS AND MATERIALS We retrospectively reviewed 175 brain metastasis resection cavities in 165 patients treated from 1998 to 2011 with postoperative SRS. The cumulative incidence rates, with death as a competing risk, of LMD, local failure (LF), and distant brain parenchymal failure (DF) were estimated. Variables associated with LMD were evaluated, including LF, DF, posterior fossa location, resection type (en-bloc vs piecemeal or unknown), and histology (lung, colon, breast, melanoma, gynecologic, other). RESULTS With a median follow-up of 12 months (range, 1-157 months), median overall survival was 17 months. Twenty-one of 165 patients (13%) developed LMD at a median of 5 months (range, 2-33 months) following SRS. The 1-year cumulative incidence rates, with death as a competing risk, were 10% (95% confidence interval [CI], 6%-15%) for developing LF, 54% (95% CI, 46%-61%) for DF, and 11% (95% CI, 7%-17%) for LMD. On univariate analysis, only breast cancer histology (hazard ratio, 2.96) was associated with an increased risk of LMD. The 1-year cumulative incidence of LMD was 24% (95% CI, 9%-41%) for breast cancer compared to 9% (95% CI, 5%-14%) for non-breast histology (P=.004). CONCLUSIONS In patients treated with SRS targeting the postoperative cavity following resection, those with breast cancer histology were at higher risk of LMD. It is unknown whether the inclusion of whole-brain irradiation or novel strategies such as preresection SRS would improve this risk or if the rate of LMD is inherently higher with breast histology.

Targeting a Glioblastoma Cancer Stem-Cell Population Defined by EGF Receptor Variant III

The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII(+)/CD133(+) defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII(+) cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII(-) cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII(+)/CD133(+) coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII(+)/CD133(+) population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population.

Radiosurgery of Glomus Jugulare Tumors: A Meta-analysis

PURPOSE During the past two decades, radiosurgery has arisen as a promising approach to the management of glomus jugulare. In the present study, we report on a systematic review and meta-analysis of the available published data on the radiosurgical management of glomus jugulare tumors. METHODS AND MATERIALS To identify eligible studies, systematic searches of all glomus jugulare tumors treated with radiosurgery were conducted in major scientific publication databases. The data search yielded 19 studies, which were included in the meta-analysis. The data from 335 glomus jugulare patients were extracted. The fixed effects pooled proportions were calculated from the data when Cochranes statistic was statistically insignificant and the inconsistency among studies was <25%. Bias was assessed using the Egger funnel plot test. RESULTS Across all studies, 97% of patients achieved tumor control, and 95% of patients achieved clinical control. Eight studies reported a mean or median follow-up time of >36 months. In these studies, 95% of patients achieved clinical control and 96% achieved tumor control. The gamma knife, linear accelerator, and CyberKnife technologies all exhibited high rates of tumor and clinical control. CONCLUSIONS The present study reports the results of a meta-analysis for the radiosurgical management of glomus jugulare. Because of its high effectiveness, we suggest considering radiosurgery for the primary management of glomus jugulare tumors.

Immune Evasion Strategies of Glioblastoma

Glioblastoma (GBM) is the most devastating brain tumor, with associated poor prognosis. Despite advances in surgery and chemoradiation, the survival of afflicted patients has not improved significantly in the past three decades. Immunotherapy has been heralded as a promising approach in treatment of various cancers; however, the immune privileged environment of the brain usually curbs the optimal expected response in central nervous system malignancies. In addition, GBM cells create an immunosuppressive microenvironment and employ various methods to escape immune surveillance. The purpose of this review is to highlight the strategies by which GBM cells evade the host immune system. Further understanding of these strategies and the biology of this tumor will pave the way for developing novel immunotherapeutic approaches for treatment of GBM.

Development of an EGFRvIII specific recombinant antibody

BackgroundEGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community.ResultsIn this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fvs linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as determined by enzyme-linked immunosorbent assay (ELISA).ConclusionThis recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.

Targeting EGF receptor variant III: tumor-specific peptide vaccination for malignant gliomas

Glioblastoma multiforme (GBM) is the most common and deadly of the human brain cancers. The EGF receptor is often amplified in GBM and provides a potential therapeutic target. However, targeting the normal receptor is complicated by its nearly ubiquitous and high level of expression in certain tissues. A naturally occurring deletion mutant of the EGF receptor, EGFRvIII, is a constitutively active variant originally identified in a high percentage of brain cancer cases, and more importantly is rarely found in normal tissue. A peptide vaccine, rindopepimut (CDX-110, Celldex Therapeutics), is directed against the novel exon 1–8 junction produced by the EGFRvIII deletion, and it has shown high efficacy in preclinical models. Recent Phase II clinical trials in patients with newly diagnosed GBM have shown EGFRvIII-specific immune responses and significantly increased time to progression and overall survival in those receiving vaccine therapy, as compared with published results for standard of care. Rindopepimut therefore represents a very promising therapy for patients with GBM.

EGF receptor variant III as a target antigen for tumor immunotherapy

The EGF receptor (EGFR) is the first tyrosine kinase receptor ever cloned and remains at the forefront of targeted therapies against cancer. Currently, there are four US FDA-approved drugs and several more in Phase III studies that target the EGFR. These drugs, while resulting in some dramatic remissions, have not resulted in strong nor consistent improvements in survival. EGFR variant III (EGFRvIII) is the most common variant of the EGFR and is present in many different cancer types but not in normal tissue. It results from the fusion of exon 1 to exon 8 of the EGFR gene, which results in a novel glycine at the junction. This mutant receptor is constitutively active in these tumors and can lead directly to cancer phenotypes due to its oncogenic properties. EGFRvIII is an attractive target antigen for cancer immunotherapy because it is not expressed in normal tissue and because cells producing EGFRvIII have an enhanced capacity for dysregulated growth, survival, invasion and angiogenesis. In this review, we will discuss preclinical and clinical data from studies using EGFRvIII as the target antigen for immunotherapy, with a focus on the potential for greatly improved survival for patients diagnosed with glioblastoma multiforme.