Small cell architecture--a histological equivalent of EGFR amplification in glioblastoma multiforme?

Although there is much written about the molecular definitions of "primary" glioblastomas (GBM), there is little known about the histological features of this predominant subtype. We hypothesized that the "small cell architecture" would represent a histological feature of most primary GBMs. This was tested by comparing the presence of the small cell phenotype with the presence or absence of amplification of the epidermal growth factor receptor (EGFR), a common event in primary GBMs. After a pilot study that found a correlation between this small cell phenotype and EGFR amplification, we selected 9 pure small cell GBMs (SCGBM) and 12 non-SCGBMs to be studied for EGFR amplification by fluorescence in situ hybridization (FISH). In this set of 21 cases, 8 of 9 SCGBMs and 5 of 12 non-SCGBMs were amplified for EGFR. We then correlated the EGFR status of 79 GBMs unselected for their histological features from a set that had been previously characterized in regard to EGFR amplification. Fourteen of 21 (67%) exclusively small cell neoplasms, 8 of 25 (32%) GBMs with both small cell and non-small cell areas, and 3 of 33 (9%) non-small cell GBMs were amplified for EGFR (p = 0.0004 with an exact test). We conclude that EGFR amplification is associated with a small cell phenotype in GBMs and that SCGBMs are an important component of "primary" GBMs.     

Glioblastoma-related gene mutations and over-expression of functional epidermal growth factor receptors in SKMG-3 glioma cells

Amplification of the epidermal growth factor receptor (EGFR) gene is found in about 40% of glioblastomas (GBMs) but is rarely detected in GBM cell lines. We confirmed that the exceptional SKMG-3 GBM cell line retained amplified EGFR genes in vitro, and found that these sequences were concentrated on extra-chromosomal DNA particles similar to double-minute chromosomes. The cells contained two other gene mutations that are associated with high-grade astrocytic tumors: extra-chromosomal amplification of the cyclin-dependent kinase-4 (CDK4) gene and a homozygous mutation within the PTEN tumor suppressor gene. Immunoblots revealed very high levels of EGFR, moderately increased expression of CDK4, and no detectable PTEN protein. The overexpressed SKMG-3 EGFRs responded to exogenous ligand and resembled normal rather than mutant receptors. A heterozygous mutation of the p53 gene (p53R282W) correlated with failure of radiation to induce the expression of cyclin-dependent kinase inhibitor p21waf1 or an early G1 cell cycle arrest. Although each of these gene mutations occurs in GBMs, SKMG-3 cells had an unusual genotype in that a p53 gene mutation co-existed with amplified EGFR genes. Nonetheless, the SKMG-3 cell line can be exploited as a model to study how oncogenic EGFR signals in GBM cells interact with over-expressed CDK4 and loss of PTEN to confer the malignant phenotype.     

DJ‐1 expression in glioblastomas shows positive correlation with p53 expression and negative correlation with epidermal growth factor receptor amplification

DJ‐1, a protein that promotes the action of multiple anti‐apoptotic/pro‐survival pathways, is expressed prominently in human reactive astrocytes and in many human cancers. Glioblastomas (GBMs) are the most common adult primary brain tumor, and most show either abnormalities in p53 or epidermal growth factor receptor (EGFR) amplification, but not both. In this retrospective study of 40 surgically resected GBMs, we compared the immunohistochemical intensity of DJ‐1 expression (based on blinded scoring by independent examiners) to these and other molecular factors associated with GBM oncogenesis. We report here that: (i) most of the GBMs that we studied expressed DJ‐1 protein at significant levels, and typically in a cytoplasmic, non‐nuclear fashion; (ii) DJ‐1 staining intensity varied directly with strong nuclear p53 expression (assessed by immunostaining); and (iii) DJ‐1 staining intensity varied inversely with EGFR amplification (assessed by fluorescent in situ hybridization). Since the anti‐apoptotic/pro‐survival actions of DJ‐1 have been clearly linked in in vitro systems to p53 and receptor tyrosine kinase (i.e. EGFR) pathways that are hypothesized to be critical to GBM genesis, these observations indicate that DJ‐1 expression may play a role in the biology of some types of GBMs. Therefore, given the new associations presented here between DJ‐1, p53 and EGFR amplification in GBMs, future investigations of these tumors should include an analysis of DJ‐1 to determine whether its expression pattern is important for tumor progression, prognosis and responsiveness to therapy.     

Association of chromosome 7, chromosome 10 and EGFR gene amplification in glioblastoma multiforme

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity in both histomorphological and genetic changes, displaying a wide variety of numerical chromosome aberrations, the most common of which are trisomy 7 and monosomy 10. The amplification of the epidermal growth factor receptor (EGFR) gene is the most frequently reported genetic abnormality. The associations between these parameters and their implication in the tumoral progression are poorly understood. We performed simultaneous fluorescence in situ hybridization (FISH) with centromeric DNA probes for chromosomes 7 and 10 in smear preparations, and EGFR gene amplification by PCR from 25 cases of GBM. Trisomy/ polysomy for chromosome 7 was present in 76% of cases and monosomy 10 in 68%. Both alterations were associated in 56% of cases. The EGFR gene was amplified in 52% of tumors; in 44% associated with trisomy/ polysomy 7, and in 36% with monosomy 10. The three parameters were associated together in 28% of cases. Kaplan-Meier survival rate analysis demonstrated lower survival rates in patients with monosomy 10, trisomy 7, and monosomy associated with trisomy 7. The other combinations were not different in frequency in relation to survival. In the present study, trisomy/polysomy 7 and monosomy 10 have been found to be frequently associated. The combination of both anomalies is probably important in the tumorigenesis of glioblastoma. Moreover, this association is apparently independent of EGFR gene amplification, which could be a later event in this process.     

EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.     

Molecular analysis of microdissected de novo glioblastomas and paired astrocytic tumors

Glioblastoma multiforme (GBM) often displays morphological heterogeneity in that low-grade (LG) area with well-differentiated cells are commonly found adjacent to high-grade (HG) area with poorly-differentiated cells. This heterogeneity may cause difficulty in obtaining representative tumor samples. Nevertheless, the genetic composition of these cells has only been occasionally examined. In the present study, we examined 29 de novo glioblastomas in which distinct LG and HG areas of sufficient volumes could be identified. These areas were microdissected from paraffin-embedded tissues and analyzed for genetic alterations: p53 mutations and immunohistochemistry; allelic losses at 17p13.1, 9p21, and 10q23-25; and amplification of the epidermal growth factor receptor (EGFR) gene and immunohistochemistry. We also examined 14 paired astrocytic tumors, in which a primary Grade II astrocytoma progressed over a period of time to a Grade III or Grade IV tumor. Our findings showed that the LG areas of the de novo glioblastomas exhibited numerous genetic aberrations, the proportion of which was increased in the HG areas. Genetic abnormalities seen in the LG areas were conserved in the HG areas suggesting that these morphologically different cellular subsets were derived from a common transformed clone. Also, the LG areas were genetically different from Grade II astrocytomas of the paired tumor group, in spite of their morphological similarity. In particular, the LG areas had more deletions on 10q23-25 (75% vs 20%, p = 0.04), but fewer p53 mutations (24% vs 71%, p = 0.003) and less p53 protein labeling (45% vs 79%, p = 0.04). These differences suggest that LG and HG areas in de novo glioblastoma are genetically closer to each other compared with paired low- and high-grade tumors that have progressed over time. Moreover, only a small proportion (17%) of our de novo glioblastomas exhibited EGFR amplification while a high proportion (62%) showed either p53 mutations or allelic loss of 17p13.1. We speculate that some de novo GBMs with copious LG areas may constitute a separate group with rapid progression from Grade II astrocytomas.     

Clinical utility of fluorescence in situ hybridization (FISH) in morphologically ambiguous gliomas with hybrid oligodendroglial/astrocytic features

Gliomas with hybrid oligodendroglial/astrocytic features are diagnostically problematic, and our ability to predict tumor behavior is limited. Some likely represent intermingled mixed oligoastrocytomas (MOAs), though precise diagnostic criteria and specific markers for this lesion are lacking. From the files at Washington University (1987-2000), 155 "ambiguous" glioma/intermingled MOA candidates were independently classified and graded by 5 neuropathologists, with consensus-derived pure oligodendrogliomas and astrocytomas excluded from further study. The 90 remaining cases (grades II = 29, III = 44, IV = 17) were analyzed by FISH on formalin-fixed, paraffin-embedded sections. Detectable deletions included combined 1p/19q (9%), solitary 19q (22%), PTEN/DMBT1 (26%), and p16 (32%). EGFR amplification was found in 11%. Patients were followed until death (47%) or a median of 3.3 years. Similar to prior glioma series, patient age (p < 0.0001) and tumor grade (p < 0.0001) were strongly associated with survival times. EGFR amplification (p = 0.0007) and deletions of PTEN/ DMBT1 (p = 0.016) or p16 (p = 0.014), either individually or as a group (p = 0.04), portended a shorter median survival compared with tumors lacking these alterations. We conclude that 1) distinct genetic subsets are identifiable by FISH in morphologically ambiguous gliomas, and 2) both histological grading and molecular analysis yield prognostically useful information.     

Differential expression of sst1, sst2A, and sst3 somatostatin receptor proteins in low-grade and high-grade astrocytomas

We have previously reported that sst2A somatostatin receptors are frequently overexpressed in human meningiomas. Initial clinical observations suggest that somatostatin analogues may also be of value for imaging and treatment of other human intracranial tumors, including astrocytomas. However, contradictory results have been reported regarding the expression of somatostatin receptors in low-grade and high-grade astrocytomas. Therefore, we determined the precise pattern of somatostatin receptor protein expression in 8 diffuse astrocytoma (DA), 10 anaplastic astrocytomas (AA), and 32 glioblastoma multiforme (GBM) using immunohistochemistry and Western blot analysis. sst1 and sst2A somatostatin receptors were not present in DA and only occasionally detected in AA. In GBM, sst1 was present in 66%, and sst2A was found in 44% of the tumors. sst3 receptors were present in 38% of DA, 40% of AA, and 84% of GBM. Thus, loss of differentiation was significantly associated with increased expression of sst1, sst2A, and sst3 somatostatin receptors. In contrast, sst4 and sst5 receptors were found in 80% and 25% of all cases, respectively, in a manner independent of histological grade. No significant correlation was found between somatostatin receptor expression and the proliferation rate of the tumors as determined by MIB-I immunostaining. Furthermore, the presence or absence of the 5 somatostatin receptor subtypes did not significantly influence survival time in 14 GBM patients.     

Management,outcomes, and prognostic factors of adult primary spinal cord gliomas

PurposePrimary spinal cord tumors are rare, particularly in the adult population, and national guidelines remain ambiguous with regard to management approaches. To address this knowledge gap, we evaluated management, outcomes, and prognostic factors of these neoplasms.MethodsThe National Cancer Database was queried (2004–2016) for newly-diagnosed, histologically-confirmed WHO grades I-III astrocytomas and glioblastoma. Statistics included Kaplan-Meier overall survival (OS) analysis, along with Cox proportional hazards modeling.ResultsOf 1,033 subjects, 196 (19%) were pilocytic astrocytomas (PAs), 539 (52%) were grade II/III astrocytomas, and 298 (29%) were glioblastomas (GBMs). Respectively, 11%, 30%, and 27% did not undergo resection (biopsy only). RT was delivered to 27%, 54%, and 73%; chemotherapy was given to 5%, 21%, and 37%, respectively. The median OS was not reached for PAs, but was 101.2 months for grade II/III astrocytomas, and 23.9 months for GBMs (p < 0.001). Neither chemotherapy nor RT (or dose thereof) was associated with increased OS for grade II/III astrocytomas (p > 0.05 for all), though there was a trend toward improved OS with the use of chemotherapy for patients with GBM. Surgical resection was associated with improved OS for grade II/III astrocytomas and GBM (p < 0.05). Independent prognostic factors for survival in this cohort included histologic classification and resection (compared to biopsy only) (p < 0.05 for both).ConclusionsThis study sheds light onto the management of these rare tumors; surgery was associated with OS benefit for patients with GBM and Grade II/III astrocytomas. Neither RT nor chemotherapy were associated with OS benefit. Although not implying causation, these data can be used to guide patient counseling and therapeutic approaches.     

Molecular genetics of radiographically defined de novo glioblastoma multiforme

Glioblastoma multiforme (GBM) represents the final endpoint of anaplastic progression in astrocytomas. GBM which arise without clinical evidence of a prior low-grade astrocytoma (LGA) have been designated de novo GBM, and are thought to develop rapidly from initial tumour formation. However, a purely clinical definition of de novo GBM does not exclude a long-standing, asymptomatic low-grade tumour. This study therefore sought to determine the genetic features of a unique group of cases in which GBMs were documented to have arisen radiographically in defined period of time (radiographically defined de novo GBM). Clinical and genetic features were examined in a group of 11 patients with a histological diagnosis of high-grade astrocytoma at first biopsy and radiographically defined de novo GBM. The mean age of the patients at tumour diagnosis was 62 years (range 32-87). Six of 11 tumours arose in the temporal lobes. Eight of 11 tumours had epidermal growth factor receptor (EGFR) overexpression, and EGFR gene amplification was found in five of the six analysed cases. Overexpression of p53 was observed in only one tumour, and a TP53 mutation was present in this case. p16 immunostaining was undetectable in 10 cases, and homozygous deletion of CDKN2A was observed in four of the six studied tumours. pRb expression was lost in four tumours. Mutations in the PTEN gene were detected in two of six cases. The results in this unique group of cases confirms the prior hypothesis that the profile of genetic alterations in de novo GBM is distinct from that of GBM arising from a known LGA, and that these specific genetic pathways promote the rapid development of GBM.     

Amplification of the epidermal growth factor receptor in astrocytic tumours by chromogenic in situ hybridization: association with clinicopathological features and patient survival

Chromogenic in situ hybridization (CISH) was used to detect amplification of the epidermal growth factor receptor (EGFR) gene in tissue microarrays of tumours derived from 287 patients with grade II-IV diffuse astrocytomas. Amplification was found in 32% of the tumours with a highly significant association with histological grade (4% in grade II, 21% in grade III and 39% in grade IV; P < 0.001). Amplification of the EGFR gene was more common in primary than in secondary glioblastomas (41%vs. 16%, P = 0.033). Overexpression of EGFR mRNA and protein (wild-type and vIII variant) was found to correlate with EGFR gene amplification (P = 0.028, P = 0.035 and P = 0.014 respectively), but wild-type EGFR protein was also frequently overexpressed in tumours without EGFR gene amplification. Patients with older age (P < 0.001) and tumours with lack of p53 overexpression (P = 0.03) and higher apoptosis rate (P < 0.001) had significantly more EGFR gene amplifications than their counterparts. No such correlation with apoptosis was found in glioblastomas. The survival of patients with EGFR gene-amplified grade III tumours was significantly shorter than in those with grade III non-amplified tumours (P = 0.03). No such difference was noted in glioblastomas (grade IV tumours). Our data verify the central role of EGFR in the pathobiology of astrocytic tumours, and highlight the advantages of CISH as a simple and practical assay to screen for EGFR gene amplification in astrocytic tumours.     

Applications of emerging molecular technologies in glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Median survival from the time of diagnosis is less than a year, with less than 5% of patients surviving 5 years. These tumors are thought to arise through two different pathways. Primary GBMs represent de novo tumors, while secondary GBMs represent the malignant progression of lower-grade astrocytomas. Moreover, despite improvements in deciphering the complex biology of these tumors, the overall prognosis has not changed in the past three decades. The hope for improving the outlook for these glial-based malignancies is centered on the successful clinical application of current high-throughput technologies. For example, the complete sequencing of the human genome has brought both genomics and proteomics to the forefront of cancer research as a powerful approach to systematically identify large volumes of data that can be utilized to study the molecular and cellular basis of oncology. The organization of these data into a comprehensive view of tumor growth and progression translates into a unique opportunity to diagnose and treat cancer patients. In this review, we summarize current genomic and proteomic alterations associated with GBM and how these modalities may ultimately impact treatment and survival.     

Molecular genetic analysis of the TP53, PTEN,CDKN2A,EGFR, CDK4 and MDM2 tumour-associated genes in supratentorial primitive neuroectodermal tumours and glioblastomas of childhood

Supratentorial primitive neuroectodermal tumours (sPNETs) are malignant central nervous system tumours of childhood which are histologically characterized by poorly differentiated neuroepithelial cells with the capacity for divergent differentiation into glial, neuronal, myogenic or melanotic lines. The histological differential diagnosis between sPNET and glioblastoma multiforme (GBM) may be difficult, particularly as GBMs can sometimes demonstrate a poorly differentiated PNET-like phenotype. To identify molecular genetic markers that may distinguish sPNET and GBM, we investigated 12 cerebral sPNETs and six GBMs from paediatric patients for genetic alterations of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 genes, as well as for allelic loss on chromosome arms 10q and 17p. Mutations of the TP53 tumour suppressor gene were found in one of 12 sPNETs (8%) and two of six GBMs (33%). None of the sPNETs but two of six GBMs (33%, including one GBM with a TP53 mutation) showed allelic losses on chromosome arm 17p. PTEN mutations were detected in one of 12 sPNET (8%) and one of six GBMs (17%). None of the sPNETs and GBMs carried a homozygous deletion involving the CDKN2A tumour suppressor gene. No amplification of the EGFR, CDK4 or MDM2 proto-oncogenes was detected. Taken together, our results indicate that paediatric GBMs differ from sPNETs by a higher incidence of allelic losses on 17p and TP53 mutations. In addition, the patterns of genetic alterations in sPNETs and paediatric GBMs appear to be distinct from those in cerebellar medulloblastomas and adult GBMs, respectively.     

The complexity of the 7p12 amplicon in human astrocytic gliomas: detailed mapping of 246 tumors

Approximately 30%-35% of human glioblastomas have epidermal growth factor receptor (EGFR) gene amplification. Amplicons containing the EGFR gene frequently include several unidentified adjacent genes. Amplification of adjacent genes in the absence of EGFR amplification has been documented. To define the region in detail, we produced a YAC contig map, determining the orientation of the EGFR gene and the general order of 11 STS and EST markers. Seventy-six tumors with amplification of the region were found in a series of 246 human astrocytic gliomas. The amplicons showed amplification of contiguous or noncontiguous loci both telomeric and centromeric to the EGFR gene. Six percent (12/190) of the glioblastomas and 1 anaplastic astrocytomas had amplicons excluding the EGFR gene. These amplicons commonly contain amplified loci telomeric to the EGFR gene. Some of the amplified loci were found to be consistently overexpressed. The findings suggest that there may be other gene target(s) for amplification in the 7p12 region in addition to EGFR.     

A clinicopathological and molecular analysis of glioblastoma multiforme with long-term survival

The median survival time of patients with glioblastoma multiforme (GBM) is 12 months, and only 3-5% of patients survive longer than 3 years. We performed histomorphological and detailed molecular analyses of seven long-term survivors of GBM to identify any prognostic factors that potentially contribute to survival. Morphology and immunohistochemistry for p53, phosphatase and tensin homologue (PTEN) and epidermal growth factor receptor (EGFR) protein expression were investigated. EGFR amplification and 1p/19q deletion were assessed by fluorescent in situ hybridization. The O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status was evaluated by performing methylation-specific polymerase chain reaction assays. All tumors were classical GBMs and no significant oligodendroglial differentiation was noted. The majority showed EGFR amplification (4/7), PTEN protein expression (6/7) and MGMT promoter methylation (5/6). Immunopositivity for p53 was noted in three of seven patients. Deletion of chromosome 1p/19q, either isolated or combined, was not identified in any of the se patients. All patients were treated by gross total resection followed by radiotherapy; six patients received additional temozolomide treatment. A relatively young age of onset (48 years), with a high MGMT promoter methylation and PTEN protein expression were favorable factors for long-term survival. The presence of EGFR amplification indicates that more than a single factor determines survival in GBM.     

Glioblastoma multiforme with long term survival

Glioblastoma multiforme (GBM) Patients generally have a dismal prognosis, with median survival of 10-12 months. GBM with long-term survival (LTS) of (3) > or = 5 years is rare, and no definite markers indicating better prognosis have been identified till date. The present study was undertaken to evaluate GBMs with LTS in order to identify additional correlates associated with favourable outcome. The cases were evaluated for relevant clinicopathological data, proliferation index and expression of tumortumour suppressor gene (p53 ), cyclin-dependant kinase-inhibitors (p27 and p16 ) and epidermal growth factor receptor (EGFR) proteins. Six cases of GBM with LTS with an average survival of 9 years (range 5-15 years) were identified. All were young patients with mean age of 27 years (range 8-45 years). Histology of three cases was consistent with conventional GBM, while two showed prominent oligodendroglial component admixed with GBM areas. One was a giant cell GBM, which progressed to gliosarcoma on recurrence. The mean MIB-1LI was 12% (range 6-20%). p53 was immunopositive in 4 out of 5 cases. EGFR and p27 were immunonegative in all, whereas p16 was immunonegative in 3 out of 5 cases. Currently, in the absence of specific molecular and genetic markers, GBM in young patients should be meticulously evaluated for foci of oligodendroglial component and/or giant cell elements, in addition to proliferative index and p53 expression, since these probably have prognostic connotations, as evident in this study. The role of p16 and p27 however needs better definition with study of more number of cases.