Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions.

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.

Metronomic chemotherapy with daily, oral etoposide plus bevacizumab for recurrent malignant glioma: a phase II study.

BACKGROUND: We evaluated bevacizumab with metronomic etoposide among recurrent malignant glioma patients in a phase 2, open-label trial. METHODS: A total of 59 patients, including 27 with glioblastoma (GBM) and 32 with grade 3 malignant glioma, received 10 mg kg(-1) bevacizumab biweekly and 50 mg m(-2) etoposide daily for 21 consecutive days each month. The primary end point was a 6-month progression-free survival, and secondary end points included safety and overall survival. Vascular endothelial growth factor (VEGF), VEGFR-2, carbonic anhydrase 9 (CA9) and hypoxia-inducible factor-2alpha (HIF-2alpha) were assessed semiquantitatively in archival tumours using immunohistochemistry and were correlated with outcome. RESULTS: Among grade 3 and GBM patients, the 6-month progression-free survivals were 40.6% and 44.4%, the radiographic response rates were 22% and 37% and the median survivals were 63.1 and 44.4 weeks, respectively. Hypertension predicted better outcome among both grade 3 and GBM patients, whereas high CA9 and low VEGF were associated with poorer progression-free survival (PFS) among those with GBM. The most common grade > or = 3 adverse events included neutropaenia (24%), thrombosis (12%), infection (8%) and hypertension (3%). Two patients had asymptomatic, grade 1 intracranial haemorrhage and one on-study death occurred because of pulmonary embolism. CONCLUSION: Bevacizumab with metronomic etoposide has increased toxicity compared with previous reports of bevacizumab monotherapy. Its anti-tumour activity is similar to that of bevacizumab monotherapy or bevacizumab plus irinotecan. (ClinicalTrials.gov: NCT00612430).

Meningioma with eosinophilic granular inclusions.

Rare meningiomas have been described that contain eosinophilic inclusions that have a granular or granulofilamentous ultrastructure. We describe a 66-year-old woman who developed a planum sphenoidale meningioma. Histologically, the tumor was composed of meningothelial cells arranged in fascicles and whorls, typical of a well-differentiated meningioma. Many tumor cells contained round intracytoplasmic eosinophilic inclusions that were periodic acid Schiff-negative and red on Masson trichrome. The inclusions were immunopositive for vimentin, and were immunonegative for epithelial membrane antigen, smooth muscle actin, desmin and type IV collagen. Ultrastructural examination showed the inclusions were composed of round to oval, well-demarcated, non-membrane-bound, osmiophilic granular material. The inclusions within this tumor had histochemical, immunohistochemical and ultrastructural properties not described in other reported meningiomas with eosinophilic granular or granulofilamentous inclusions.

The pathology of extracranial scalp and skull masses in young children.

OBJECTIVE: Extracranial subcutaneous masses involving the scalp and/or skull in young children are uncommon lesions that get excised by the neurosurgeon. Although the most common reported lesion is the dermoid cyst, our experience suggests that the spectrum of pathology in these lesions can present diagnostic challenges to the pathologist. MATERIAL: We reviewed 30 consecutive extracranial masses from 29 patients between July 1998 and June 2003. METHOD: Hematoxylin and eosin-stained sections were reviewed in all cases, and immunohistochemistry was performed in select cases. RESULTS: Twenty-three were within the scalp, 5 involved the scalp and skull and 2 were within the limits of the inner and outer tables of the skull. There were 8 dermoid cysts, 2 epidermoid cysts, 6 post-traumatic lesions including 3 calcified cephalhematomas and 3 pseudocysts, 5 vascular lesions including 3 capillary hemangiomas, 1 venous angioma and 1 lymphangioma, 2 cases of cranial fasciitis and 1 case each of benign teratoma, deep granuloma annulare, benign fibrous histiocytoma, congenital melanocytic nevus, hamartoma with ectopic meningothelial elements, cutaneous hyalinised ectopic meningioma and a meningocele with a fibrohistiocytic reaction. No lesions have recurred or exhibited malignant features. CONCLUSIONS: Surgical pathologists and neuropathologists should be aware that the differential diagnosis of "lumps and bumps on babie's heads" is quite varied and can be histologically challenging.

Biphasic malignant meningioma: a comparative genomic hybridization study.

To ascertain if a carcinoma-like component within a fibroblastic meningioma represented a metastatic carcinoma to a meningioma or malignant progression, we employed traditional immunohistochemical methods as well as comparative genomic hybridization (CGH) which compares chromosomal alterations. Vimentin and epithelial membrane antigen were strongly immunoreactive in both the fibroblastic and carcinoma-like components. The CGH profile in both components had similar chromosomal alterations, including losses of 1p, 14, 16p13-->p10 and 22. However, the CGH profiles from the fibroblastic component showed losses of 4p, 10q23-->q24 and 18, along with gains of 1q, 6q25-->qter and 13q32-->qter. The profile of the carcinoma-like component showed losses of chromosome 4, in addition to gains of 3p12-->q13.11, 5q14.3-->q23.2, 6pter-->p23, and 13q14.2-->qter. CGH analysis of a biphasic malignant meningioma confirmed that the disparate histologic components were genetically related and likely derivative from a common precursor, demonstrating genetic instability and clonal expansion. Furthermore, CGH showed that the histologically appearing low-grade fibroblastic component had not solely the characteristic alterations of a benign meningioma but had already progressed to an atypical meningioma.

Phase I study of Gliadel wafers plus temozolomide in adults with recurrent supratentorial high-grade gliomas.

Both Gliadel wafers [1,3-bis(2-chloroethyl)-1-nitrosourea] and temozolomide (TEMO) have been shown in independent studies to prolong survival of patients with recurrent malignant glioma following surgery and radiotherapy. On the basis of preclinical evidence of synergism between Gliadel wafers and TEMO, a phase I study was designed to evaluate the toxicity of combining these 2 agents in the treatment of patients with recurrent supratentorial malignant glioma. All patients had surgical resection of the tumor at relapse, and up to 8 Gliadel (3.85%) wafers were placed in the surgical cavity following resection. Two weeks after surgery, TEMO was given orally daily for 5 days. Cohorts of 3 patients received TEMO at daily doses of 100 mg/m2, 150 mg/m2, and 200 mg/m2, respectively. Patients were assessed for toxicity 4 weeks after start of the first course of TEMO. Contrast-enhanced MRI of the brain was used to assesstumor response after the first cycle of TEMO. Patients with stable disease or response after the first cycle of TEMO were allowed to continue treatment at the same dose every 4 weeks for 12 cycles or until disease progression or unacceptable toxicity. Ten patients with a median age of 47 years (range, 22-66 years) were enrolled in this study. There were 7 patients with glioblastoma multiforme and 3 patients with anaplastic astrocytoma. Three patients were treated with TEMO at the first dose level of 100 mg/m2, 4 at the second dose level of 150 mg/m2, and 3 at the third dose level of 200 mg/m2. The 10 patients received a median of 3 cycles (range, 1-12 cycles) of TEMO following placement of Gliadel wafers. The treatment was well tolerated, with only 1 patient suffering grade III thrombocytopenia at the highest dose level. Two patients at each dose level had no evidence of disease progression after treatment. Four patients suffered progressive disease on therapy. Our study demonstrates that TEMO can be given safely after placement of Gliadel (3.85%) wafers. The recommended dosage for TEMO for a phase II study of this combination is 200 mg/m2 per day for 5 days.

CD34 and dural fibroblasts: the relationship to solitary fibrous tumor and meningioma.

Intracranial solitary fibrous tumors (SFTs) are typically dural-based, CD34-positive neoplasms of uncertain histogenesis. We examined ten cases of meninges obtained at autopsy from patients with no history of neurological illness, head trauma, or neurosurgical intervention, and ten cases of typical meningiomas with attached dural margins not involved by tumor. All cases were immunostained with CD34. CD34 reactivity was noted in the long, thin delicate processes of dural fibroblasts preferentially located in the meningeal portion of the dura rather than the periosteal portion. No CD34 reactivity was identified in the arachnoid or pia mater, except in some endothelial cells. One supratentorial dural-based fibrous nodule and one SFT within the confines of the fourth ventricle showed strong and diffuse reactivity to CD34, bcl-2, and vimentin, and were negative for epithelial membrane antigen (EMA), S-100 protein, glial fibrillary acidic protein, smooth muscle actin, and desmin. We also describe a meningothelial meningioma within which a well circumscribed SFT-like nodule was embedded. The SFT-like nodule was strongly CD34 positive and EMA negative, and the meningioma was strongly EMA positive and CD34 negative. Fibroblasts of the dural border cell layer are attached to the underlying arachnoid, and their inclusion with arachnoidal stromal elements and pial-based tela choroidea during formation of choroid plexus interstitium may account for intraventricular SFTs. Our results suggest that SFTs and dural-based fibrous nodules derive from CD34-positive dural-based fibroblasts, and that CD34 reactivity in meningiomas may result from inclusion of dural fibroblasts within the neoplasm.

March 2000: A 16 year old female with a cerebellar mass.

The March COM: A 16 year old female presented with headaches and cerebellar dysfunction. MR images showed a mass lesion of the right cerebellar hemisphere with mass effect on the medulla. The mass exhibited a striated pattern of alternating isointense and hypointense zones on T1-weighted images that did not contrast enhance. The lesion was hyperintense on T2-weighted images, and also showed a striated appearance. A suboccipital craniotomy and resection of the lesion was performed. Microscopically, the specimen consisted of widened folia and a disorganized cerebellar architectonic pattern in which the internal granular cell layer was occupied by a population of large dysmorphic nerve cell bodies. Patient's diagnosed with Lhermitte-Duclos disease must be adequately evaluated for Cowden's syndrome.

An anatomical investigation of the human cervical facet capsule, quantifying muscle insertion area.

Facet capsule injury has been hypothesised as a mechanism for neck pain. While qualitative studies have demonstrated the proximity of neck muscles to the cervical facet capsule, the magnitude of their forces remains unknown owing to a lack of quantitative muscle geometry. In this study, histological techniques were employed to quantify muscle insertions on the human cervical facet capsule. Computerised image analysis of slides stained with Masson's trichrome was performed to characterise the geometry of the cervical facet capsule and determine the total insertion area of muscle fibres into the facet capsule for the C4-C5 and C5-C6 joints. Muscle insertions were found to cover 22.4+/-9.6% of the capsule area for these cervical levels, corresponding to a mean muscle insertion area of 47.6+/-21.8 mm2. The magnitude of loading to the cervical facet capsule due to eccentric muscle contraction is estimated to be as high as 51 N. When taken in conjunction with the forces acting on the capsular ligament due to vertebral motions, these forces can be as high as 66 N. In that regard, these anatomical data provide quantitative evidence of substantial muscle insertions into the cervical facet capsular ligament and provide a possible mechanism for injury to this ligament and the facet joint as a whole.

Ham56-immunoreactive macrophages in untreated infiltrating gliomas.

CONTEXT: Classic diagnostic neuropathologic teachings have cautioned against making the diagnosis of neoplasia in the presence of a macrophage population. The knowledge of macrophage distribution should prove useful when confronted with an infiltrating glioma containing macrophages. OBJECTIVE: To identify macrophages in untreated, infiltrating gliomas using the monoclonal antibody HAM56, and to confirm their presence in an untreated glioblastoma multiforme (GBM) with the serial analysis of gene expression (SAGE) method. METHODS: We evaluated the presence of macrophages in 16 cases of untreated, supratentorial infiltrating gliomas with the macrophage monoclonal antibody HAM56. We performed SAGE for one case of GBM and for normal brain tissue. RESULTS: In World Health Organization (WHO) grade II well-differentiated astrocytoma and oligodendroglioma, HAM56 reactivity was noted only in endothelial cells, and unequivocal macrophages were not identified. In WHO grade III anaplastic astrocytoma and anaplastic oligodendroglioma, rare HAM56-positive macrophages were noted in solid areas of tumor. In WHO grade IV GBM, HAM56-positive macrophages were identified in areas of solid tumor (mean labeling index, 8.6%). In all cases of GBM, nonquantitated HAM56-positive macrophages were identified in foci of pseudopalisading cells abutting necrosis and in foci of microvascular proliferations. In none of the cases were granulomas or microglial nodules found, and there was no prior history of surgical intervention, radiation therapy, chemotherapy, or head trauma in these cases. By SAGE, the macrophage-related proteins osteopontin and macrophage-capping protein were overexpressed 12-fold and eightfold, respectively, in one untreated GBM compared with normal brain tissue. In this case, numerous HAM56-positive macrophages (labeling index, 24.5%) were present in the solid portion of tumor, and abundant nonquantified macrophages were identified in foci of pseudopalisading cells abutting necrosis and in foci of microvascular proliferations. CONCLUSIONS: This study confirms the utility of the monoclonal antibody HAM56 in identifying macrophages within untreated infiltrating gliomas. The overexpression of macrophage-related proteins in one case of GBM as detected by SAGE signifies that macrophages may be present in untreated GBMs.

A genetically tractable model of human glioma formation.

Gliomas remain one of the deadliest forms of cancer. Improved therapeutics will require a better understanding of the molecular nature of these tumors. We, therefore, mimicked the most common genetic changes found in grade III-IV gliomas, disruption of the p53 and RB pathways and activation of telomere maintenance and independence from growth factors, through the ectopic expression of the SV40 T/t-Ag oncogene, an oncogenic form of H-ras (H-ras(V12G)), and the human telomerase catalytic subunit hTERT in normal human astrocytes. The resulting cells displayed many of the hallmarks of grade III-IV gliomas, including greatly expanded life span and growth in soft agar and, most importantly, were tumorigenic with pathology consistent with grade III-IV neuroectodermal tumors in mice. This model system will, for the first time, allow the biological significance of selected genetic alterations to be studied in human gliomas.

Endodermal cyst of the oculomotor nerve.

Endodermal cysts are rare congenital intracranial lesions. Although histologically benign, they can become symptomatic as a result of mass effect and cause neurological deficits. We report a 30-year-old woman who presented with paresis of her right oculomotor nerve. Magnetic resonance imaging showed a 13 x 8-mm cystic lesion originating from the right oculomotor nerve at its exit from the mesencephalon. She underwent craniotomy, biopsy, slit resection, and drainage of the cyst. To our knowledge, endodermal cysts have not been previously described in relation to the oculomotor nerve.

Phase I trial results of iodine-131-labeled antitenascin monoclonal antibody 81C6 treatment of patients with newly diagnosed malignant gliomas.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine-131 ((131)I)-labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment. PATIENTS AND METHODS: In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of (131)I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment. RESULTS: We treated 42 patients with (131)I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively. CONCLUSION: The MTD for administration of (131)I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.

Phase I trial of carmustine plus O6-benzylguanine for patients with recurrent or progressive malignant glioma.

PURPOSE: The major mechanism of resistance to alkylnitrosourea therapy involves the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), which removes chloroethylation or methylation damage from the O(6) position of guanine. O(6)-benzylguanine (O(6)-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial of carmustine (BCNU) plus O(6)-BG to define the toxicity and maximum-tolerated dose (MTD) of BCNU in conjunction with the preadministration of O(6)-BG with recurrent or progressive malignant glioma. PATIENTS AND METHODS: Patients were treated with O(6)-BG at a dose of 100 mg/m(2) followed 1 hour later by BCNU. Cohorts of three to six patients were treated with escalating doses of BCNU, and patients were observed for at least 6 weeks before being considered assessable for toxicity. Plasma samples were collected and analyzed for O(6)-BG, 8-oxo-O(6)-BG, and 8-oxoguanine concentration. RESULTS: Twenty-three patients were treated (22 with glioblastoma multiforme and one with anaplastic astrocytoma). Four dose levels of BCNU (13.5, 27, 40, and 55 mg/m(2)) were evaluated, with the highest dose level being complicated by grade 3 or 4 thrombocytopenia and neutropenia. O(6)-BG rapidly disappeared from plasma (elimination half-life = 0. 54 +/- 0.14 hours) and was converted to a longer-lived metabolite, 8-oxo-O(6)-BG (elimination half-life = 5.6 +/- 2.7 hours) and further to 8-oxoguanine. There was no detectable O(6)-BG 5 hours after the start of the O(6)-BG infusion; however, 8-oxo-O(6)-BG and 8-oxoguanine concentrations were detected 25 hours after O(6)-BG infusion. The mean area under the concentration-time curve (AUC) of 8-oxo-O(6)-BG was 17.5 times greater than the mean AUC for O(6)-BG. CONCLUSION: These results indicate that the MTD of BCNU when given in combination with O(6)-BG at a dose of 100 mg/m(2) is 40 mg/m(2) administered at 6-week intervals. This study provides the foundation for a phase II trial of O(6)-BG plus BCNU in nitrosourea-resistant malignant glioma.

Temozolomide delivered by intracerebral microinfusion is safe and efficacious against malignant gliomas in rats.

Intracerebral microinfusion (ICM) is an innovative technique of delivering therapeutic agents throughout large portions of the brain that circumvents the blood-brain barrier, minimizes systemic toxicity, and provides a homogeneous distribution of the infused agent. Temozolomide is a novel methylating agent with proven efficacy against malignant gliomas (MGs) after systemic administration but with dose-limiting myelotoxicity. Because MGs rarely metastasize, systemic drug delivery is unnecessary. Therefore, we evaluated the efficacy and toxicity of ICM with temozolomide in an athymic rat model of human MGs. Treatment of rats by ICM with temozolomide 3 days after intracerebral challenge with D54 human MG xenograft increased median survival by 128% compared with rats treated by ICM with saline, by 113% compared with rats treated with i.p. saline, and by 100% compared with rats treated with i.p. temozolomide (P < 0.001). Delay of treatment until 9 days after tumor challenge still resulted in a 23% increase in median survival in rats treated by ICM of temozolomide compared with rats treated with i.p. temozolomide. In addition, overall, 21.7% of rats treated by ICM with temozolomide survived for > 100 days without clinical or histological evidence of tumor. The dose of temozolomide delivered by ICM in this study was limited only by drug solubility, and no neurological or systemic toxicity could be attributed to ICM with temozolomide. Therefore, ICM of temozolomide may offer significant advantages in the treatment of MGs.

Castleman's disease confined to the leptomeninges.

We report a rare case of the plasma cell variant of Castleman's disease confined to the leptomeninges in a 42-year-old female. Flow cytometry demonstrated a minor monoclonal kappa light chain population, and conventional Southern blotting confirmed clonal rearrangement of the J(H) immunoglobulin heavy-chain gene. Polymerase chain reaction for Epstein-Barr virus and Kaposi's sarcoma-associated herpes virus was negative. The patient is disease-free five years after surgical resection. To our knowledge, clonal gene rearrangement has not been previously reported in the plasma cell variant of localized intracranial Castleman's disease.