Adoptive cell therapy for high grade gliomas using simultaneous temozolomide and intracranial mgmt-modified γδ t cells following standard post-resection chemotherapy and radiotherapy: current strategy and future directions.

Cellular therapies, including chimeric antigen receptor T cell therapies (CAR-T), while generally successful in hematologic malignancies, face substantial challenges against solid tumors such as glioblastoma (GBM) due to rapid growth, antigen heterogeneity, and inadequate depth of response to cytoreductive and immune therapies, We have previously shown that GBM constitutively express stress associated NKG2D ligands (NKG2DL) recognized by gamma delta (γδ) T cells, a minor lymphocyte subset that innately recognize target molecules via the γδ T cell receptor (TCR), NKG2D, and multiple other mechanisms. Given that NKG2DL expression is often insufficient on GBM cells to elicit a meaningful response to γδ T cell immunotherapy, we then demonstrated that NKG2DL expression can be transiently upregulated by activation of the DNA damage response (DDR) pathway using alkylating agents such as Temozolomide (TMZ). TMZ, however, is also toxic to γδ T cells. Using a p140K/MGMT lentivector, which confers resistance to TMZ by expression of O(6)-methylguanine-DNA-methyltransferase (MGMT), we genetically engineered γδ T cells that maintain full effector function in the presence of therapeutic doses of TMZ. We then validated a therapeutic system that we termed Drug Resistance Immunotherapy (DRI) that combines a standard regimen of TMZ concomitantly with simultaneous intracranial infusion of TMZ-resistant γδ T cells in a first-in-human Phase I clinical trial (NCT04165941). This manuscript will discuss DRI as a rational therapeutic approach to newly diagnosed GBM and the importance of repeated administration of DRI in combination with the standard-of-care Stupp regimen in patients with stable minimal residual disease.

Phase I trial of sorafenib in patients with recurrent or progressive malignant glioma.

Sorafenib is an inhibitor of multiple kinases that has demonstrated antiproliferative and antiangiogenic activity in a number of in vitro and in vivo model systems. A phase I study was conducted to determine the maximum tolerated dose (MTD) of sorafenib in patients with recurrent malignant glioma. Sorafenib was given orally, twice a day (BID), continuously in 28-day cycles. The dose was escalated in 2 groups of patients stratified by use of enzyme-inducing antiseizure drugs (± EIASDs). Dose-limiting toxicity (DLT) was defined as any grades 3-4 nonhematological toxicity, grade 4 hematological toxicity, and febrile neutropenia. The number of evaluable patients enrolled in the +EIASD and -EIASD arms were 23 and 24, respectively. DLTs were predominantly dermatological and gastrointestinal effects, as observed in previous clinical trials of sorafenib. The MTD was 600 mg BID for patients receiving EIASDs and 800 mg BID for those who were not. The plasma pharmacokinetics of sorafenib were not significantly affected by the concurrent administration of EIASDs. The MTD of sorafenib given orally BID on a continuous basis was established as 600 mg BID in patients with malignant glioma who were concurrently receiving EIASDs and 800 mg BID in those who were not. Further evaluation is warranted of sorafenib at the recommended MTD against recurrent or progressive malignant glioma in combination with other molecularly targeted drugs or in the newly diagnosed setting concurrent with chemoradiation.

Cancer susceptibility variants and the risk of adult glioma in a US case-control study.

Malignant gliomas are the most common and deadly brain tumors. Although their etiology remains elusive, recent studies have narrowed the search for genetic loci that influence risk. We examined variants implicated in recent cancer genome-wide association studies (GWAS) for associations with glioma risk in a US case-control study. Cases were identified from neurosurgical and neuro-oncology clinics at major academic centers in the Southeastern US. Controls were identified from the community or were friends or other associates of cases. We examined a total of 191 susceptibility variants in genes identified in published cancer GWAS including glioma. A total of 639 glioma cases and 649 controls, all Caucasian, were included in analysis. Cases were enrolled a median of 1 month following diagnosis. Among glioma GWAS-identified variants, we detected associations in CDKN2B, RTEL1, TERT and PHLDB1, whereas we did not find overall associations for CCDC26. Results showed clear heterogeneity according to histologic subtypes of glioma, with TERT and RTEL variants a feature of astrocytic tumors and glioblastoma (GBM), CCDC26 and PHLDB1 variants a feature of astrocytic and oligodendroglial tumors, and CDKN2B variants most prominent in GBM. No examined variant in other cancer GWAS was found to be related to risk after adjustment for multiple comparisons. These results suggest that GWAS-identified SNPs in glioma mark different molecular etiologies in glioma. Stratification by broad histological subgroups may shed light on molecular mechanisms and assist in the discovery of novel loci in future studies of genetic susceptibility variants in glioma.

Amyotrophic lateral sclerosis-linked mutant SOD1 sequesters Hu antigen R (HuR) and TIA-1-related protein (TIAR): implications for impaired post-transcriptional regulation of vascular endothelial growth factor.

Down-regulation of vascular endothelial growth factor (VEGF) in the mouse leads to progressive and selective degeneration of motor neurons similar to amyotrophic lateral sclerosis (ALS). In mice expressing ALS-associated mutant superoxide dismutase 1 (SOD1), VEGF mRNA expression in the spinal cord declines significantly prior to the onset of clinical manifestations. In vitro models suggest that dysregulation of VEGF mRNA stability contributes to that decline. Here, we show that the major RNA stabilizer, Hu Antigen R (HuR), and TIA-1-related protein (TIAR) colocalize with mutant SOD1 in mouse spinal cord extracts and cultured glioma cells. The colocalization was markedly reduced or abolished by RNase treatment. Immunoanalysis of transfected cells indicated that colocalization occurred in insoluble aggregates and inclusions. RNA immunoprecipitation showed a significant loss of VEGF mRNA binding to HuR and TIAR in mutant SOD1 cells, and there was marked depletion of HuR from polysomes. Ectopic expression of HuR in mutant SOD1 cells more than doubled the mRNA half-life of VEGF and significantly increased expression to that of wild-type SOD1 control. Cellular effects produced by mutant SOD1, including impaired mitochondrial function and oxidative stress-induced apoptosis, were reversed by HuR in a gene dose-dependent pattern. In summary, our findings indicate that mutant SOD1 impairs post-transcriptional regulation by sequestering key regulatory RNA-binding proteins. The rescue effect of HuR suggests that this impairment, whether related to VEGF or other potential mRNA targets, contributes to cytotoxicity in ALS.

Isolated central nervous system posttransplant lymphoproliferative disorder treated with high-dose intravenous methotrexate.

Posttransplant lymphoproliferative disorder (PTLD) is an uncommon neoplastic complication of kidney transplantation, affecting about 1% of recipients. It is generally associated with Epstein-Barr virus (EBV) infection of B-lineage lymphocytes. Central nervous system (CNS) involvement is rare. There is little clinical experience with treatment of CNS PTLD due to the relative rarity of the disease other than reduction or withdrawal of immunosuppression, but it is usually fatal. We describe six patients with renal allografts and histologically proven isolated CNS PTLD. Tissue analysis from the biopsy specimens was positive for EBV material in five of the six patients. All six patients were treated with high-dose intravenous methotrexate (HD IV MTX). Methotrexate was initiated at 8 g/m2, with later adjustments for creatinine clearance. With MTX therapy, four patients have had a sustained complete response, and two had progressive disease and were referred for radiation therapy. This finding suggests a subgroup of patients may benefit from MTX but our case series is inadequate to describe overall efficacy. No unexpected toxicities were encountered in 37 courses of treatment. HD IV MTX chemotherapy should be considered as an alternative for treatment of CNS PTLD.

Distress and quality of life in primary high-grade brain tumor patients.

GOALS OF WORK: We report on the routine use of the NCCN Distress Thermometer and the Functional Assessment of Cancer Therapy-Brain (FACT-Br) to assess patient distress and quality of life in GBM patients. The purpose of this study was to examine the relationship between patient quality of life and distress. MATERIALS AND METHODS: Data from 50 GBM patients presenting to a neuro-oncology clinic were evaluated. Descriptive statistics and correlations between the distress score and the FACT-Br subscale scores were generated. MAIN RESULTS: The mean distress score was 2.15 (std 2.66), and 28.9% of brain tumor patients identified a distress score of 4 or above. The mean FACT-Br total was 127.34 (std 21.29), with patients scoring lowest in the EWB (18.95 std 4.4) and FWB (15.06 std 6.80) subscales. No differences between demographic groups were identified with regard to distress or quality of life. Statistically significant correlations were identified between the distress score and the SWB (R = -0.46, P = 0.001) and EWB (R = -0.56, P = 0.001) subscales of the FACT-Br. Fifty percent of participants who did not complete the FACT-Br reported clinically significant distress, but this did not differ significantly from participants who completed it. CONCLUSIONS: Assessment of distress in brain tumor patients provides clinically relevant information and suggests interventions that may support quality of life. Further research is needed to explore the relationship between distress and quality of life. Current approaches to measuring quality of life in brain tumor patients may systematically undersample patients with advanced illness or significant psychosocial distress.

High-resolution longitudinal assessment of flow and permeability in mouse glioma vasculature: Sequential small molecule and SPIO dynamic contrast agent MRI.

The poor prognosis associated with malignant glioma is largely attributable to its invasiveness and robust angiogenesis. Angiogenesis involves host-tumor interaction and requires in vivo evaluation. Despite their versatility, few studies have used mouse glioma models with perfusion MRI approaches, and generally lack longitudinal study design. Using a micro-MRI system (8.5 Tesla), a novel dual bolus-tracking perfusion MRI strategy was implemented. Using the small molecule contrast agent Magnevist, dynamic contrast enhanced MRI was implemented in the intracranial 4C8 mouse glioma model to determine K(trans) and v(e), indices of tumor vascular permeability and cellularity, respectively. Dynamic susceptibility contrast MRI was subsequently implemented to assess both cerebral blood flow and volume, using the macromolecular superparamagnetic iron oxide, Feridex, which circumvented tumor bolus susceptibility curve distortions from first-pass extravasation. The high-resolution parametric maps obtained over 4 weeks, indicated a progression of tumor vascularization, permeability, and decreased cellularity with tumor growth. In conclusion, a comprehensive array of key parameters were reliably quantified in a longitudinal mouse glioma study. The syngeneic 4C8 intracerebral mouse tumor model has excellent characteristics for studies of glioma angiogenesis. This approach provides a useful platform for noninvasive and highly diagnostic longitudinal investigations of anti-angiogenesis strategies in a relevant orthotopic animal model.

Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells.

Cyclooxygenase-2 (COX-2) expression is normally tightly regulated. However, constitutive overexpression plays a key role in colon carcinogenesis. To understand the molecular nature of enhanced COX-2 expression detected in colon cancer, we examined the ability of the AU-rich element-containing (ARE-containing) 3' untranslated region (3'UTR) of COX-2 mRNA to regulate rapid mRNA decay in human colon cancer cells. In tumor cells displaying enhanced growth and tumorigenicity that is correlated with elevated COX-2, vascular endothelial growth factor (VEGF), and IL-8 protein levels, the corresponding mRNAs were transcribed constitutively and turned over slowly. The observed mRNA stabilization is owing to defective recognition of class II-type AREs present within the COX-2, VEGF, and IL-8 3'UTRs; c-myc mRNA, containing a class I ARE decayed rapidly in the same cells. Correlating with cellular defects in mRNA stability, the RNA-binding of trans-acting cellular factors was altered. In particular, we found that the RNA-stability factor HuR binds to the COX-2 ARE, and overexpression of HuR, as detected in tumors, results in elevated expression of COX-2, VEGF, and IL-8. These findings demonstrate the functional significance rapid mRNA decay plays in controlling gene expression and show that dysregulation of these trans-acting factors can lead to overexpression of COX-2 and other angiogenic proteins, as detected in neoplasia.

HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs.

Tumors of the central nervous system (CNS) often have sustained expression of labile genes, including angiogenic growth factors and immunosuppressive cytokines, which promote tumor progression. Stabilization of the RNA transcripts for these genes, such as vascular endothelial growth factor (VEGF), is an important molecular pathway for this up-regulation. HuR, a member of the Elav family of RNA-binding proteins, has been implicated in this pathway through its binding to adenine and uridine (AU)-rich stability elements (ARE) located in the 3' untranslated regions (3'-UTRs) of the mRNA. Whereas three of the Elav family members (Hel-N1, HuC, and HuD) are restricted to young and mature neurons, HuR is more broadly expressed, including proliferating cells of the developing CNS. Because RNA stabilization of labile genes may promote tumor growth, we analyzed and compared the expression pattern of HuR in 35 freshly resected and cultured CNS tumors to determine whether there was any correlation with tumor grade or histological type. We found that HuR mRNA was consistently expressed in all of the tumors, regardless of cell origin or degree of malignancy. Using a novel HuR-specific polyclonal antibody, we found that strong HuR protein expression was limited to high-grade malignancies (glioblastoma multiforme and medulloblastoma). Within the glioblastoma multiforme, prominent HuR expression was also detected in perinecrotic areas in which angiogenic growth factors are up-regulated. To further define its role as a potential RNA stabilizer, we analyzed whether HuR could bind to the stability motifs within the 3'-UTRs of cytokines and growth factors linked to brain tumor progression. We used a novel ELISA-based RNA binding assay and focused on the 3'-UTRs of angiogenic factors VEGF, COX-2, and (interleukin) IL-8 as well as the immunomodulating factors IL-6, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha as potential RNA ligands. Our results indicated overall a very high binding affinity to these RNA targets. A comparison of these ligands revealed a hierarchy of binding affinities with the angiogenic factors, and TGF-beta showing the highest (Kd of 1.8-3.4 nM), and TNF-alpha the lowest (Kd of 18.3 nM). The expression pattern of HuR, coupled with the RNA binding data, strongly suggests a role for this protein in the posttranscriptional regulation of these genes in CNS tumors.

Analysis of the 5' end of the mouse Elavl1 (mHuA) gene reveals a transcriptional regulatory element and evidence for conserved genomic organization.

mHuA (Elavl1) belongs to a highly conserved family of genes encoding RNA-binding proteins and has been linked to cell growth and proliferation through its regulation of mRNA stability. Here, we use an RNase protection assay to demonstrate that the mHuA transcript is relatively abundant in a range of mouse tissues, with the highest levels being found in lung and embryonic stem cells. We then cloned and mapped an 18 kb DNA fragment which encompasses the 5' end of the mHuA gene. The genomic organization in this region is similar to the neural-restricted family members, Hel-N1 (ELAVL2) and mHuD (Elavl4). The first exon is lengthy and untranslated, and the second exon, which includes the methionine start site, ends between the ribonucleoprotein motifs of the first RNA binding domain. Mapping of the mHuA transcript by primer extension demonstrated three potential transcription-initiation sites which were detected consistently among different tissues and cell lines. Analysis of the sequence flanking these sites revealed the presence of transcriptional elements including TATA, CREB, c-ets, and AP1 sites. Transfection analysis of this promoter region using a luciferase-reporter-gene assay indicated strong transcriptional activity both in HeLa and in mouse macrophage (RAW) cells which is consistent with the ubiquitous expression pattern of mHuA. Thus, while the genomic organization of mHuA is similar to the neural-restricted members of the Elav family, the promoter element differs substantially both by sequence analysis and transcriptional activity in non-neural cell types.

Hu antigen specificities of ANNA-I autoantibodies in paraneoplastic neurological disease.

Despite a broad clinical spectrum, paraneoplastic enecephalomyelitis/sensory neuronopathy (PEM/SSN) is characterized by the presence of a common autoantibody, referred to as anti-Hu or type I anti-neuronal nuclear antibody (ANNA-1). The target of these antibodies is a family of four Hu antigens: three (Hel-N1, HuC, HuD) are neural-specific, while the fourth (HuR) is ubiquitous. Here, we have analysed by enzyme-linked immunosorbent assay (ELISA) the immunoreactivity of all four Hu antigens in serum from 75 patients with ANNA-1 autoantibodies and looked for clinical correlations. IgG in all the patients' sera bound to each of the four antigens, and the titers correlated with those of the ANNA-I immunofluorescence assay. Median titers for the neural-specific antigens (range: 56, 892-90,051) were significantly higher than for HuR (36,799). Patients with gastrointestinal dysmotility or subacute sensory neuronopathy had the highest median titers to all four antigens, while patients with sensorineural deafness had the lowest titers. The results indicate a heterogeneous immune response to individual Hu antigens in patients with PEM/SSN, and that the titers to these antigens as a group, rather than individually, correlate with clinical profile. Furthermore, these results suggest that ELISA analysis of a single neural-specific Hu antigen is sufficient for serological screening in PEM/SSN.

HuR, a novel target of anti-Hu antibodies, is expressed in non-neural tissues.

Paraneoplastic encephalomyelitis (PEM) is characterized by a diverse set of clinical signs that are limited to the nervous system. The serologic hallmark of PEM is the presence of circulating autoantibodies, collectively referred to as 'anti-Hu,' which immunoreact specifically with members of the Elav protein family. Until recently, the ELAV antigens were only detected in neurons, thus strongly supporting a role for anti-Hu antibodies in the selective neural tissue injury in PEM. The identification of HuR, however, a new member with a broad, non-neural pattern of RNA expression, raises several fundamental questions regarding PEM. First, why are non-neural tissues spared in PEM? Second, why is PEM predominantly associated with neuroendocrine tumors? To begin addressing these questions, we sought to determine whether the antibody response to HuR differs from the neural-specific counterparts in patients with PEM, and to characterize the protein expression pattern of this novel antigen in peripheral tissues and tumors. Using sera from 11 patients with Hu-positive PEM, we found that the majority of samples (73%) were weakly or non-reactive for recombinant HuR on Western blot, in contrast to consistently strong immunoreactivity with the neural-specific members HuD and Hel-N1. We also demonstrate that HuR is expressed at the protein level in both non-neural tissues and non-neuroendocrine tumors. These findings suggest that immunoreactive differences among Elav family members may contribute to the neural-restrictive pattern of tissue injury in patients with PEM.

Magnetic resonance cisternography in the diagnosis of delayed iatrogenic cerebrospinal fistula: a case report.

A 52-year-old woman presented with a clinical picture consistent with bacterial meningitis 3 years after functional endoscopic sinus surgery. Diagnosis of a cerebrospinal fluid (CSF) fistula was made clinically, and the site of the fistula was confirmed using magnetic resonance cisternography. The utilization of this technique in the diagnosis of CSF disorders is gaining popularity. Its usefulness in the context of other imaging modalities is discussed.

A unique neuronal organization in the cat pretectum revealed by antibodies to the calcium-binding protein calbindin-D 28K.

The pretectum is an important center for visual reflexes. However, the location, boundaries, and connections of individual nuclei of the pretectum are incompletely understood. In cat, the traditionally defined nuclear boundaries have been placed in doubt by recent evidence showing that the retinal input to the pretectum forms four continuous projection zones that do not match the cytoarchitectural boundaries of individual pretectal nuclei defined by previous studies. We now show that antibodies to the calcium-binding protein calbindin-D 28K (CaBP) label clusters of neurons within the pretectum that match the zones of retinal termination. Four obvious cell clusters within the pretectum were labeled by CaBP antisera. Computer three-dimensional reconstruction of these cell clusters revealed that they form four distinct but continuous zones that run the rostrocaudal length of the pretectum in a medial-to-lateral direction. By combining anterograde HRP labeling of retinal terminals with CaBP immunocytochemistry, these CaBP-labeled cell clusters were found virtually to overlap the retinal projection zones. The CaBP-labeled neurons included both multipolar and fusiform morphologies, and most were medium- to large-sized cells. HRP retrograde transport studies showed that many CaBP-labeled neurons in the clusters projected to the LGN, while none projected to the inferior olive (IO). GABA-immunoreactive neurons were also found within the CaBP cell clusters, but these neurons were smaller than most CaBP-labeled neurons, and none were retrogradely labeled following HRP injections into the LGN or IO. Two-color antibody double-labeling experiments did not reveal any GABA neurons within the clusters that colocalized CaBP. In summary, calbindin is a precise marker of neuron clusters that overlap the retinal projection zones in the cat pretectum. Many of these CaBP neurons project to the LGN, and none contain GABA.

Quantitative immunocytochemistry using an image analyzer. I. Hardware evaluation, image processing, and data analysis.

In this review we describe how video-based image analysis systems are used to measure immunocytochemically labeled tissue. The general principles underlying hardware and software procedures are emphasized. First, the characteristics of image analyzers are described, including the densitometric measure, spatial resolution, gray scale resolution, dynamic range, and acquisition and processing speed. The errors produced by these instruments are described and methods for correcting or reducing the errors are discussed. Methods for evaluating image analyzers are also presented, including spatial resolution, photometric transfer function, short- and long-term temporal variability, and measurement error. The procedures used to measure immunocytochemically labeled cells and fibers are then described. Immunoreactive profiles are imaged and enhanced using an edge sharpening operator and then extracted using segmentation, a procedure which captures all labeled profiles above a threshold gray level. Binary operators, including erosion and dilation, are applied to separate objects and to remove artifacts. The software then automatically measures the geometry and optical density of the extracted profiles. The procedures are rapid and efficient methods for measuring simultaneously the position, geometry, and labeling intensity of immunocytochemically labeled tissue, including cells, fibers, and whole fields. A companion paper describes non-biological standards we have developed to estimate antigen concentration from the optical density produced by antibody labeling (Nabors et al., 1988).

Quantitative immunocytochemistry using an image analyzer. II. Concentration standards for transmitter immunocytochemistry.

Image analyzers can measure both the optical density and geometry of immunocytochemically labeled cells and fibers, as reviewed in a companion paper (Mize et al., 1988). In this paper, we report a procedure which allows us to estimate the concentration of a neurotransmitter based upon the optical density of antibody labeling produced by immunocytochemistry. To accomplish this, we developed a standard which binds conjugated neurotransmitters. Several artificial media for the standard were compared, including agar, gelatin, and agar-gelatin. A 3% agar matrix was found to be most suitable because it cut well and was nearly transparent. The agar sections were activated with cyanogen bromide/acetonitrile to promote coupling to the antigen. To test the standard, we used gamma-aminobutyric acid (GABA) conjugated to bovine serum albumin (BSA) as the antigen. The antibody was directed against this conjugate. Activated agar sections were incubated in serial dilutions of the tritium-labeled GABA/BSA conjugate. The radioactivity of some of these sections was measured to estimate the amount of coupled antigen. The remaining sections were incubated in the GABA antibody and processed for immunocytochemistry. The optical density of these sections was measured with an image analyzer. A linear relationship was found between GABA concentration and optical density over a range of at least 0.01 to 1 nmol/mg of agar. These results show that the concentration of bound GABA can be estimated from the optical density of sections labeled by antibody immunocytochemistry. The applicability of this technique to fixed brain tissue is discussed.