Growth characteristics of glioblastoma spheroids.

Cell spheroids have been proposed as models of early tumor growth from which a better understanding of tumor cell heterogeneity and its effects on treatment response might be gained. Results of experiments performed to understand the underlying dynamics of cell growth within a spheroid formed by SNB19, a high-grade glioblastoma cell line, are presented. We discuss the spatiotemporal distribution of the cells and their cell cycle status based on physical measurements, immunohistochemistry, and flow cytometry analysis. The size of the spheroids and their growth rates were dependent on the initial cell number, the proliferation was mostly limited to the outermost region as the spheroids grew in size, and the number of dead cells increased with age and size as well. Interestingly, though the population of the proliferating cells became localized to the outer rim as spheroids grew, the fraction of proliferating cells did not change drastically. Also, our data reveal that the calculated density varied with respect to age of the spheroid as well as position within the spheroid. We show that a simple exponential model is not adequate for modelling the growth characteristics that have been seen by these experiments. In contradiction to available studies, we report that an acellular (necrotic) center appeared and then disappeared during the period of investigation. Furthermore, after the acellular region disappeared, a few proliferative cells appeared in the center area, raising many questions about the growth-related dynamics of the spheroids formed by this particular cell type.

Radiation-induced morphologic changes in the rhesus monkey (Macaca mulatta) brain.

The right cerebral hemisphere of 24 rhesus monkeys scheduled for necropsy at the completion of another project were studied histopathologically 1-30 days after a single dose of 60Co-irradiation. Histopathologically, inflammation and gliosis consistently occurred at specific time points but varied in severity between individuals. Multifocal hemorrhage, edema, and an acute neutrophilic inflammatory response were observed initially whereas perivascular accumulations of lymphocytes were observed in specimens at the end of the study. Microglia/macrophages were most prominent during the first week after irradiation, whereas astrocytes were reactive throughout the observation period. The early clinical manifestations of the central nervous system (CNS), because of brain irradiation in humans, correspond temporally with acute vascular responses, acute and subacute inflammatory cell responses, and subacute demyelination and reactive astrocytic and microglial responses observed in the rhesus monkey. Initial responses of the CNS to gamma-irradiation may have potential implications for the development of radiation-induced late injury of the CNS.

Differential expression of MMAC/PTEN in glioblastoma multiforme: relationship to localization and prognosis.

MMAC/PTEN, a tumor suppressor gene located on chromosome 10q, has recently been shown to act as a phosphatidylinositol 3,4,5-triphosphate phosphatase and to modulate cell growth and apoptosis. Somatic mutations of MMAC/PTEN have been reported in a number of human cancers, especially in glioblastoma multiforme (GBM), although the number of identified mutations (approximately 10-35%) is significantly lower than the frequency of LOH affecting the MMAC/PTEN locus in the specimens (approximately 75-95%). To further investigate the possible alterations that may affect MMAC/PTEN, we examined the expression of the gene by reverse transcription-PCR in a series of gliomas. A significant difference (P < 0.001) was observed between the expression of MMAC/PTEN in GBMs versus lower grades of gliomas, thus mimicking the difference in allelic deletion associated with the locus in these tumors. Furthermore, Kaplan-Meier survival plots, adjusted for age and tumor grade, showed a significantly better prognosis for patients whose tumors expressed high levels of MMAC/PTEN. Additionally, immunostaining of GBMs revealed little or no MMAC/PTEN expression in about two-thirds of the tumors, whereas the other approximately one-third of tumors had significantly higher levels of expression. However, in about two-thirds of the high-expressing specimens, a heterogeneous pattern of expression was observed, indicating that certain cells within the tumor failed to express MMAC/PTEN. The combination of these results suggest that, in addition to molecular alterations affecting the gene, altered expression of MMAC/PTEN may play a significant role in the progression of GBM and patient outcome.

Functional and molecular analyses of 10q deletions in human gliomas.

Extensive genomic deletions involving chromosome 10 are the most common genetic alteration in glioblastoma multiforme (GBM). To localize and examine the potential roles of two chromosome arm 10q tumor suppressor regions, we used two independent strategies: mapping of allelic deletions, and functional analysis of phenotypic suppression after transfer of chromosome 10 fragments. By allelic deletion analysis, the region of 10q surrounding the MMAC/PTEN locus was shown to be frequently lost in GBMs but maintained in most low-grade astrocytic tumors. An additional region at 10q25 containing the DMBT1 locus was lost in all grades of gliomas examined. The potential biological significance of these two regions was further assessed by examining microcell hybrids that contained various fragments of 10q. Somatic cell hybrid clones that retained the MMAC/PTEN locus have a less transformed phenotype with clones exhibiting an inability to grow in soft agarose. However, presence or absence of DMBT1 did not correlate with any in vitro phenotype assessed in our model system. These results support a model of molecular progression in gliomas in which the frequent deletion of 10q25-26 is an early event and is followed by the deletion of the MMAC/PTEN during the progression to high-grade GBMs.

Allelic deletion analyses of MMAC/PTEN and DMBT1 loci in gliomas: relationship to prognostic significance.

The frequency of loss of heterozygosity (LOH) around MMAC/PTEN and DMBT1 loci and survival analyses based on the LOH status were assessed in 110 patients with different histological groups of gliomas. Twenty-six of the patients had anaplastic oligodendrogliomas, 31 had anaplastic astrocytomas, and 53 had glioblastomas multiforme (GM). At the DMBT1 locus, LOH was observed very frequently in all three histological groups, with no significant difference in the frequency of LOH among the three histological groups. At the MMAC/PTEN locus, patients with GM exhibited a significantly increased frequency of LOH (72%) compared with patients with anaplastic astrocytomas (29%) or anaplastic oligodendrogliomas (31%) (P < 0.0001). Kaplan-Meier survival plots showed that patients with LOH at the MMAC/PTEN locus had a significantly worse prognosis than did patients without LOH at the MMAC/PTEN locus [hazard ratio (LOH versus non-LOH), 2.65; 95% confidence interval (CI), 1.69-4.46; P < 0.0001]. Cox proportional hazards regression analysis, adjusted for age at surgery and histological grades (GM and non-GM), showed that LOH at the MMAC/PTEN locus was a significant predictor of shorter survival [hazard ratio (LOH versus non-LOH), 2.01; 95% CI, 1.1-3.5; P = 0.018). Our analysis failed to indicate a similar association between the frequency of LOH at the DMBT1 locus and patient survival [hazard ratio (LOH versus non-LOH), 2; 95% CI, 0.37-3.13; P = 0.2]. These results suggest that the DMBT1 gene may be involved early in the oncogenesis of gliomas, whereas alterations in the MMAC/PTEN gene may be a late event in the oncogenesis related to progression of gliomas and provide a significant prognostic marker for patient survival.

Sulfonylurea receptor mRNA expression in pituitary macroadenomas.

ATP-sensitive K+ (KATP) channels modulated by sulfonylurea compounds have been previously identified in the anterior pituitary of the rat and have been demonstrated to influence GH release. Recently, a sulfonylurea receptor (SUR) has been cloned from an islet cell tumor and identified as a member of the ATP binding cassette superfamily capable to coupling with inwardly rectifying potassium channels. To determine if the same receptor is expressed in pituitary tumors, SUR mRNA levels were measured in 28 human macroadenoma specimens using an RNase protection assay. All immunonegative, corticotrophin (ACTH), growth hormone (GH), and GH/prolactin (GH/Prl) immunostaining tumors expressed detectable amounts of SUR message. Among these tumors, only the GH and GH/ Prl adenomas were functional. Of the tumors immunostaining for luteinizing hormone (LH), follicle-stimulating hormone (FSH), or both, SUR mRNA was present in small amounts in 5/11. Only 1/3 Prl immunostaining tumors contained SUR mRNA. In summary, we have demonstrated that SUR mRNA expression is common in several types of silent pituitary adenomas and in functional tumors that secrete GH. Lower levels are seen in some gonadotrophin immunostaining tumors.

Ependymomas: MIB-1 proliferation index and survival.

The biologic behavior of ependymomas is highly variable, and its correlation with histologic features is at best imprecise. This retrospective study attempted to correlate the malignant histologic characteristics of ependymomas with MIB-1 proliferation index and survival. Biopsy and resection specimens taken from 34 patients who received treatment 1972 to 1996 were histologically examined. The patients' ages range was 1 to 59 years. The histologic specimens were assessed for anaplastic features (necrosis, mitosis, vascular proliferation, cellular pleomorphism, and overlapping of nuclei) and an MIB-1 (Ki-67 antigen) proliferation index was also determined. The overall median MIB-1 proliferation index was 7.8% (range 0.1 - 62.5%). An MIB-1 of 20% was significant for a decrease in survival (RR = 5.7) (p = 0.0013). The median MIB-1 for patients < 20 years old was 20.6% with range (0.1, 43%), while that for patients > 20 years was 5.1% (range 0.2, 9.4%) (KW p = 0.055). Three of 5 histological features evaluated were significantly associated with outcome: > 5 mitotic figures per high-power field, necrosis, and vascular proliferation, but not nuclear overlap or pleomorphism. All pathologic factors except pleomorphism were significantly related to the MIB-1 proliferation index. In brief, our data support the association of poor prognoses in ependymomas with young age, the presence of three to four anaplastic histologic features, and an MIB-1 proliferation index > 20%.

MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines.

A candidate tumor suppressor gene, MMAC1/PTEN, located in human chromosome band 10q23, was recently identified based on sequence alterations observed in several glioma, breast, prostate, and kidney tumor specimens or cell lines. To further investigate the mutational profile of this gene in human cancers, we examined a large set of human tumor specimens and cancer cell lines of many types for 10q23 allelic losses and MMAC1 sequence alterations. Loss of heterozygosity (LOH) at the MMAC1 locus was observed in approximately one-half of the samples examined, consistent with the high frequency of 10q allelic loss reported for many cancers. Of 124 tumor specimens exhibiting LOH that have been screened for MMAC1 alterations to date, we have detected variants in 13 (approximately 10%) of these primary tumors; the highest frequency of variants was found in glioblastoma specimens (approximately 23%). Novel alterations identified in this gene include a missense variant in a melanoma sample and a splicing variant and a nonsense mutation in pediatric glioblastomas. Of 76 tumor cell lines prescreened for probable LOH, microsequence alterations of MMAC1 were detected in 12 (approximately 16%) of the lines, including those derived from astrocytoma, leukemia, and melanoma tumors, as well as bladder, breast, lung, prostate, submaxillary gland, and testis carcinomas. In addition, in this set of tumor cell lines, we detected 11 (approximately 14%) homozygous deletions that eliminated coding portions of MMAC1, a class of abnormality not detected by our methods in primary tumors. These data support the occurrence of inactivating MMAC1 alterations in multiple human cancer types. In addition, we report the discovery of a putative pseudogene of MMAC1 localized on chromosome 9.

Telomerase activity in ordinary meningiomas predicts poor outcome.

Telomerase, the enzyme that stabilizes telomere length, is reactivated with almost all cancer types, and it may be necessary for unlimited cell proliferation. Assessment of malignancy in ordinary meningiomas is inconclusive because no clear-cut correlation exists between aggressive clinical behavior and histological features or karyotypic abnormalities. We analyzed telomerase activity in 52 cases of meningioma by using the highly sensitive telomeric repeat amplification protocol and then compared clinical behavior in telomerase-positive and -negative ordinary meningiomas. Twenty-six of the 52 tumors (50%) had detectable telomerase activity. Twenty-one of the 22 neoplasms classified as malignant or atypical (95%) had detectable telomerase activity, and these tumors generally had a poor outcome. Interestingly, 5 of 30 ordinary (morphologically benign) meningiomas (17%) also showed detectable telomerase activity. Of the 5 patients with telomerase-positive ordinary meningiomas, 3 had rapid regrowth of the tumor despite gross total resection. The remaining 2 patients also had other primary malignancies. We observed a highly significant correlation in ordinary meningiomas between the presence of telomerase activity and a poor prognosis for the patient (P = .0002). Telomerase activity in benign meningiomas is clinically relevant because the presence of the enzyme suggests that these benign-appearing tumors may contain a population of immortal cells. The detection of telomerase activity may help to identify benign meningiomas that would be more likely to continue to grow and to recur clinically if surgical resection were incomplete.

Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.

Deletions involving regions of chromosome 10 occur in the vast majority (> 90%) of human glioblastoma multiformes. A region at chromosome 10q23-24 was implicated to contain a tumour suppressor gene and the identification of homozygous deletions in four glioma cell lines further refined the location. We have identified a gene, designated MMAC1, that spans these deletions and encodes a widely expressed 5.5-kb mRNA. The predicted MMAC1 protein contains sequence motifs with significant homology to the catalytic domain of protein phosphatases and to the cytoskeletal proteins, tensin and auxilin. MMAC1 coding-region mutations were observed in a number of glioma, prostate, kidney and breast carcinoma cell lines or tumour specimens. Our results identify a strong candidate tumour suppressor gene at chromosome 10q23.3, whose loss of function appears to be associated with the oncogenesis of multiple human cancers.

Late sequelae of treated pleomorphic xanthoastrocytoma: malignant brain stem astrocytoma occurring 15 years after radiation therapy.

The pleomorphic xanthoastrocytoma (PXA) is a unique astrocytic neoplasm with an unexpectedly favorable prognosis despite striking pleomorphism of the cellular constituents. Although a majority of patients experience extended survival, these tumors may recur and some cases progress to high-grade astrocytoma. Recurrence inevitably involves the anatomic vicinity of the primary tumor. In this report, we describe a malignant brain stem astrocytoma that occurred 15 years after surgery and radiation treatment of a 16-year-old patient who had a temporal lobe PXA. To our knowledge, this is the first reported case of a malignant astrocytoma arising outside the primary anatomic site of a previously treated PXA and likely represents a radiation-induced secondary neoplasm.

Tanycytic ependymoma.

Tanycytic ependymoma is an uncommon fibrillar variant of ependymoma characterized by streams of piloid, or hair-like, cells having "ependymal" nuclei. True ependymal rosettes are absent, and perivascular rosettes are inconspicuous. Misinterpretation as schwannoma or astrocytoma is a diagnostic problem and well-documented cases are scarce. The purpose of this report is to document the ependymal features of the neoplasm and to increase awareness of the entity's existence. Biopsy tissues from three patients with tanycytic ependymoma were examined. All tumors consisted of sheets of spindle cells that were positive for glial fibrillary acidic and S-100 proteins. Ultrastructural examination showed characteristic ependymal features, including intracytoplasmic intermediate filaments, prominent intercellular junctions, numerous slender surface microvilli, and microvilli-lined lumina. Accurate recognition of the ependymal nature of this spindle neoplasm requires a high index of suspicion. Because the spindle cells are immunoreactive with antibodies to both glial fibrillary acidic and S-100 proteins, ultrastructural confirmation of ependymal features is necessary.

Diagnostic discrepancies and their clinical impact in a neuropathology referral practice.

BACKGROUND: During the course of their neuropathology practice, the authors received cases to review in consultation. In some cases, the patients came to the authors' hospital for therapy; in others, the primary pathologists requested a consultation. Because changes in diagnosis might significantly alter patient management, protocol entry, care costs, or the potential for physician liability, the authors determined the frequency and degrees of their disagreements with the original diagnoses submitted to them. METHODS: The authors reviewed the first 500 brain or spinal cord biopsy cases that were submitted to their neuropathology consultation service for a second opinion in 1995. Disagreements were coded into 10 categories, but were grouped for this analysis as follows: serious (having immediate significance for therapy or intervention), less serious but potentially substantial (calling for a change in type or grade of glioma), minor (adding or deleting information), and those in which the authors made the first diagnosis themselves. RESULTS: There was some degree of disagreement between the original and review diagnoses in 214 (42.8%) of the 500 cases. Disagreements were counted as serious in 44 cases (8.8%), less serious but substantial in 96 cases (19.2%), and minor in 50 cases (10.0%); the authors made the first diagnosis in 24 cases (4.8%). CONCLUSIONS: Clinically important diagnostic errors that can affect immediate patient care decisions occur in a substantial number of brain and spinal cord biopsy cases. Thus, seeking expert neuropathology consultation is prudent and cost-effective for pathologists who are less experienced with these types of cases. Cost savings in case management might result from confirmation of diagnosis before definitive therapy is administered to the patients. The rates of discrepancy between original diagnoses and second opinions in other subspecialties of pathology should be examined.

Resolution of recurrent malignant ganglioglioma after treatment with cis-retinoic acid.

Ganglioglioma is an uncommon brain tumor with glial and neuronal cellular components and a somewhat benign course. We are presenting an unusual case of ganglioglioma with malignant transformation in both cellular components associated with an aggressive clinical course. An almost complete resolution of the recurrent progressing tumor was achieved after treatment with cis-retinoic acid (cRA) as a single agent. A possible differential effect of cRA on the neuronal component of the tumor is suggested.

Pathology of meningiomas.

Because meningiomas arise from arachnoid cells present in the meninges, they can occur in any location where meninges or ectopic meninges exist, such as the nasal cavity, the paranasal sinuses, the middle ear, and even the mediastinum. Although the tumors may range in appearance from epithelial to mesenchymal, they are characterized by a uniform distribution of cells with shapes ranging from polygonal epithelial-like to spindled and fusiform. Historically, classification of meningiomas has been based upon cell shapes, cell patterns, cell products, or stroma, implying clinicopathologic differences among the types. Numerous observations have shown that certain conditions may indicate a predisposition for developing meningiomas, prompting extensive studies of meningiomas using cytogenetic techniques. Meningiomas are common neoplasms arising from the central nervous system meninges. They are important because of the morbidity they produce. Their critical intracranial and intraspinal locations make diagnosis and surgical removal difficult.

Glioblastoma multiforme arising in the irradiated spinal cord of a rhesus monkey (Macaca mulatta).

An adult female rhesus monkey that had received 44.0 Gy of cobalt 60 radiation to 8 cm of the cervical and upper thoracic spinal cord approximately 2.8 years postirradiation developed a sudden onset of self-mutilation and loss of function of the right arm followed progressively by loss of function of the left arm and terminally bilateral paresis of the legs. Histopathologic examination of the cervical spinal cord revealed a glioblastoma multiforme that extended from the cervical medullary junction to the sixth cervical vertebrae. Because of the infrequent occurrence of spontaneous neoplasia in rhesus monkeys and the location in the radiation field, the glioblastoma is believed to be radiation induced.

Comparison of MIB-1 (Ki-67) antigen and bromodeoxyuridine proliferation indices in meningiomas.

Meningiomas from 40 adult patients were labeled immunohistochemically with monoclonal antibodies to bromodeoxyuridine (BUdR) and the Ki-67 antigen, MIB-1. The meningiomas were classified as classical, or benign (n = 31); atypical (n = 4); or malignant (n = 5). Meningeal sarcomas and hemangiopericytomas were excluded. The patient population consisted of 26 women and 14 men, ranging in age from 26 to 75 years. BUdR proliferation indices ranged from 0% to 5.8%, measurements that were expectedly lower than those for MIB-1, which ranged from 1.5% to 19.3%. MIB-1 proliferation indices were not significantly affected regarding steroid pretreatment or age. These results show a good correlation between the BUdR and MIB-1 proliferation markers (rs = 0.72; P < .0001), which supports the use of anti-MIB-1 as an alternative labeling tool to BUdR for the determination of the proliferation index in meningiomas, thus avoiding the administration of a potentially mutagenic drug.

Central nervous system neoplasms: indications for electron microscopy.

Diagnostic dilemmas of biopsy specimens in the central nervous system (CNS) tumors are often the result of multiple factors, including fixation artifact, biopsy size, lack of immunohistochemical techniques to distinguish cell types, and unawareness of rare entities. Correct diagnosis and confirmation of diagnosis of primary CNS neoplasms is imperative and may require electron microscopic examination. In some instances, use of electron microscopy may be the only approach for accurate recognition of an entity. Although diagnostic electron microscopy is expensive and cost cutting is encouraged in today's practice of medicine, cost must be weighed against the consequences of even 1 patient developing CNS treatment-related necrosis or a radiation-induced neoplasm secondary to misdiagnosis of a benign entity. This study reviews the ultrastructural differences of three groups of diagnostically difficult CNS lesions: clear cell neoplasms (ependymoma, oligodendroglioma, central neurocytoma), rare entities containing astrocytes invested by a basal lamina (pleomorphic xanthoastrocytoma, the desmoplastic neuroepithelial tumors of infancy), and benign entities characterized by transitional cell forms (subependymoma, subependymal giant cell astrocytoma).

Telomerase activity in human brain tumours.

Malignant gliomas are invasive into surrounding brain and are refractory to therapy. Telomerase stabilises telomere length and may immortalise cells to allow unlimited proliferation. Our analysis of telomerase activity in 90 human gliomas showed that 19 of 19 oligodendrogliomas and 38 of 51 glioblastoma multiformes have detectable telomerase activity. The absence of telomerase activity in anaplastic astrocytomas (2/20 positive) and in one-quarter (13/51) of the glioblastomas suggests that these tumours follow different pathways of neoplastic progression. Thus we have found that a distinct subgroup of brain tumour consists of transformed yet pre-immortal cells.