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.

Unmyelinated axons in the posterior funiculi.

Electron microscopy of the dorsal funiculus in the rat reveals that most axons in this pathway are unmyelinated. These axons have not previously been counted, nor are they considered in modern studies on the organization of the dorsal funiculus. Because of the importance of this pathway in somatic sensation, it is important to understand that these fibers exist and that they are present in greater numbers than the well-studied myelinated axons.

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.

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.

Increased levels of p21WAF1/Cip1 in human brain tumors.

The cdk inhibitor p21WAF1/Cip1 (p21), which can be transcriptionally activated by p53, functions to block cell cycle progression. In this study, we analysed the expression of p21 in normal and reactive brain and in gliomas of various malignancy grades. Southern blotting showed no p21 gene deletion. Western blotting and immunohistochemical assay showed that the levels of p21 protein in normal and reactive brain tissue were very low; however, p21 was elevated in a majority of gliomas tested, regardless of their malignancy grades. In glioblastoma multiforme, marked elevation of p21 was observed in samples harboring either wild-type or mutant p53. But, in anaplastic astrocytomas, the level of p21 was not elevated in samples harboring mutant-type p53. Immunohistochemical staining of paraffin-embedded astrocytomas and glioblastomas showed that tumor cells and not contaminating normal cells were positive for p21. Therefore, overexpression of p21 appears to be an early event in the development of glial neoplasms and p53-dependent p21 expression appears to be tumor grade specific.

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.

Branching of sensory axons in the dorsal root and evidence for the absence of dorsal root efferent fibers.

The present study demonstrates that there are more dorsal root axons than dorsal root ganglion cells in the L6-S1 dorsal roots of the rat. The excess fibers do not come from aberrant dorsal root ganglion cells and our control procedures indicate that there are no extraneous fibers in these dorsal roots. Accordingly many dorsal root axons must branch in the dorsal root.

Branching of sensory axons in the peripheral nerve of the rat.

The currently accepted concept of a primary sensory cell is a cell that gives rise to a central process which passes through the dorsal root to the spinal cord and a peripheral process which passes to the periphery via a peripheral nerve. If this is correct, then there should be equal numbers of sensory axons in the dorsal root, dorsal root ganglion cells, and sensory axons in the proximal peripheral nerve. The present study obtains these counts in animals in which extraneous axons have been removed from the peripheral nerve and root. The counts indicate that there are approximately 2.3 sensory axons in the dorsal root and proximal peripheral nerve for each ganglion cell in the sacral segments of the rat. We interpret these data as indicating that there is significant branching of sensory axons in the dorsal root and proximal peripheral nerve and thus the generally accepted picture of a dorsal root ganglion cell is not correct for some, perhaps all, of these cells. We offer the speculation that this peripheral branching may be an indication of single sensory neurons having receptive fields in two separate locations, and thus this may be an anatomical explanation for certain types of referred pain.

Primary afferent and propriospinal fibers in the rat dorsal and dorsolateral funiculi.

The purpose of this study is to determine the numbers of primary afferent and propriospinal fibers in the dorsal and dorsolateral funiculi of the rat. The reason for concentrating on these areas is that they contain large numbers of unmyelinated axons. Our data are axonal numbers from the S2 segment of spinal cord in animals that had unilateral dorsal rhizotomies or spinal cord isolations. The major conclusions are 1) that 23% of the primary afferent fibers in the dorsal funiculus are unmyelinated; 2) that there are approximately 12,500 unmyelinated primary afferent fibers in the dorsolateral funiculus, which is more than the number of primary afferent fibers in the dorsal funiculus and tract of Lissauer combined, and 3) that approximately 25% of the axons in the dorsal funiculus and 44% of the axons in the dorsolateral funiculus are propriospinal. These data modify and extend previous ideas of the organization of spinal white matter.

Primary afferent axons in the tract of Lissauer in the monkey.

The main thrust of the present study was to determine the numbers of primary afferent fibers in the tract of Lissauer in the monkey. The findings are that approximately 40% of the axons in the tract of Lissauer are primary afferent axons from a single segment, and approximately another 40% are primary afferents from segments cranial and caudal to the segment under consideration. Presumably, the remaining 20% are propriospinal axons. There is relatively little difference in the proportions of primary afferents in medial as opposed to lateral parts of the tract, and in this respect the monkey differs somewhat from the rat and cat. Thus in the monkey the tract of Lissauer should probably be regarded as a primary afferent pathway with the propriospinal forming a distinct but relatively minor component of the tract.

Unmyelinated sensory and preganglionic fibers in rat L6 and S1 ventral spinal roots.

Approximately 30% of the axons in the L6 and S1 ventral roots in the rat are unmyelinated. Appropriate surgical procedures show that 30% of these unmyelinated axons arise from dorsal root ganglion cells and are therefore presumably sensory. Thus approximately 10% of the total number of axons in these ventral roots are sensory. The other 70% of the unmyelinated axons come from the spinal cord and are therefore presumably preganglionic parasympathetic fibers. Thus approximately 20% of the axons in these ventral roots are unmyelinated preganglionic fibers. The implications of these findings are discussed.

Primary afferent fibers in the tract of Lissauer in the rat.

More than two-thirds of the axons in the tract of Lissauer at mid-thoracic and lumbosacral levels of the rat spinal cord are primary afferent fibers. The proportions of primary afferents in the tract are approximately the same at the two spinal levels. A slightly higher percentage of the unmyelinated, as opposed to the myelinated, fibers are primary afferents. There is a somewhat greater percentage of primary afferent axons in medial parts of the mid-thoracic levels, but all areas of the tract that were examined contain a majority of primary afferent fibers. The primary afferent axons appear to travel less than a segment in the tract at mid-thoracic levels but for several segments in the tract at lumbo-sacral levels. These data indicate that the tract of Lissauer is predominately a primary afferent fiber system in these segments of the rat.

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.

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.

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.

Spinal cord protection with intravenous nimodipine. A functional and morphologic evaluation.

The purpose of this study is to determine the effects of ischemia in the spinal cord when a calcium channel blocker, nimodipine, is administered intravenously before, during, and after crossclamping of the thoracic aorta. In this series of experiments, 18 pigs underwent thoracotomies and had 17.5 to 18.0 cm of the thoracic aorta clamped for 30 minutes. By random selection, nine animals received intravenous nimodipine (2 micrograms/kg per minute) and nine control animals received only a carrier solution. Of the nine animals that received nimodipine, eight walked after the operation. In contrast, in the control group only two of nine animals walked. The ninth nimodipine-treated animals walked but had a severe delayed deterioration response. All animals, except one control animal, had a negative central spinal perfusion pressure. Morphologic examination of serial sections of spinal cords from control animals showed diffuse neuronal necrosis. In comparison, cords from the nimodipine group had swollen neurons accompanied by an inflammatory infiltrate and only occasional necrotic neurons. With this data, we conclude that certain calcium channel blockers, when administered in sufficient doses, can lend a protective effect to the spinal cord during ischemic events even when cord perfusion pressure has dropped to dangerously low levels.

Discharge characteristics of fine medial articular afferents at rest and during passive movements of inflamed knee joints.

After induction of an experimental knee joint inflammation, the activity of single Group III and IV afferent units in the medial articular nerve of the cat was recorded at rest and during passive movements. The properties of these units were compared to those sampled from normal knee joints. The proportion of units displaying resting discharges was higher in the inflamed group. The frequency of discharges was also higher. The receptive fields were larger than those in the control units. Passive movements in the normal working range of the joint activated many more units in the inflamed joint than in the control sample. We conclude that joint inflammation sensitizes articular nociceptors to be active not only at rest, but also during normally innocuous joint movements.

Unmyelinated axon ratios in cat motor, cutaneous and articular nerves.

The goal of this electron microscopic study is to determine ratios of unmyelinated to myelinated axons and afferent unmyelinated to postganglionic autonomic (sympathetic) efferent axons in representative cat motor, cutaneous and articular nerves. Of the total axons in the tibial, medial gastrocnemius, sural and medial and posterior articular nerves, 70% or more are unmyelinated. The afferent unmyelinated axon components range from 49% and 45% in the medial and posterior articular nerves, respectively, to 58% in the medial gastrocnemius nerve and 61% in the sural nerve.

Primary xanthoma of thoracic spine presenting with myelopathy.

STUDY DESIGN: This retrospective case study reports on a patient with an isolated primary xanthoma arising in the second thoracic vertebra with paravertebral and spinal canal extension. OBJECTIVE: The possibility of this lesion's occurrence in the spine is presented with radiologic and pathologic findings appropriate for the diagnosis of spinal xanthoma. SUMMARY OF BACKGROUND DATA: Primary xanthoma of bone is an extremely rare but benign entity. It has not been previously described in this location, and has been described only once in the spine at all. METHODS: The mass was resected by curettage, and posterior instrumentation was successfully performed. Preoperative radiographs and magnetic resonance imaging of the thoracic spine were performed, as was histologic examination of the lesion. RESULTS: Radiographs of the upper thoracic spine revealed a lytic defect. Magnetic resonance imaging revealed a heterogeneous lesion that was enhanced upon the administration of gadolinium-diethylenetriaminetetraacetic acid. Histologic examination revealed a cellular lesion consisting of lipid-laden macrophages, fibroblasts, and scattered Touton giant cells. These features correspond to descriptions of primary xanthoma of bone. Two years after surgery, the patient was neurologically intact with no evidence the lesion would recur. CONCLUSIONS: Primary xanthoma of bone is considered a benign lesion and can be successfully treated by local resection without adjuvant therapy. It should be considered part of the differential diagnosis of a mass lesion, with appropriate signal characteristics presenting in a thoracic vertebra.

Histopathologic correlation of magnetic resonance imaging signal patterns in a spinal cord injury model.

Magnetic resonance imaging (MRI) provides a noninvasive method of monitoring the pathologic response to spinal cord injury. Specific MR signal intensity patterns appear to correlate with degrees of improvement in the neurologic status in spinal cord injury patients. Histologic correlation of two types of MR signal intensity patterns are confirmed in the current study using a rat animal model. Adult male Sprague-Dawley rats underwent spinal cord trauma at the midthoracic level using a weight-dropping technique. After laminectomy, 5- and 10-gm brass weights were dropped from designated heights onto a 0.1-gm impounder placed on the exposed dura. Animals allowed to regain consciousness demonstrated variable recovery of hind limb paraplegia. Magnetic resonance images were obtained from 2 hours to 1 week after injury using a 2-tesla MRI/spectrometer. Sacrifice under anesthesia was performed by perfusive fixation; spinal columns were excised en bloc, embedded, sectioned, and observed with the compound light microscope. Magnetic resonance axial images obtained during the time sequence after injury demonstrate a distinct correlation between MR signal intensity patterns and the histologic appearance of the spinal cord. Magnetic resonance imaging delineates the pathologic processes resulting from acute spinal cord injury and can be used to differentiate the type of injury and prognosis.