Connectomic Basis for Tremor Control in Stereotactic Radiosurgical Thalamotomy.

BACKGROUND AND PURPOSE: Given the increased use of stereotactic radiosurgical thalamotomy and other ablative therapies for tremor, new biomarkers are needed to improve outcomes. Using resting-state fMRI and MR tractography, we hypothesized that a "connectome fingerprint" can predict tremor outcomes and potentially serve as a targeting biomarker for stereotactic radiosurgical thalamotomy. MATERIALS AND METHODS: We evaluated 27 patients who underwent unilateral stereotactic radiosurgical thalamotomy for essential tremor or tremor-predominant Parkinson disease. Percentage postoperative improvement in the contralateral limb Fahn-Tolosa-Marin Clinical Tremor Rating Scale (TRS) was the primary end point. Connectome-style resting-state fMRI and MR tractography were performed before stereotactic radiosurgery. Using the final lesion volume as a seed, "connectivity fingerprints" representing ideal connectivity maps were generated as whole-brain R-maps using a voxelwise nonparametric Spearman correlation. A leave-one-out cross-validation was performed using the generated R-maps. RESULTS: The mean improvement in the contralateral tremor score was 55.1% (SD, 38.9%) at a mean follow-up of 10.0 (SD, 5.0) months. Structural connectivity correlated with contralateral TRS improvement (r = 0.52; P = .006) and explained 27.0% of the variance in outcome. Functional connectivity correlated with contralateral TRS improvement (r = 0.50; P = .008) and explained 25.0% of the variance in outcome. Nodes most correlated with tremor improvement corresponded to areas of known network dysfunction in tremor, including the cerebello-thalamo-cortical pathway and the primary and extrastriate visual cortices. CONCLUSIONS: Stereotactic radiosurgical targets with a distinct connectivity profile predict improvement in tremor after treatment. Such connectomic fingerprints show promise for developing patient-specific biomarkers to guide therapy with stereotactic radiosurgical thalamotomy.

KLF6 depletion promotes NF-κB signaling in glioblastoma.

Dysregulation of the NF-κB transcription factor occurs in many cancer types. Krüppel-like family of transcription factors (KLFs) regulate the expression of genes involved in cell proliferation, differentiation and survival. Here, we report a new mechanism of NF-κB activation in glioblastoma through depletion of the KLF6 tumor suppressor. We show that KLF6 transactivates multiple genes negatively controlling the NF-κB pathway and consequently reduces NF-κB nuclear localization and downregulates NF-κB targets. Reconstitution of KLF6 attenuates their malignant phenotype and induces neural-like differentiation and senescence, consistent with NF-κB pathway inhibition. KLF6 is heterozygously deleted in 74.5% of the analyzed glioblastomas and predicts unfavorable patient prognosis suggesting that haploinsufficiency is a clinically relevant means of evading KLF6-dependent regulation of NF-κB. Together, our study identifies a new mechanism by which KLF6 regulates NF-κB signaling, and how this mechanism is circumvented in glioblastoma through KLF6 loss.

DNA topoisomerase IIalpha expression in optic pathway gliomas of childhood.

DNA topoisomerase IIalpha (Topo IIalpha) is linked to tumour cell growth and chemoresistance. We examined immunohistochemically Topo IIalpha expression levels in a series of 36 consecutive paediatric optic pathway glioma (OPG) patients. Topo IIalpha labelling index (LI) ranged from 0.0 to 11.6 and was significantly associated with patient age, with higher levels of Topo IIalpha in children < or = 3 years (P=0.031). Topo IIalpha expression did not correlate with patient survival. Topo IIalpha LI was not significantly increased in specimens of repeat surgery. Topo IIalpha LI closely correlated with MIB-1 LI (R=0.781, P<0.001). We conclude that Topo IIalpha expression correlates with tumour cell proliferation in paediatric OPGs. Assessment of cell proliferation, however, does not assist in refining prognostic predictions. Enhanced Topo IIalpha expression in children < or = 3.0 years suggests that Topo IIalpha-interfering anticancer compounds for adjuvant treatment of OPGs may be of particular benefit to young children.

Anticancer drug resistance in primary human brain tumors.

The difficult clinical situation still associated with most types of primary human brain tumors has fostered significant interest in defining novel therapeutic modalities for this heterogeneous group of neoplasms. Beginning in the 1980s chemotherapy has been incorporated into the treatment protocol of a number of intractable brain tumors. However, it has predominantly failed to improve patient outcome. The unsatisfactory results with chemotherapeutic intervention have chiefly been attributed to tumor cell resistance. In recent years, there has been a literal explosion in our understanding about the mechanisms by which cancer cells become chemoresistant. During the course of their evolution (intrinsic resistance) or in response to chemotherapy (acquired resistance) these cells may follow a number of pathways of genetic alterations to possess a common (multidrug) or drug-specific (individual drug) resistant phenotype. Genomic aberrations, deregulation of membrane transporting proteins and cellular enzymes, and an altered susceptibility to commit to apoptosis are among the steps on the way that contribute to the genesis of chemotherapeutic treatment failure. Although, through the years we have come to yield information and inferences as to the roles that different molecular events may have in the resistance phenotype of cancer cells, the actual involvement of single genetic alterations in conferring drug resistance in primary brain tumors remains debatable. This uncertainty and, besides, the lack of proper drug resistance diagnostics, in a vicious circle, hinder the development of effective resistance-modulation strategies. Clinical non-responsiveness to chemotherapy remains a formidable obstacle to the successful treatment of brain tumors and one of the most serious problems to be solved in the therapy of these lesions. Future advances in the chemotherapeutic management of these neoplasms will come with an improved understanding of the significance and interrelationship of the multiple biological systems operative in promoting resistance to this treatment modality. The focus of this review is to summarize current knowledge concerning major drug resistance-related markers, to describe their functional interaction en route to chemoresistance, and to discuss their implication in rendering human brain tumor cells resistant to chemotherapy.

Inhibition of Ras and related guanosine triphosphate-dependent proteins as a therapeutic strategy for blocking malignant glioma growth: II--preclinical studies in a nude mouse model.

OBJECTIVE: Preliminary studies have demonstrated that the Ras family and related guanosine triphosphate-dependent proteins are overactivated in malignant gliomas and that inhibition of the activation of such proteins, by blockade of their post-translational processing, reduces tumor cell growth in vitro. The current study evaluates the utility of this therapeutic strategy in vivo, using preclinical glioma model systems. METHODS: We examined the efficacy against U-87 human malignant glioma cells, in both subcutaneous and intracranial nude mouse models, of selective peptidomimetic inhibitors of farnesyltransferase (FTI-276) and geranylgeranyltransferase (GGTI-297), which are involved in critical steps in the post-translational processing of Ras and related guanosine triphosphate-dependent proteins. For the subcutaneous model, 2 x 10(5) U-87 cells were implanted; after measurable tumors were detected on Day 7, animals were treated with either FTI-276, GGTI-297, or vehicle, administered by continuous infusion for 7 days. Differences in tumor volumes among the treatment groups were examined for significance using a Student's t test. For the intracranial model, 2 x 10(5) U-87 cells were implanted in the right frontal lobe and treatment was initiated on Day 7. In initial studies, animals received a 7-day course of either FTI-276, GGTI-297, or vehicle. In subsequent studies, a 28-day treatment period was used. Comparisons of survival times among treatment groups were performed using a rank-sum test. RESULTS: Although the two agents exhibited comparable antiproliferative activities in previous in vitro studies, an obvious difference in efficacy was apparent in this study. Whereas the geranylgeranyltransferase inhibitor failed to improve survival rates, compared with those observed for control animals, in either the subcutaneous or intracranial model, the farnesyltransferase inhibitor produced objective regression of tumor growth in the subcutaneous model and significant prolongation of survival times in the intracranial model, without apparent toxicity. In the subcutaneous model, tumor volumes for the control, GGTI-297-treated, and FTI-276-treated animals on Day 28 after implantation were 621+/-420, 107+/-104, and 18.5+/-12.7 mm3, respectively (P < 0.05). In the 7-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 27.7+/-2.9, 29.8+/-2.1, and 43.6+/-2.7 days, respectively (P < 0.001). In the 28-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 29.2+/-3.7, 28.3+/-3.9, and 58.7+/-6.2 days, respectively, with five of six animals in the latter group surviving more than 55 days after tumor implantation (P < 0.001). CONCLUSION: These studies demonstrate that farnesyltransferase inhibition is effective in diminishing the growth of human glioma cells in vivo. Evaluation of this treatment approach in clinical trials is warranted.

Epidermal growth factor receptor expression and gene amplification in high-grade non-brainstem gliomas of childhood.

Epidermal growth factor receptor (EGFR) is commonly overexpressed in adult high-grade gliomas. Forty to 50% of such tumors demonstrate amplification of the EGFR gene, often with rearrangement and constitutive activation of the gene product, suggesting that EGFR might play a role in the malignant progression of a subset of these neoplasms. In this regard, several groups have shown that overexpression of EGFR is associated with an adverse outcome in adult gliomas. In contrast to the extensive studies of EGFR status that have been performed in adult high-grade gliomas, little information has been reported about EGFR expression and amplification, as well as their prognostic relevance in high-grade gliomas of childhood, which carry a somewhat more favorable prognosis than their adult counterparts. To address this issue, we examined the expression of EGFR using immunohistochemistry and screened for amplification of the EGFR gene using a competitive PCR in a series of 27 archival pediatric high-grade nonbrainstem gliomas treated consecutively at our institution between 1975 and 1992. Tumors were categorized based on protein expression patterns, and the association between expression status and outcome was examined. Although elevated immunoreactivity for EGFR was observed in 80% of tumors, only two of the cases had gene amplification. No difference in outcome was observed between tumors that exhibited extensive EGFR immunoreactivity and those that did not (P > 0.3). Although EGFR expression did not seem to be of prognostic relevance for the outcome of pediatric patients harboring high-grade nonbrainstem gliomas, the consistently high levels of expression of EGFR in these neoplasms suggest that this receptor plays a role in the malignant phenotype of these tumors. Accordingly, treatment approaches targeting EGFR might be of potential therapeutic benefit for high-grade gliomas of childhood.

Protein kinase C inhibition by UCN-01 induces apoptosis in human glioma cells in a time-dependent fashion.

Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly, we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3-6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2-4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.

The p21-Ras signal transduction pathway and growth regulation in human high-grade gliomas.

Deregulated p21-Ras function, as a result of mutation, overexpression or growth factor-induced overactivation, contributes to at least 30% of human cancer. This article reviews the potential role of the p21-Ras family of GTPases in the regulation of growth of high-grade gliomas and describes how targeting this oncoprotein clinically may provide a novel strategy to counteract glioma proliferation. The application of strategies directed at selectively opposing the deregulated signal transduction pathway of high-grade gliomas may be of potential therapeutic benefit and may offer a whole new arsenal of antineoplastic agents to be included in the multimodal treatment of these challenging neoplasms.

Inhibition of Ras and related G-proteins as a therapeutic strategy for blocking malignant glioma growth.

OBJECTIVE: Preliminary studies have demonstrated that the Ras family and related guanosine 5'-triphosphate-dependent proteins (G-proteins) are overactivated in malignant gliomas and may function as indirect mediators of glial transformation initiated by deregulated upstream signaling elements. We postulated that inhibiting the activation of such proteins might represent a promising strategy for blocking the aberrant proliferation of these tumors. METHODS AND RESULTS: Accordingly, we examined the therapeutic efficacy against malignant glioma cells in vitro of a series of selective peptidomimetic inhibitors of farnesylation (FTI-277) and geranylgeranylation (GGTI-286 and GGTI-298), which are critical steps in the post-translational processing (prenylation) of these proteins. We first defined concentration-response relationships for each of these agents, using MTS-based cell proliferation assays in the established malignant glioma cell lines U-87 and LN-Z308 and the low-passage malignant glioma cell line SG-388. FTI-277, GGTI-286, and GGTI-298 each produced a striking concentration-dependent antiproliferative effect on the glioma cell lines, with the median effective dose ranging from 2.5 to 15.5 micromol/L. We then assessed the effect of prenylation inhibition on cell viability using clonogenic growth assays. This demonstrated a steady drop in the number of colonies with increasing drug concentrations for all three inhibitors. Third, we examined whether the cytotoxic effects of one of these inhibitors (GGTI-298) were associated with the induction of apoptosis using a terminal transferase-catalyzed in situ end-labeling technique. This approach showed a time-dependent increase in apoptotic cell numbers, which correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. CONCLUSION: Our studies demonstrated that FTI-277, GGTI-286, and GGTI-298 each yielded significant antiproliferative effects in human malignant glioma cells in vitro at low micromolar concentrations, which have been achievable in vivo without major systemic toxicity. Extended periods of drug treatment produced cytotoxicity in the tumor cells, which correlated with the induction of apoptosis. We conclude that inhibition of Ras and related G-proteins offers a promising approach for blocking glioma proliferation that justifies further investigation in vivo.

Application of signal transduction inhibition as a therapeutic strategy for central nervous system tumors.

During the last decade, rapid progress has been made in understanding the molecular pathways underlying the proliferation of both normal and neoplastic cells. The processes by which messages to initiate protein synthesis, cell cycle progression, and even cell death are transmitted from the cell surface or the cytoplasm to the nucleus are broadly referred to as 'signal transduction'. These multistep pathways involve a host of proteins that interact with other proteins in overlapping cascades that flow downstream in a stepwise fashion from the cell membrane to the nucleus. Inappropriate overactivation or underactivation of various components of such signaling pathways can contribute to pathological processes, such as neoplasia. Conversely, molecular and pharmacological interventions that target and attempt to reverse the aberrant state of activation can potentially be of therapeutic benefit. The present article provides a background for understanding the contribution of signal transduction pathways to the proliferation of normal and neoplastic cells and describes ways in which targetted inhibition of selected signaling pathway components has been exploited to inhibit tumor growth in vitro and in vivo. Because most studies to date involving central nervous system (CNS) tumors have focused on gliomas, in view of their frequency in both the pediatric and adult age groups, the discussion of therapeutic applications for CNS neoplasia will deal primarily with these lesions. However, the basic concepts presented are generalizable to most tumor types and have been successfully applied in vitro in medulloblastomas as well. Ultimately, the translation of such strategies to the treatment of patients with malignant brain tumors may provide novel approaches for improving the poor outlook associated with these neoplasms.

The role of protein kinase C (PKC) in the evolution and proliferation of malignant gliomas, and the application of PKC inhibition as a novel approach to anti-glioma therapy.

The present article reviews the role of the second messenger enzyme protein kinase C (PKC) in the growth regulation of high-grade gliomas, and evaluates the efficacy of therapeutic strategies directed against PKC for blocking the proliferation of these malignancies in in vitro and in vivo models. The translation of such strategies to the treatment of patients with malignant gliomas may provide a novel approach for improving the otherwise grim outlook associated with these neoplasms.

Basic fibroblast growth factor expression as a predictor of prognosis in pediatric high-grade gliomas.

Clinical and histopathological factors fail to adequately predict outcomes in children with high-grade gliomas, indicating a need to identify relevant biological markers of tumor behavior to guide therapeutic decision-making. Basic fibroblast growth factor (bFGF) is a mitogenic and angiogenic factor that has been observed to be overexpressed in a significant percentage of malignant gliomas, although the prognostic significance of this expression is unknown. To address this issue, the expression status of bFGF was examined immunohistochemically in a series of 27 archival pediatric malignant non-brainstem gliomas treated consecutively at our institution between 1975 and 1992. Tumors were categorized based on expression levels, and the association between expression status and outcome was examined. Sixteen cases showed high levels of expression of bFGF, and 11 showed low levels. There was no correlation between expression status and either tumor histology, patient age, or tumor location. However, there was a significant difference in outcome between patients with high levels of bFGF immunoreactivity and those with low expression. Median progression-free survival was >66 months in the low bFGF group as compared to 6 months in the high bFGF group (P = 0.006). Median overall survival was >66 months in the low bFGF group as compared to 18 months in the high bFGF group (P = 0.03). Tumor bFGF expression seems to be strongly associated with outcome in children with high-grade gliomas and, consequently, may serve as a biological correlate of patient prognosis in conjunction with other prognostic variables.