Keith L. Black

The somatic genomic landscape of glioblastoma.

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

Analysis of gene expression and chemoresistance of CD133 + cancer stem cells in glioblastoma

BackgroundRecently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown.ResultsIn this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol) and etoposide (VP16) compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors.ConclusionOur study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumors resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in tumors to improve the survival of brain tumor patients.

Isolation of cancer stem cells from adult glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common adult primary brain tumor and is comprised of a heterogeneous population of cells. It is unclear which cells within the tumor mass are responsible for tumor initiation and maintenance. In this study, we report that brain tumor stem cells can be identified from adult GBMs. These tumor stem cells form neurospheres, possess the capacity for self-renewal, express genes associated with neural stem cells (NSCs), generate daughter cells of different phenotypes from one mother cell, and differentiate into the phenotypically diverse populations of cells similar to those present in the initial GBM. Having a distinguishing feature from normal NSCs, these tumor stem cells can reform spheres even after the induction of differentiation. Furthermore, only these tumor stem cells were able to form tumors and generate both neurons and glial cells after in vivo implantation into nude mice. The identification of tumor stem cells within adult GBM may represent a major step forward in understanding the origin and maintenance of GBM and lead to the identification and testing of new therapeutic targets.

Vaccination with Tumor Lysate-Pulsed Dendritic Cells Elicits Antigen-Specific, Cytotoxic T-Cells in Patients with Malignant Glioma

The primary goal of this Phase I study was to assess the safety and bioactivity of tumor lysate-pulsed dendritic cell (DC) vaccination to treat patients with glioblastoma multiforme and anaplastic astrocytoma. Adverse events, survival, and cytotoxicity against autologous tumor and tumor-associated antigens were measured. Fourteen patients were thrice vaccinated 2 weeks apart with autologous DCs pulsed with tumor lysate. Peripheral blood mononuclear cells were differentiated into phenotypically and functionally confirmed DCs. Vaccination with tumor lysate-pulsed DCs was safe, and no evidence of autoimmune disease was noted. Ten patients were tested for the development of cytotoxicity through a quantitative PCR-based assay. Six of 10 patients demonstrated robust systemic cytotoxicity as demonstrated by IFN-γ expression by peripheral blood mononuclear cells in response to tumor lysate after vaccination. Using HLA-restricted tetramer staining, we identified a significant expansion in CD8+ antigen-specific T-cell clones against one or more of tumor-associated antigens MAGE-1, gp100, and HER-2 after DC vaccination in four of nine patients. A significant CD8+ T-cell infiltrate was noted intratumorally in three of six patients who underwent reoperation. The median survival for patients with recurrent glioblastoma multiforme in this study (n = 8) was 133 weeks. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous tumor lysate-pulsed DC vaccine for patients with malignant glioma. We demonstrate for the first time the ability of an active immunotherapy strategy to generate antigen-specific cytotoxicity in brain tumor patients.

Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme

PURPOSE This open-label, prospective, multicenter single-arm phase II study combined bevacizumab (BV) with radiation therapy (RT) and temozolomide (TMZ) for the treatment of newly diagnosed glioblastoma (GBM). The objectives were to determine the efficacy of this treatment combination and the associated toxicity. PATIENTS AND METHODS Seventy patients with newly diagnosed GBM were enrolled between August 2006 and November 2008. Patients received standard RT starting within 3 to 6 weeks after surgery with concurrent administration of daily TMZ and biweekly BV. After completion of RT, patients resumed TMZ for 5 days every 4 weeks and continued biweekly BV. MGMT promoter methylation was assessed on patient tumor tissue. A University of California, Los Angeles/Kaiser Permanente Los Angeles (KPLA) control cohort of newly diagnosed patients treated with first-line RT and TMZ who had mostly received BV at recurrence was derived for comparison. RESULTS The overall survival (OS) and progression-free survival (PFS) were 19.6 and 13.6 months, respectively, compared to 21.1 and 7.6 months in the University of California, Los Angeles/KPLA control cohort, and 14.6 and 6.9 months in the European Organisation for Research and Treatment of Cancer-National Cancer Institute of Canada cohort. Correlation of MGMT promoter methylation and improved OS and PFS was retained in the study group. Comparative subset analysis showed that poor prognosis patients (recursive partitioning analysis class V/VI) may derive an early benefit from the use of first-line BV. Toxicity attributable to RT/TMZ was similar, and additional toxicities were consistent with those reported in other BV trials. CONCLUSION Patients treated with BV and TMZ during and after RT showed improved PFS without improved OS compared to the University of California, Los Angeles/KPLA control group. Additional studies are warranted to determine if BV administered first-line improves survival compared to BV at recurrence.

Identification of amyloid plaques in retinas from Alzheimer's patients and noninvasive in vivo optical imaging of retinal plaques in a mouse model.

Noninvasive monitoring of β-amyloid (Aβ) plaques, the neuropathological hallmarks of Alzheimers disease (AD), is critical for AD diagnosis and prognosis. Current visualization of Aβ plaques in brains of live patients and animal models is limited in specificity and resolution. The retina as an extension of the brain presents an appealing target for a live, noninvasive optical imaging of AD if disease pathology is manifested there. We identified retinal Aβ plaques in postmortem eyes from AD patients (n=8) and in suspected early stage cases (n=5), consistent with brain pathology and clinical reports; plaques were undetectable in age-matched non-AD individuals (n=5). In APP(SWE)/PS1(∆E9) transgenic mice (AD-Tg; n=18) but not in non-Tg wt mice (n=10), retinal Aβ plaques were detected following systemic administration of curcumin, a safe plaque-labeling fluorochrome. Moreover, retinal plaques were detectable earlier than in the brain and accumulated with disease progression. An immune-based therapy effective in reducing brain plaques, significantly reduced retinal Aβ plaque burden in immunized versus non-immunized AD mice (n=4 mice per group). In live AD-Tg mice (n=24), systemic administration of curcumin allowed noninvasive optical imaging of retinal Aβ plaques in vivo with high resolution and specificity; plaques were undetectable in non-Tg wt mice (n=11). Our discovery of Aβ specific plaques in retinas from AD patients, and the ability to noninvasively detect individual retinal plaques in live AD mice establish the basis for developing high-resolution optical imaging for early AD diagnosis, prognosis assessment and response to therapies.

Vaccination elicits correlated immune and clinical responses in glioblastoma multiforme patients.

Cancer vaccine trials have failed to yield robust immune-correlated clinical improvements as observed in animal models, fueling controversy over the utility of human cancer vaccines. Therapeutic vaccination represents an intriguing additional therapy for glioblastoma multiforme (GBM; grade 4 glioma), which has a dismal prognosis and treatment response, but only early phase I vaccine trial results have been reported. Immune and clinical responses from a phase II GBM vaccine trial are reported here. IFN-gamma responsiveness was quantified in peripheral blood of 32 GBM patients given therapeutic dendritic cell vaccines. Posttreatment times to tumor progression (TTP) and survival (TTS) were compared in vaccine responders and nonresponders and were correlated with immune response magnitudes. GBM patients (53%) exhibited >or=1.5-fold vaccine-enhanced cytokine responses. Endogenous antitumor responses of similar magnitude occurred in 22% of GBM patients before vaccination. Vaccine responders exhibited significantly longer TTS and TTP relative to nonresponders. Immune enhancement in vaccine responders correlated logarithmically with TTS and TTP spanning postvaccine chemotherapy, but not with initial TTP spanning vaccination alone. This is the first report of a progressive correlation between cancer clinical outcome and T-cell responsiveness after therapeutic vaccination in humans and the first tracing of such correlation to therapeutically exploitable tumor alteration. As such, our findings offer unique opportunities to identify cellular and molecular components of clinically meaningful antitumor immunity in humans.

Clinical Responsiveness of Glioblastoma Multiforme to Chemotherapy after Vaccination

Purpose: Although the development of immune-based therapies for various cancers including malignant glioma has been heralded with much hope and optimism, objective clinical improvements in most vaccinated cancer patients have not been realized. To broaden the search for vaccine-induced benefits, we examined synergy of vaccines with conventional chemotherapy. Experimental Design: Survival and progression times were analyzed retrospectively in 25 vaccinated (13 with and 12 without subsequent chemotherapy) and 13 nonvaccinated de novo glioblastoma (GBM) patients receiving chemotherapy. Immune responsiveness and T-cell receptor excision circle (TREC) content within CD8+ T cells (CD8+ TRECs) was determined in vaccinated patients. Results: Vaccinated patients receiving subsequent chemotherapy exhibited significantly longer times to tumor recurrence after chemotherapy relative to their own previous recurrence times, as well as significantly longer postchemotherapy recurrence times and survival relative to patients receiving isolated vaccination or chemotherapy. Patients exhibiting objective (>50%) tumor regression, extremely rare in de novo GBM, were also confined to the vaccine + chemotherapy group. Prior tumor behavior, demographic factors, other treatment variables, distribution of vaccine responders, and patients with high CD8+ TRECs all failed to account for these differences in clinical outcome. Within all GBM patients receiving post-vaccine chemotherapy, however, CD8+ TRECs predicted significantly longer chemotherapeutic responses, revealing a strong link between the predominant T-cell effectors in GBM and tumor chemosensitivity. Conclusions: We propose that therapeutic vaccination synergizes with subsequent chemotherapy to elicit tangible clinical benefits for GBM patients.

Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme (GBM)

The proteasome plays a pivotal role in controlling cell proliferation, apoptosis, and differentiation in a variety of normal and tumor cells. PS-341, a novel boronic acid dipeptide that inhibits 26S proteasome activity, has prominent effects in vitro and in vivo against several solid tumors. We examined its antiproliferation, proapoptotic effects using three human glioblastoma multiforme (GBM) cell lines and five primary GBM explants. PS-341 markedly inhibited proliferation of GBM cell lines and explants in liquid and soft agar culture. These cells developed a G2/M cell cycle arrest with a concomitant decreased percentage of cells in S phase (≈2-fold), associated with an increased expression of p21WAF1, p27KIP1, as well as cyclin B1 and decreased levels of CDK2, CDK4, and E2F4. About 35–40% of the cells became apoptotic when exposed to PS-341 (10−7 M, 24–48 h) as shown by Annexin V analysis; in concert with these findings, immunobloting showed a C-terminal 85 kDa apoptotic fragment of poly ADP-ribose polymerase (PARP), and a decreased level of Bcl2 and Bcl-xl. PS-341 downregulated the expression of Bcl-2 and Bcl-xl in protein levels at an early time of treatment. These changes occurred irrespective of the p53 mutational status of the cells. PS-341 activated JNK/c-Jun signaling in GBM cells, and the JNK inhibitor SP600125 blocked the JNK signaling to reverse partially the PS-341 growth inhibition. PS-341 (10−7 M, 24 h) decreased nuclear NF-κB levels as shown by Western blot, and reduced transcriptional activity of NF-κB as measured by reporter assays in these transformed cells. Also, PS-341 enhanced TRAIL (TNF-related apoptosis-inducing ligand) and TNFα (tumor necrosis factor alpha) induced cell death and apoptosis (two- to five-fold) in GBM cells. In summary, PS-341 has profound effects on growth and apoptosis of GBM cells, suggesting that PS-341 may be an effective therapy for patients with gliomas.

Levels of Expression of CYR61 and CTGF Are Prognostic for Tumor Progression and Survival of Individuals with Gliomas

The biological properties of CCN proteins include stimulation of cell proliferation, migration, and adhesion, as well as angiogenesis and tumorigenesis. We quantified CYR61, CTGF, WISP-1, and NOV mRNA expression levels in samples from sixty-six primary gliomas and five normal brain samples using quantitative real-time PCR assay. Statistical analysis was performed to explore the links between expression of the CCN genes and clinical and pathological parameters. Overexpression of CYR61, CTGF, WISP-1, and NOV occurred in 48% (32 of 66), 58% (38 of 66), 36% (24 of 66), and 15% (10 of 66) of primary gliomas, respectively. Interestingly, significant associations were found between CYR61 expression versus tumor grade, pathology, gender, and age at diagnosis. Also, a significant correlation existed between CTGF mRNA levels versus tumor grade, gender, and pathology. In contrast to CYR61 and CTGF, no significant association was found between expression of either WISP-1 or NOV versus any of the pathological features. Furthermore, Cox regression analysis showed that CYR61 and CTGF expression had a significant correlation with patient survival. These results suggest that CYR61 and CTGF may play a role in the progression of gliomas; their levels at diagnosis may have prognostic significance; and these proteins might serve as valuable targets for therapeutic intervention.