Despite extensive study, few therapeutic targets have been identified for glioblastoma (GBM). Here we show that patient-derived glioma sphere cultures (GSCs) that resemble either the proneural (PN) ...or mesenchymal (MES) transcriptomal subtypes differ significantly in their biological characteristics. Moreover, we found that a subset of the PN GSCs undergoes differentiation to a MES state in a TNF-α/NF-κB-dependent manner with an associated enrichment of CD44 subpopulations and radioresistant phenotypes. We present data to suggest that the tumor microenvironment cell types such as macrophages/microglia may play an integral role in this process. We further show that the MES signature, CD44 expression, and NF-κB activation correlate with poor radiation response and shorter survival in patients with GBM.
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•Patient-derived GSCs differ in their molecular features compared to the parental GBM•PN GSCs undergo differentiation to a MES state in a TNF-α/NF-κB-dependent manner•Macrophages/microglia infiltration correlates with the MES signature in human GBM•NFκB activation, MES state, and CD44 levels associate with radio resistance in GBM
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We have profiled promoter DNA methylation alterations in 272 glioblastoma tumors in the context of The Cancer Genome Atlas (TCGA). We found that a distinct subset of samples displays concerted ...hypermethylation at a large number of loci, indicating the existence of a glioma-CpG island methylator phenotype (G-CIMP). We validated G-CIMP in a set of non-TCGA glioblastomas and low-grade gliomas. G-CIMP tumors belong to the proneural subgroup, are more prevalent among lower-grade gliomas, display distinct copy-number alterations, and are tightly associated with
IDH1 somatic mutations. Patients with G-CIMP tumors are younger at the time of diagnosis and experience significantly improved outcome. These findings identify G-CIMP as a distinct subset of human gliomas on molecular and clinical grounds.
► Identification of a CpG island methylator phenotype (G-CIMP) in gliomas ► G-CIMP is tightly associated with
IDH1 mutation ► G-CIMP patients are younger at diagnosis and display improved survival ► G-CIMP is more prevalent among low- and intermediate-grade gliomas
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hypermethylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) gene has been shown to be associated with improved outcome in glioblastoma (GBM) and may be a predictive marker of sensitivity ...to alkylating agents. However, the predictive utility of this marker has not been rigorously tested with regard to sensitivity to other therapies, namely radiation. To address this issue, we assessed MGMT methylation status in a cohort of patients with GBM who underwent radiation treatment but did not receive chemotherapy as a component of adjuvant treatment. Formalin-fixed, paraffin-embedded tumor samples from 225 patients with newly diagnosed GBM were analyzed via methylation-specific, quantitative real-time polymerase chain reaction following bisulfite treatment on isolated DNA to assess MGMT promoter methylation status. In patients who received radiotherapy alone following resection, methylation of the MGMT promoter correlated with an improved response to radiotherapy. Unmethylated tumors were twice as likely to progress during radiation treatment. The median time interval between resection and tumor progression of unmethylated tumors was also nearly half that of methylated tumors. Promoter methylation was also found to confer improved overall survival in patients who did not receive adjuvant alkylating chemotherapy. Multivariable analysis demonstrated that methylation status was independent of age, Karnofsky performance score, and extent of resection as a predictor of time to progression and overall survival. Our data suggest that MGMT promoter methylation appears to be a predictive biomarker of radiation response. Since this biomarker has also been shown to predict response to alkylating agents, perhaps MGMT promoter methylation represents a general, favorable prognostic factor in GBM.
The transcription factor Forkhead box M1 (FoxM1) is overexpressed in malignant glioma. However, the functional importance of this factor in human glioma is not known. In the present study, we found ...that FoxM1B was the predominant FoxM1 isoform expressed in human glioma but not in normal brain tissue. The level of FoxM1 protein expression in human glioma tissues was directly correlated with the glioma grade. The level of FoxM1 protein expression in human glioblastoma tissues was inversely correlated with patient survival. Enforced FoxM1B expression caused SW1783 and Hs683 glioma cells, which do not form tumor xenografts, to regain tumorigenicity in nude mouse model systems. Moreover, gliomas that arose from FoxM1B-transfected anaplastic astrocytoma SW1783 cells displayed glioblastoma multiforme phenotypes. Inhibition of FoxM1 expression in glioblastoma U-87MG cells suppressed their anchorage-independent growth in vitro and tumorigenicity in vivo. Furthermore, we found that FoxM1 regulates the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27(Kip1). These results showed that FoxM1 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity. Therefore, FoxM1 might be a new potential target of therapy for human malignant gliomas.
Deregulation of the mTOR pathway is closely associated with tumorigenesis. Accordingly, mTOR inhibitors such as rapamycin and mTOR-selective kinase inhibitors have been tested as cancer therapeutic ...agents. Inhibition of mTOR results in sensitization to DNA-damaging agents; however, the molecular mechanism is not well understood. We found that an mTOR-selective kinase inhibitor, AZD8055, significantly enhanced sensitivity of a pediatric rhabdomyosarcoma xenograft to radiotherapy and sensitized rhabdomyosarcoma cells to the DNA interstrand cross-linker (ICL) melphalan. Sensitization correlated with drug-induced downregulation of a key component of the Fanconi anemia pathway, FANCD2 through mTOR regulation of FANCD2 gene transcripts via mTORC1-S6K1. Importantly, we show that FANCD2 is required for the proper activation of ATM-Chk2 checkpoint in response to ICL and that mTOR signaling promotes ICL-induced ATM-Chk2 checkpoint activation by sustaining FANCD2. In FANCD2-deficient lymphoblasts, FANCD2 is essential to suppress endogenous and induced DNA damage, and FANCD2-deficient cells showed impaired ATM-Chk2 and ATR-Chk1 activation, which was rescued by reintroduction of wild-type FANCD2. Pharmacologic inhibition of PI3K-mTOR-AKT pathway in Rh30 rhabdomyosarcoma cells attenuated ICL-induced activation of ATM, accompanied with the decrease of FANCD2. These data suggest that the mTOR pathway may promote the repair of DNA double-strand breaks by sustaining FANCD2 and provide a novel mechanism of how the Fanconi anemia pathway modulates DNA damage response and repair.
Only a subset of patients with newly diagnosed glioblastoma (GBM) exhibit a response to standard therapy. To date, a biomarker panel with predictive power to distinguish treatment sensitive from ...treatment refractory GBM tumors does not exist. An analysis was performed using GBM microarray data from 4 independent data sets. An examination of the genes consistently associated with patient outcome, revealed a consensus 38-gene survival set. Worse outcome was associated with increased expression of genes associated with mesenchymal differentiation and angiogenesis. Application to formalin fixed-paraffin embedded (FFPE) samples using real-time reverse-transcriptase polymerase chain reaction assays resulted in a 9-gene subset which appeared robust in these samples. This 9-gene set was then validated in an additional independent sample set. Multivariate analysis confirmed that the 9-gene set was an independent predictor of outcome after adjusting for clinical factors and methylation of the methyl-guanine methyltransferase promoter. The 9-gene profile was also positively associated with markers of glioma stem-like cells, including CD133 and nestin. In sum, a multigene predictor of outcome in glioblastoma was identified which appears applicable to routinely processed FFPE samples. The profile has potential clinical application both for optimization of therapy in GBM and for the identification of novel therapies targeting tumors refractory to standard therapy.
The clinical significance of epidermal growth factor receptor variant III (EGFRvIII) expression in glioblastoma multiforme (GBM) and its relationship with other key molecular markers are not clear. ...We sought to evaluate the clinical significance of GBM subtypes as defined by EGFRvIII status.
The expression of EGFRvIII was assessed by immunohistochemistry in 649 patients with newly diagnosed GBM. These data were then examined in conjunction with the expression of phospho-intermediates (in a subset of these patients) of downstream AKT and Ras pathways and YKL-40 as well as with known clinical risk factors, including the Radiation Therapy Oncology Group's recursive partitioning analysis (RTOG-RPA) class.
The RTOG-RPA class was highly predictive of survival in EGFRvIII-negative patients but much less predictive in EGFRvIII-positive patients. These findings were seen in both an initial test set (n = 268) and a larger validation set (n = 381). Similarly, activation of the AKT/MAPK pathways and YKL-40 positivity were predictive of poor outcome in EGFRvIII-negative patients but not in EGFRvIII-positive patients. Pair-wise combinations of markers identified EGFRvIII and YKL-40 as prognostically important. In particular, outcome in patients with EGFRvIII-negative/YKL-40-negative tumors was significantly better than the outcome in patients with the other three combinations of these two markers.
Established prognostic factors in GBM were not predictive of outcome in the EGFRvIII-positive subset, although this requires confirmation in independent data sets. GBMs negative for both EGFRvIII and YKL-40 show less aggressive behavior.
To compare CT-based volumetric calculations and International Commission on Radiation Units and Measurements (ICRU) reference-point estimates of radiation doses to the bladder and rectum in patients ...with carcinoma of the uterine cervix treated with definitive low-dose-rate intracavitary radiotherapy (ICRT).
Between November 2001 and March 2003, 60 patients were prospectively enrolled in a pilot study of ICRT with CT-based dosimetry. Most patients underwent two ICRT insertions. After insertion of an afterloading ICRT applicator, intraoperative orthogonal films were obtained to ensure proper positioning of the system and to facilitate subsequent planning. Treatments were prescribed using standard two-dimensional dosimetry and planning. Patients also underwent helical CT of the pelvis for three-dimensional reconstruction of the radiation dose distributions. The systems were loaded with 137Cs sources using the Selectron remote afterloading system according to institutional practice for low-dose-rate brachytherapy. Three-dimensional dose distributions were generated using the Varian BrachyVision treatment planning system. The rectum was contoured from the bottom of the ischial tuberosities to the sigmoid flexure. The entire bladder was contoured. The minimal doses delivered to the 2 cm3 of bladder and rectum receiving the highest dose (DBV2 and DRV2, respectively) were determined from dose-volume histograms, and these estimates were compared with two-dimensionally derived estimates of the doses to the corresponding ICRU reference points.
A total of 118 unique intracavitary insertions were performed, and 93 were evaluated and the subject of this analysis. For the rectum, the estimated doses to the ICRU reference point did not differ significantly from the DRV2 (p = 0.561); the mean (+/- standard deviation) difference was 21 cGy (+/- 344 cGy). The median volume of the rectum that received at least the ICRU reference-point dose was 2.1 cm3. In 66 (71%) of 93 cases, <5 cm3 was treated to this dose. However, for the bladder, the estimated doses to the ICRU reference point were significantly lower than the D(BV2) (p <0.001); the mean difference was 680 cGy (+/- 543 cGy). The median volume of the bladder that received at least the ICRU reference-point dose was 13.0 cm3.
Our data suggest that the estimated dose to the ICRU rectal point may be a reasonable surrogate for the DRV2. However, this result may not be applicable to other treatment guidelines and ICRT applicator systems. In contrast, the dose to the ICRU bladder point does not appear to be a reasonable surrogate for the DBV2. Correlation with late complications are needed to define the role of three-dimensional dosimetry in treatment planning.
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GEOZS, IJS, NUK, OILJ, UL, UM, UPUK
Purpose: Activation of mitogen-activated protein kinase (MAPK) and members of the Akt pathway have been shown to promote cell proliferation,
survival, and resistance to radiation. This study was ...conducted to determine whether any of these markers are associated with
survival time and response to radiation in glioblastoma.
Experimental Design: The expression of phosphorylated (p-)Akt, mammalian target of rapamycin (p-mTOR), p-p70S6K, and p-MAPK were assessed by immunohistochemical
staining in 268 cases of newly diagnosed glioblastoma. YKL-40, a prognostic marker previously examined in these tumors, was
also included in the analysis. Expression data were tested for correlations with response to radiation therapy in 131 subtotally
resected cases and overall survival (in all cases). Results were validated in an analysis of 60 patients enrolled in clinical
trials at a second institution.
Results: Elevated p-MAPK expression was most strongly associated with poor response to radiotherapy, a finding corroborated in the
validation cohort. For survival, higher expressions of p-mTOR, p-p70S6K, and p-MAPK were associated with worse outcome (all
P < 0.03). YKL-40 expression was associated with the expressions of p-MAPK, p-mTOR, and p-p70S6K (all P < 0.02), with a trend toward association with p-Akt expression ( P = 0.095). When known clinical variables were added to a multivariate analysis, only age, Karnofsky performance score, and
p-MAPK expression emerged as independent prognostic factors.
Conclusions: p-MAPK and activated members of the Akt pathway are markers of outcome in glioblastoma. Elevated expression of p-MAPK is
associated with increased radiation resistance and represents an independent prognostic factor in these tumors.
Purpose: YKL-40 is a secreted protein that has been reported to be overexpressed in epithelial cancers and gliomas, although its function
is unknown. Previous data in a smaller sample set suggested ...that YKL-40 was a marker associated with a poorer clinical outcome
and a genetically defined subgroup of glioblastoma. Here we test these findings in a larger series of patients with glioblastoma,
and in particular, determine if tumor YKL-40 expression is associated with radiation response.
Experimental Design: Patients ( n = 147) with subtotal resections were studied for imaging-assessed changes in tumor size in serial studies following radiation
therapy. An additional set ( n = 140) of glioblastoma patients who underwent a gross-total resection was tested to validate the survival association and
extend them to patients with minimal residual disease.
Results: In the subtotal resection group, higher YKL-40 expression was significantly associated with poorer radiation response, shorter
time to progression and shorter overall survival. The association of higher YKL-40 expression with poorer survival was validated
in the gross-total resection group. In multivariate analysis with both groups combined ( n = 287), YKL-40 was an independent predictor of survival after adjusting for patient age, performance status, and extent of
resection. YKL-40 expression was also compared with genetically defined subsets of glioblastoma by assessing epidermal growth
factor receptor amplification and loss at chromosome 10q, two of the common recurring aberrations in these tumors, using fluorescent
in situ hybridization. YKL-40 was significantly associated with 10q loss.
Conclusions: The findings implicate YKL-40 as an important marker of therapeutic response and genetic subtype in glioblastomas and suggest
that it may play an oncogenic role in these tumors.
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