Kevin Yagle

Regional Hypoxia in Glioblastoma Multiforme Quantified with [18F]Fluoromisonidazole Positron Emission Tomography before Radiotherapy: Correlation with Time to Progression and Survival

Purpose: Hypoxia is associated with resistance to radiotherapy and chemotherapy and activates transcription factors that support cell survival and migration. We measured the volume of hypoxic tumor and the maximum level of hypoxia in glioblastoma multiforme before radiotherapy with [18F]fluoromisonidazole positron emission tomography to assess their impact on time to progression (TTP) or survival. Experimental Design: Twenty-two patients were studied before biopsy or between resection and starting radiotherapy. Each had a 20-minute emission scan 2 hours after i.v. injection of 7 mCi of [18F]fluoromisonidazole. Venous blood samples taken during imaging were used to create tissue to blood concentration (T/B) ratios. The volume of tumor with T/B values above 1.2 defined the hypoxic volume (HV). Maximum T/B values (T/Bmax) were determined from the pixel with the highest uptake. Results: Kaplan-Meier plots showed shorter TTP and survival in patients whose tumors contained HVs or tumor T/Bmax ratios greater than the median (P ≤ 0.001). In univariate analyses, greater HV or tumor T/Bmax were associated with shorter TTP or survival (P < 0.002). Multivariate analyses for survival and TTP against the covariates HV (or T/Bmax), magnetic resonance imaging (MRI) T1Gd volume, age, and Karnovsky performance score reached significance only for HV (or T/Bmax; P < 0.03). Conclusions: The volume and intensity of hypoxia in glioblastoma multiforme before radiotherapy are strongly associated with poorer TTP and survival. This type of imaging could be integrated into new treatment strategies to target hypoxia more aggressively in glioblastoma multiforme and could be applied to assess the treatment outcomes.

Quantitative Imaging of Estrogen Receptor Expression in Breast Cancer with PET and 18F-Fluoroestradiol

The PET compound 18F-fluoroestradiol (18F-FES) has been developed and tested as an agent for the imaging of estrogen receptor (ER) expression in vivo. 18F-FES uptake has been shown to correlate with ER expression assayed in vitro by radioligand binding; however, immunohistochemistry (IHC) rather than radioligand binding is used most often to measure ER expression in clinical practice. We therefore compared 18F-FES uptake with ER expression assayed in vitro by IHC with both qualitative and semiquantitative measures. Methods: Seventeen patients with primary or metastatic breast cancer were studied with dynamic 18F-FES PET; cancer tissue samples, collected close to the time of imaging, were assayed for ER expression by IHC. For each tumor, partial-volume-corrected measures of 18F-FES uptake were compared with ER expression measured by 3 different ER scoring methods: qualitative scoring (0–3+), the Allred score (0–10), and a computerized IHC index. Results: There was excellent agreement (r = 0.99) between observers using IHC as well as the different methods of measuring ER content (P < 0.001). ER-negative tumors had 18F-FES partial-volume-corrected standardized uptake values of less than 1.0, whereas ER-positive tumors had values above 1.1. Correlation coefficients for the different measures of ER content and the different measures of 18F-FES uptake ranged from 0.57 to 0.73, with the best correlation being between the computerized IHC index and 18F-FES partial-volume-corrected standardized uptake values. Conclusion: Our results showed good agreement between 18F-FES PET and ER expression measured by IHC. 18F-FES imaging may be a useful tool for aiding in the assessment of ER status, especially in patients with multiple tumors or for tumors that are difficult to biopsy.

Site-Specific Labeling of Annexin V with F-18 for Apoptosis Imaging

Annexin V is useful in detecting apoptotic cells by binding to phosphatidylserine (PS) that is exposed on the outer surface of the cell membrane during apoptosis. In this study, we examined the labeling of annexin V-128, a mutated form of annexin V that has a single cysteine residue at the NH2 terminus, with the thiol-selective reagent 18F-labeling agent N-[4-[(4-[18F]fluorobenzylidene)aminooxy]butyl]maleimide ([18F]FBABM). We also examined the cell binding affinity of the 18F-labeled annexin V-128 ([18F]FAN-128). [18F]FBABM was synthesized in two-step, one-pot method modified from literature procedure. (Toyokuni et al., Bioconjugate Chem. 2003, 14, 1253−1259). The average yield of [18F]FBABM was 23 ± 4% (n = 4, decay-corrected) and the specific activity was ∼6000 Ci/mmol. The total synthesis time was ∼92 min. The critical improvement of this study was identifying and then developing a purification method to remove an impurity N-[4-[(4-dimethylaminobenzylidene)aminooxy]butyl]maleimide 4, whose presence dramatically decreased the yield of protein labeling. Conjugation of [18F]FBABM with the thiol-containing annexin V-128 gave [18F]FAN-128 in 37 ± 9% yield (n = 4, decay corrected). Erythrocyte binding assay of [18F]FAN-128 showed that this modification of annexin V-128 did not compromise its membrane binding affinity. Thus, an in vivo investigation of [18F]FAN-128 as an apoptosis imaging agent is warranted.

Activation of mitogen-activated protein kinase by muscarinic receptors in astroglial cells: Role in DNA synthesis and effect of ethanol

Mitogen‐activated protein kinase (MAPK) can be phosphorylated by mitogens binding to G‐protein‐coupled receptors and is considered a major pathway involved in cell proliferation. In this study, we report on the activation of MAPK by muscarinic acetylcholine receptors in astroglial cells, namely the 1321N1 human astrocytoma cell line, primary rat cortical astrocytes, and fetal human astrocytes. Carbachol caused a rapid and transient phorphorylation of MAPK (ERK1/2) in all cell types, with an increase in MAPK activity, without changing the levels of MAPK proteins. Human astrocytoma cells were used to characterize the effect of carbachol on MAPK. Experiments with M2‐ and M3‐receptor subtype‐selective antagonists, and with pertussis toxin, indicated that the M3 subtype is responsible for activating MAPK in glial cells. Pretreatment of cells with the protein kinase C (PKC) inhibitor bisindolylmaleimide I, or downregulation of PKC by 24‐h treatment with the phorbol ester TPA inhibited carbachol‐induced MAPK activation. Additional experiments with PKC α‐ or PKC ϵ‐specific compounds indicated that the ϵ isozyme of PKC is primarily involved in MAPK activation by carbachol. Chelation of calcium also inhibited MAPK activation by carbachol. Two MEK (MAPK kinase) inhibitors inhibited carbachol‐induced DNA synthesis but only at concentrations that exceeded those sufficient to block carbachol‐induced MAPK activation. Ethanol (≤200 mM) had no effect on MAPK when present alone and did not affect carbachol‐induced MAPK activation under various experimental conditions, although it inhibits carbachol‐induced DNA synthesis at low concentrations (10–100 mM). These results suggest that activation of MAPK by carbachol may be necessary but not sufficient for its mitogenic effect in astroglial cells, and that does not represent a target for ethanol‐induced inhibition of DNA synthesis elicited by muscarinic receptors. GLIA 35:111–120, 2001.

A robust automated measure of average antibody staining in immunohistochemistry images

Identifying and scoring cancer markers plays a key role in oncology, helping to characterize the tumor and predict the clinical course of the disease. The current method for scoring immunohistochemistry (IHC) slides is labor intensive and has inherent issues of quantitation. Although multiple attempts have been made to automate IHC scoring in the past decade, a major limitation in these efforts has been the setting of the threshold for positive staining. In this report, we propose the use of an averaged threshold measure (ATM) score that allows for automatic threshold setting. The ATM is a single multiplicative measure that includes both the proportion and intensity scores. It can be readily automated to allow for large-scale processing, and it is applicable in situations in which individual cells are hard to distinguish. The ATM scoring method was validated by applying it to simulated images, to a sequence of images from the same tumor, and to tumors from different patient biopsies that showed a broad range of staining patterns. Comparison between the ATM score and manual scoring by an expert pathologist showed that both methods resulted in essentially identical scores when applied to these patient biopsies. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Invasion and proliferation kinetics in enhancing gliomas predict IDH1 mutation status

BACKGROUND Glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than gliomas with wild-type IDH1. METHODS Here we compare the IDH1 mutational status in 172 contrast-enhancing glioma patients with the invasion profile generated by a patient-specific mathematical model we developed based on MR imaging. RESULTS We show that IDH1-mutated contrast-enhancing gliomas were relatively more invasive than wild-type IDH1 for all 172 contrast-enhancing gliomas as well as the subset of 158 histologically confirmed glioblastomas. The appearance of this relatively increased, model-predicted invasive profile appears to be determined more by a lower model-predicted net proliferation rate rather than an increased model-predicted dispersal rate of the glioma cells. Receiver operator curve analysis of the model-predicted MRI-based invasion profile revealed an area under the curve of 0.91, indicative of a predictive relationship. The robustness of this relationship was tested by cross-validation analysis of the invasion profile as a predictive metric for IDH1 status. CONCLUSIONS The strong correlation between IDH1 mutation status and the MRI-based invasion profile suggests that use of our tumor growth model may lead to noninvasive clinical detection of IDH1 mutation status and thus lead to better treatment planning, particularly prior to surgical resection, for contrast-enhancing gliomas.

Modulation of DNA synthesis by muscarinic cholinergic receptors

Abstract Acetylcholine muscarinic receptors are a family of five G-protein—coupled receptors widely distributed in the central nervous system and in peripheral organs. Activation of certain subtypes of muscarinic receptors (M1, M3, M5) has been found to modulate DNA synthesis in a number of cell types. In several cell types acetylcholine, by activating endogenous or transfected muscarinic receptors, can indeed elicit cell proliferation. In other cell types, however, or under different experimental conditions, activation of muscarinic receptors has no effect, or inhibits DNA synthesis. A large number of intracellular pathways are being investigated to define the mechanisms involved in these effects of muscarinic receptors; these include among others, phospholipase D, protein kinases C and mitogen-activated-protein kinases. The ability of acetylcholine to modulate DNA synthesis through muscarinic receptors may be relevant in the context of brain development and neoplastic growth.

Contrasting brain patterns of writing-related DTI parameters, fMRI connectivity, and DTI-fMRI connectivity correlations in children with and without dysgraphia or dyslexia.

Based on comprehensive testing and educational history, children in grades 4–9 (on average 12 years) were diagnosed with dysgraphia (persisting handwriting impairment) or dyslexia (persisting word spelling/reading impairment) or as typical writers and readers (controls). The dysgraphia group (n = 14) and dyslexia group (n = 17) were each compared to the control group (n = 9) and to each other in separate analyses. Four brain region seed points (left occipital temporal gyrus, supramarginal gyrus, precuneus, and inferior frontal gyrus) were used in these analyses which were shown in a metaanalysis to be related to written word production on four indicators of white matter integrity and fMRI functional connectivity for four tasks (self-guided mind wandering during resting state, writing letter that follows a visually displayed letter in alphabet, writing missing letter to create a correctly spelled real word, and planning for composing after scanning on topic specified by researcher). For those DTI indicators on which the dysgraphic group or dyslexic group differed from the control group (fractional anisotropy, relative anisotropy, axial diffusivity but not radial diffusivity), correlations were computed between the DTI parameter and fMRI functional connectivity for the two writing tasks (alphabet and spelling) by seed points. Analyses, controlled for multiple comparisons, showed that (a) the control group exhibited more white matter integrity than either the dysgraphic or dyslexic group; (b) the dysgraphic and dyslexic groups showed more functional connectivity than the control group but differed in patterns of functional connectivity for task and seed point; and (c) the dysgraphic and dyslexic groups showed different patterns of significant DTI–fMRI connectivity correlations for specific seed points and written language tasks. Thus, dysgraphia and dyslexia differ in white matter integrity, fMRI functional connectivity, and white matter–gray matter correlations. Of clinical relevance, brain differences were observed in dysgraphia and dyslexia on written language tasks yoked to their defining behavioral impairments in handwriting and/or in word spelling and on the cognitive mind wandering rest condition and composition planning.

Efficacy of cabazitaxel in mouse models of pediatric brain tumors.

BACKGROUND There is an unmet need in the treatment of pediatric brain tumors for chemotherapy that is efficacious, avoids damage to the developing brain, and crosses the blood-brain barrier. These experiments evaluated the efficacy of cabazitaxel in mouse models of pediatric brain tumors. METHODS The antitumor activity of cabazitaxel and docetaxel were compared in flank and orthotopic xenograft models of patient-derived atypical teratoid rhabdoid tumor (ATRT), medulloblastoma, and central nervous system primitive neuroectodermal tumor (CNS-PNET). Efficacy of cabazitaxel and docetaxel were also assessed in the Smo/Smo spontaneous mouse medulloblastoma tumor model. RESULTS This study observed significant tumor growth inhibition in pediatric patient-derived flank xenograft tumor models of ATRT, medulloblastoma, and CNS-PNET after treatment with either cabazitaxel or docetaxel. Cabazitaxel, but not docetaxel, treatment resulted in sustained tumor growth inhibition in the ATRT and medulloblastoma flank xenograft models. Patient-derived orthotopic xenograft models of ATRT, medulloblastoma, and CNS-PNET showed significantly improved survival with treatment of cabazitaxel. CONCLUSION These data support further testing of cabazitaxel as a therapy for treating human pediatric brain tumors.

Brain’s functional network clustering coefficient changes in response to instruction (RTI) in students with and without reading disabilities: Multi-leveled reading brain’s RTI

Abstract In students in grades 4 to 9 (22 males, 20 females), two reading disability groups—dyslexia (n = 20) or oral and written language learning disability (OWL LD) (n = 6)—were compared to each other and two kinds of control groups—typical readers (n = 6) or dysgraphia (n = 10) on word reading/spelling skills and fMRI imaging before and after completing 18 computerized reading lessons. Mixed ANOVAs showed significant time effects on repeated measures within participants and between groups effects on three behavioral markers of reading disabilities—word reading/spelling: All groups improved on the three behavioral measures, but those without disabilities remained higher than those with reading disabilities . On fMRI reading tasks, analyzed for graph theory derived clustering coefficients within a neural network involved in cognitive control functions, on a word level task the time x group interaction was significant in right medial cingulate; on a syntax level task the time x group interaction was significant in left superior frontal and left inferior frontal gyri; and on a multi-sentence text level task the time x group interaction was significant in right middle frontal gyrus. Three white matter-gray matter correlations became significant only after reading instruction: axial diffusivity in left superior frontal region with right inferior frontal gyrus during word reading judgments; mean diffusivity in left superior corona radiata with left middle frontal gyrus during sentence reading judgments; and mean diffusivity in left anterior corona radiata with right middle frontal gyrus during multi-sentence reading judgments. Significance of results for behavioral and brain response to reading instruction (RTI) is discussed.