Catherine L. Nutt

A multigene predictor of outcome in glioblastoma

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.

MSH6 Mutations Arise in Glioblastomas during Temozolomide Therapy and Mediate Temozolomide Resistance

Purpose: Over the past few years, the alkylating agent temozolomide has become the standard-of-care therapy for patients with glioblastoma, the most common brain tumor. Recently, large-scale cancer genome sequencing efforts have identified a hypermutation phenotype and inactivating MSH6 mismatch repair gene mutations in recurrent, post-temozolomide glioblastomas, particularly those growing more rapidly during temozolomide treatment. This study aimed to clarify the timing and role of MSH6 mutations in mediating glioblastoma temozolomide resistance. Experimental Design:MSH6 sequence and microsatellite instability (MSI) status were determined in matched prechemotherapy and postchemotherapy glioblastomas identified by The Cancer Genome Atlas (TCGA) as having posttreatment MSH6 mutations. Temozolomide-resistant lines were derived in vitro through selective growth under temozolomide, and the MSH6 gene was sequenced in resistant clones. The role of MSH6 inactivation in mediating resistance was explored using lentiviral short hairpin RNA knockdown and MSH6 reconstitution. Results:MSH6 mutations were confirmed in posttreatment TCGA glioblastomas but absent in matched pretreatment tumors. The posttreatment hypermutation phenotype displayed a signature bias toward CpC transitions and was not associated with MSI. In vitro modeling through exposure of an MSH6 wild-type glioblastoma line to temozolomide resulted in resistant clones; one clone showed an MSH6 mutation, Thr1219Ile, that had been independently noted in two treated TCGA glioblastomas. Knockdown of MSH6 in the glioblastoma line U251 increased resistance to temozolomide cytotoxicity and reconstitution restored cytotoxicity in MSH6-null glioma cells. Conclusions:MSH6 mutations are selected in glioblastomas during temozolomide therapy both in vitro and in vivo and are causally associated with temozolomide resistance.

Activation of STAT3, MAPK, and AKT in Malignant Astrocytic Gliomas: Correlation With EGFR Status, Tumor Grade, and Survival

Diffuse astrocytic gliomas are the most common human glial tumors with glioblastoma being the most malignant form. Epidermal growth factor receptor (EGFR) gene amplification is one of the most common genetic changes in glioblastoma and can lead to the activation of various downstream signaling molecules, including STAT3, MAPK, and AKT. In this study, we investigated the activation status of these 3 signaling molecules as well as wild-type (EGFRwt) and mutant (EGFRvIII) EGFR in 82 malignant astrocytic gliomas (55 glioblastomas and 27 anaplastic astrocytomas) using immunohistochemistry. The presence of EGFRwt, but not EGFRvIII, immunopositivity correlated significantly with prevalent EGFR gene amplification in glioblastomas. STAT3 and AKT activation correlated significantly with EGFR status, although the correlation for p-STAT3 was attributed exclusively to EGFRvIII. The distribution of these 3 activated molecules varied significantly with tumor grade; although activation of STAT3 was essentially identical between anaplastic astrocytomas and glioblastomas, an increase in the activation of MAPK and AKT appeared to correlate with the progression of anaplastic astrocytoma to glioblastoma. Finally, activated STAT3 and AKT were marginally predictive of improved and worse prognosis, respectively. Taken together, these findings begin to elucidate the interrelationship between these signaling pathways in astrocytic gliomas in vivo.

Deregulation of a STAT3–Interleukin 8 Signaling Pathway Promotes Human Glioblastoma Cell Proliferation and Invasiveness

Inactivation of the tumor suppressor phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) is recognized as a major event in the pathogenesis of the brain tumor glioblastoma. However, the mechanisms by which PTEN loss specifically impacts the malignant behavior of glioblastoma cells, including their proliferation and propensity for invasiveness, remain poorly understood. Genetic studies suggest that the transcription factor signal transducers and activators of transcription 3 (STAT3) harbors a PTEN-regulated tumor suppressive function in mouse astrocytes. Here, we report that STAT3 plays a critical tumor suppressive role in PTEN-deficient human glioblastoma cells. Endogenous STAT3 signaling is specifically inhibited in PTEN-deficient glioblastoma cells. Strikingly, reactivation of STAT3 in PTEN-deficient glioblastoma cells inhibits their proliferation, invasiveness, and ability to spread on myelin. We also identify the chemokine interleukin 8 (IL8) as a novel target gene of STAT3 in human glioblastoma cells. Activated STAT3 occupies the endogenous IL8 promoter and directly represses IL8 transcription. Consistent with these results, IL8 is upregulated in PTEN-deficient human glioblastoma tumors. Importantly, IL8 repression mediates STAT3 inhibition of glioblastoma cell proliferation, invasiveness, and spreading on myelin. Collectively, our findings uncover a novel link between STAT3 and IL8, the deregulation of which plays a key role in the malignant behavior of PTEN-deficient glioblastoma cells. These studies suggest that STAT3 activation or IL8 inhibition may have potential in patient-tailored treatment of PTEN-deficient brain tumors.

Genetic Alterations of Phosphoinositide 3‐kinase Subunit Genes in Human Glioblastomas

Genetic alterations of PI3K (phosphoinositide 3‐kinase) subunits have been documented in a number of tumor types, with increased PI3K activity linked to gene amplification and mutation of catalytic subunits, as well as mutations of regulatory subunits. Among high grade gliomas, activation of the PI3K‐AKT signaling pathway through loss of PTEN function is common. We therefore investigated whether genetic alteration of class IA PI3Ks might provide a mechanism for deregulation of this pathway in glioblastomas. We studied a series of glioblastomas with FISH to assess copy number of catalytic subunits (PIK3CA and PIK3CD) and with PCR‐SSCP to screen for somatic mutations of conserved regions of both catalytic and regulatory subunits. FISH revealed frequent balanced copy number increases of both PIK3CA and PIK3CD, and one case showed an extra copy limited to PIK3CA. One glioblastoma exhibited a 9‐bp deletion that encompassed the exon‐intron junction of exon 12 of PIK3R1, documenting for the first time a mutation within a PI3K regulatory subunit in human glioblastoma. This deletion would be predicted to yield a truncated protein that lacks the inhibitory domain, resulting in increased PI3K activity. Furthermore, the case with selected PIK3CA copy number gain and the case with a truncating PIK3R1 mutation both featured AKT activation without PTEN mutation. These results suggest that genetic alterations of class IA PI3K subunit genes can occasionally play a role in human glioblastoma by activating the PI3K‐AKT signaling pathway independently of PTEN mutation.

Downregulation of RUNX3 and TES by hypermethylation in glioblastoma

Glioblastoma, the most aggressive and least treatable form of malignant glioma, is the most common human brain tumor. Although many regions of allelic loss occur in glioblastomas, relatively few tumor suppressor genes have been found mutated at such loci. To address the possibility that epigenetic alterations are an alternative means of glioblastoma gene inactivation, we coupled pharmacological manipulation of methylation with gene profiling to identify potential methylation-regulated, tumor-related genes. Duplicates of three short-term cultured glioblastomas were exposed to 5 μM 5-aza-dC for 96 h followed by cRNA hybridization to an oligonucleotide microarray (Affymetrix U133A). We based candidate gene selection on bioinformatics, reverse transcription-polymerase chain reaction (RT–PCR), bisulfite sequencing, methylation-specific PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two genes identified in this manner, RUNX3 and Testin (TES), were subsequently shown to harbor frequent tumor-specific epigenetic alterations in primary glioblastomas. This overall approach therefore provides a powerful means to identify candidate tumor-suppressor genes for subsequent evaluation and may lead to the identification of genes whose epigenetic dysregulation is integral to glioblastoma tumorigenesis.

Efficacy of Systemically Administered Oncolytic Vaccinia Virotherapy for Malignant Gliomas Is Enhanced by Combination Therapy with Rapamycin or Cyclophosphamide

Purpose: The oncolytic effects of a systemically delivered, replicating, double-deleted vaccinia virus has been previously shown for the treatment of many cancers, including colon, ovarian, and others. The purpose of this study was to investigate the oncolytic potential of double-deleted vaccinia virus alone or in combination with rapamycin or cyclophosphamide to treat malignant gliomas in vitro and in vivo. Experimental Design: Rat (RG2, F98, C6) and human (A172, U87MG, U118) glioma cell lines were cultured in vitro and treated with live or UV-inactivated vaccinia virus. Viral gene [enhanced green fluorescent protein (EGFP)] expression by fluorescence-activated cell sorting, relative cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and assays for cytopathic effects were examined. S.c. murine tumor xenografts (U87MG, U118, C6) and i.c. (RG2, F98) tumor models in immunocompetent rats were treated with systemic administration of EGFP-expressing vaccinia virus (vvDD-EGFP), alone or in combination with rapamycin or cyclophosphamide, or controls. Tumor size, viral biodistribution, and animal survival were assessed. Lastly, the oncolytic effects of vvDD-EGFP on human malignant glioma explants were evaluated. Results: vvDD-EGFP was able to infect and kill glioma cells in vitro. A single systemic dose of vvDD-EGFP significantly inhibited the growth of xenografts in athymic mice. Systemic delivery of vvDD-EGFP alone was able to target solitary and multifocal i.c. tumors and prolong survival of immunocompetent rats, whereas combination therapy with rapamycin or cyclophosphamide enhanced viral replication and further prolonged survival. Finally, vvDD-EGFP was able to infect and kill ex vivo primary human malignant gliomas. Conclusions: These results suggest that vvDD-EGFP is a promising novel agent for human malignant glioma therapy, and in combination with immunosuppressive agents, may lead to prolonged survival from this disease.

YKL-40 Is a Differential Diagnostic Marker for Histologic Subtypes of High-Grade Gliomas

Purpose and Experimental Design: In modern neuro-oncology, no variable affects therapeutic decisions and prognostic estimation more than tumor classification. We showed recently that class prediction models, based on gene expression profiles, classify diagnostically challenging malignant gliomas in a manner that better correlates with clinical outcome than standard pathology. In the present study, we used immunohistochemistry to investigate YKL-40 protein expression in independent sets of glioblastomas and anaplastic oligodendrogliomas to determine whether this single marker can aid classification of these high-grade gliomas. Results and Conclusions: Glioblastomas show strikingly more YKL-40 expression than anaplastic oligodendrogliomas. Only 2 of 37 glioblastomas showed completely negative YKL-40 staining in both tumor cells and extracellular matrix, whereas 18 of 29 anaplastic oligodendrogliomas were completely negative in non-microgemistocytic tumor cells and extracellular matrix. Tumor cell staining intensity was also markedly different: 84% of glioblastomas showed strong staining intensities of 2+ or 3+ whereas 76% of anaplastic oligodendrogliomas either did not stain or stained at only 1+. YKL-40 staining provided a better class distinction of glioblastoma versus anaplastic oligodendroglioma than glial fibrillary acidic protein, the current standard immunohistochemical marker used to distinguish diagnostically challenging gliomas. Moreover, a combination of YKL-40 and glial fibrillary acidic protein immunohistochemistry afforded even greater diagnostic accuracy in anaplastic oligodendrogliomas.

Increased Expression of Angiogenic Genes in the Brains of Mouse Meg3-Null Embryos

Maternally expressed gene 3 (MEG3) is a noncoding RNA highly expressed in the normal human brain and pituitary. Expression of MEG3 is lost in gonadotroph-derived clinically nonfunctioning pituitary adenomas. Meg3 knockout mice were generated to identify targets and potential functions of this gene in embryonic development and tumorigenesis. Gene expression profiles were compared in the brains of Meg3-null embryos and wild-type littermate controls using microarray analysis. Microarray data were analyzed with GeneSifter, which uses Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology classifications to identify signaling cascades and functional categories of interest within the dataset. Differences were found in signaling pathways and ontologies related to angiogenesis between wild-type and knockout embryos. Quantitative RT-PCR and immunohistological staining showed increased expression of some Vascular Endothelial Growth Factor pathway genes and increased cortical microvessel density in the Meg3-null embryos. In conclusion, Meg3 may play an important role in control of vascularization in the brain and may function as a tumor suppressor in part by inhibiting angiogenesis.

Mutant IDH1-specific immunohistochemistry distinguishes diffuse astrocytoma from astrocytosis

One of the most vexing issues in diagnostic neuropathology relates to the distinction of diffuse astrocytomas (and other diffuse gliomas) from astrocytosis (gliosis) on biopsies, particularly small biopsies. This challenging differential diagnosis arises in two general situations: (1) low cellularity edges of infiltrating astrocytomas versus mild astrocytosis from a nearby reactive condition; and (2) florid astrocytosis (e.g., near a vascular malformation) versus more cellular astrocytomas. The “holy grail” sought in such diagnostic dilemmas is a tumor-specific marker. To date, the most widely used marker for this purpose has been p53 detection by immunohistochemistry; since mutant p53 has a longer half-life than wild-type p53, it can be more readily detected immunohistochemically than wild-type protein [3, 9]. However, p53 immunohistochemistry is not an entirely accurate marker since: (1) it may show light labeling of non-neoplastic cells; (2) not all TP53 gene mutations result in immunohistochemically detectable p53; and (3) some reactive conditions (notably progressive multifocal leukoencephalopathy) may be strongly positive [8]. Another immunohistochemical marker of tumor cells is the vIII mutant of the epidermal growth factor receptor (EGFR) protein. However, this marker is not of diagnostic utility in the above differential diagnosis, since EGFRvIII is primarily expressed in glioblastomas rather than lower-grade astrocytomas. Moreover, antibodies are not widely available or readily optimized for standard immunohistochemistry, again limiting its differential diagnostic utility. Recently, isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations have been demonstrated in a variety of diffuse gliomas, with IDH1 mutations occurring commonly in lower-grade gliomas [1, 2, 10, 12]. Notably, nearly all IDH1 mutations are the same, with CGT–CAT transition causing a specific amino acid change from arginine to histidine at codon 132 (R132H). As a result, the detection of IDH1 mutations may be a specific means to aid in differentiating between glioma and gliosis. Indeed, one recent paper utilized a PCR-based assay to show that IDH mutations are found in astrocytomas but not in reactive conditions [6]. Of 57 non-neoplastic conditions, none showed IDH1/2 mutations. In contrast, 67.3% of grade II and grade III diffuse gliomas did; in addition, in a small subset of gliomas, IDH mutations were demonstrated in the infiltrative edge of the tumor, an area represented in the “near miss” scenario in stereotactic biopsy. Despite the promise of IDH mutations as a tumor-specific marker, not all institutions have ready access to mutation detection methods, and DNA extraction followed by sequencing may be problematic in very small biopsies. Furthermore, IDH1 immunohistochemistry, using an antibody specific to the common R132H mutant form of IDH1, may be more sensitive than sequencing to detect tumors with mutations [4]. Using R132H mutant IDH1 and p53 immunohistochemistry, we therefore studied 21 samples of WHO grade II diffuse astrocytoma and 20 samples of reactive conditions (10 resections for epilepsy, 7 infarcts, 2 evacuated hematomas and 1 traumatic brain injury), all surgical biopsy specimens. The mean patient age for low-grade astrocytomas and reactive cases was 33.4 and 32.5 years, respectively. Immunohistochemical staining for IDH1 was done on BenchMark XT automated tissue staining systems (Ventana Medical Systems, Inc., Tucson, AZ) using validated protocols. Endogenous peroxidase activity was blocked by H2O2 and antigen retrieved using CC1 reagent (Ventana Medical Systems). After washing, tissue sections were incubated with mouse monoclonal anti-R132H-IDH1 antibody culture supernatant, followed by incubation with UltraView HRP-conjugated multimer antibody reagent (Ventana Medical Systems). Antigen detection was performed using UltraView diaminobenzidine chromogen step (Ventana Medical Systems). Tissues were counterstained with hematoxylin and scored independently by two investigators (SC-P, MJ). Immunohistochemical staining for p53 was performed using a mouse monoclonal antibody (Santa Cruz, CA; # SC47698) using standard protocol. Positive granular cytoplasmic staining of tumor cells for mutant IDH1 was found in 9 out of 21 (42.9%) WHO grade II astrocytomas, but was entirely absent in all 20 reactive samples (Fig. 1). Positive nuclear staining of tumor cells with p53 was found in 10 out of 21 (47.6%) astrocytomas; of 20 reactive cases, none showed nuclear staining in astrocytes, but one showed positive nuclear signal in macrophages (CD68-positive). Five tumors showed co-expression of mutant IDH1 and p53. When used together, mutant IDH1 and p53 demonstrated the presence of tumor in 14 out of 21 (66.7%) cases. Fig. 1 R132H mutant IDH1 immunohistochemistry in WHO grade II astrocytoma and astrocytosis. Strong granular cytoplasmic mutant IDH1 staining in cellular area of astrocytoma (a HE b mutant IDH1) and in infiltrating tumor cells (c HE d mutant ... To date, detection of IDH mutations in low-grade astrocytomas (WHO grade II) ranges from 59 to 88% [1, 5, 7, 11, 12]; as expected, the use of an antibody specific only for the R132H mutant IDH1 resulted in a slightly lower detection rate. However, in positive cases, tumor cells demonstrated staining both in the densely cellular areas of the tumor, as well as in the less cellular infiltrating tumor edges. This latter finding is important since the less cellular areas of tumors can be the most difficult to differentiate from non-neoplastic conditions in a stereotactic biopsy and may not yield sufficient tumor DNA after extraction to allow mutant IDH1 detection by sequencing. We therefore demonstrate, for the first time, that use of immunohistochemistry with an antibody specific for the common mutant form of IDH1 is a powerful and easy adjunct to practical neuropathological diagnosis. The antibody is likely to find its place quickly alongside that of p53 in such cases. Indeed, our data further suggests that when p53 is used concomitantly with mutant IDH1, the ability of immunohistochemistry to confirm the morphologic impression of glioma is enhanced. These findings also illustrate the increasing rapidity with which molecular assays are being converted to immunohistochemical stains. In the diagnosis of atypical teratoid/rhabdoid tumor, the transition from fluorescence in situ hybridization for chromosome 22q loss, to INI1 gene sequencing, to INI1 immunohistochemistry took well over 10 years; today, INI1 immunohistochemistry represents the commonly and widely used method. In the case of IDH1, scarcely more than one year has passed between discovery of mutations in diffuse gliomas and the implementation of diagnostic IDH1 immunohistochemistry—attesting to the quickening pace of diagnostic change.