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Tuberous sclerosis (TS) is a congenital neurocutaneous syndrome (or phacomatosis) characterized by widespread development of hamartomas in multiple organs. For affected individuals, neurological and psychiatric complications are the most disabling and lethal features. Although the clinical phenotype of TS is complex, only three lesions characterize the neuropathological features of the disease: cortical tubers, subependymal nodules, and subependymal giant cell astrocytomas. The latter is a benign brain tumor of mixed neuronal and glial origin. Tuberous sclerosis is caused by loss-of-function mutations in one of two genes, TSC1 or TSC2. The normal cellular proteins encoded by these genes, hamartin and tuberin, respectively, form a heterodimer that suppresses cell growth in the central nervous system by dampening the phosphatidylinositol 3-kinase signal transduction pathway. The authors review the clinical and neuropathological features of TS as well as recent research into the molecular biology of this disease. Through this work, investigators are beginning to resolve the paradoxical findings that malignant cancers seldom arise in patients with TS, even though the signaling molecules involved are key mediators of cancer cell growth.
Tuberous sclerosis is a congenital neurocutaneous syndrome (or phacomatosis) characterized by widespread development of hamartomas in multiple organs. The French neurologist Désiré-Magloire Bourneville coined the term "tuberous sclerosis" in 1880 when he described the brain lesions found on postmortem examination of a 15-year-old patient who had suffered seizures since infancy as well as mental retardation and hemiplegia. Tuberous sclerosis has an incidence of 1 in 6000 to 1 in 10,000 live births, with no ethnic clustering. Approximately two thirds of cases are sporadic; that is, affected individuals have no family history of the disease. Familial cases show an autosomal-dominant pattern of inheritance. The cloning of two different disease-causing genes (TSC1 and TSC2) has accelerated our understanding of the molecular pathogenesis of TS. Control of cell growth in the central nervous system is markedly perturbed in TS, but malignant brain tumors rarely occur. Nevertheless, this syndrome is a topic of clinical relevance to neurosurgeons because TS-related space-occupying lesions and intractable epilepsy may require timely surgical intervention.
The characteristic lesions of TS are hamartomas, which are congenitally misplaced groups of cells that form disorganized, tumor-like masses. Hamartomas occur in the brain (discussed later), kidneys, lung, heart, eyes, and skin. Skin hamartomas include facial angiofibromas (also known as adenoma sebaceum), subungual fibromas, and shagreen patches. The growth rate of visceral hamartomas can accelerate spontaneously to create expanding tumors (SEGAs of the brain, angiomyolipomas of the kidney, lymphangioleiomyomatosis of the lung, and rhabdomyomas of the heart). On histopathological examination, these tumors are almost always benign. The molecular signals that trigger this transition from quiescent hamartoma to enlarging tumor are not known. Surprisingly, patients with TS rarely present with malignant neoplasms even though they have numerous hamartomas and benign tumors, which are considered premalignant lesions. The most common malignant tumor associated with TS is renal cell carcinoma.
The clinical presentation of TS is determined by the specific organs affected. The severity of presenting symptoms is highly variable, ranging from minor skin lesions to in tractable epilepsy and debilitating cognitive impairment. The classic symptom triad of adenoma sebaceum, epilepsy, and mental retardation comprised the first diagnostic criteria for TS. In 1998, the National Institutes of Health convened a consensus conference to standardize diagnostic criteria for the TSC. The published set of criteria was composed of clinical and radiographic features, which were divided into major and minor categories ( Table 1 ). A definitive diagnosis of TS requires that a patient present with two of the major criteria shown in Table 1 , or one major and two minor criteria. Notably, certain clinical signs that once were regarded as pathognomonic for TS, like mental retardation and epilepsy, are now considered nonspecific. Furthermore, no single criterion, found either clinically or radiographically, is present in all patients.
For affected individuals, neurological and psychiatric symptoms are the most disabling features. In fact, neurological complications are the leading cause of death for patients with TS, followed by renal disease and pulmonary lymphangioleiomyomatosis. The most common neurological symptoms are seizures, mental retardation, autism, hyperactivity, spastic paralysis, involuntary movements, ataxia, dementia, and ophthalmoplegia. More than 75% of patients suffer from seizures, and 68% have mild to severe cognitive impairment. As a general rule, larger and more numerous cortical tubers are associated with earlier seizure onset and more severe mental retardation. The types of seizures found in patients with TS are highly variable and include tonic-clonic, atonic, myoclonic, atypical absence, partial, and partial complex seizures.
Although the clinical phenotype of TS is complex, only three lesions characterize the neuropathology of the disease: cortical tubers and SENs, both of which are hamartomas, and SEGAs, which are histopathologically benign neoplasms. All three lesions occur predominantly in the brain. Hamartomas in the spinal cord have been reported, but no lesions occur in the peripheral nervous system. The fact that cortical tubers and SEGAs have been reported in spontaneously aborted fetuses indicates that the lesions of TS originate during fetal development.
Cortical tubers occur in approximately 80% of patients with TS. Grossly, they appear as hard, wide gyri with smooth, flat tops or as rounded nodules with rough surfaces. Most cortical tubers occur in the frontal and parietal lobes, but some arise in the cerebellum, brainstem, and spinal cord. Microscopically, cortical tubers consist of interlacing fascicles of normal and abnormal neurons, astrocytes, and giant cells (Fig. 1A-C). Subependymal nodules are distributed along the sulcus terminalis throughout both lateral ventricles. Microscopically they resemble cortical tubers, except that SENs have a higher cellular packing density and sometimes contain polygonal and spindle-shaped epithelioid cells and mast cells (Fig. 1D).
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Photomicrographs showing histopathological findings in a SEGA. A and B: Low-power photomicrographs showing loosely distributed giant cells (A) and densely packed, interlacing fascicles of spindle-shaped cells (B). H & E, original magnification Ă— 200. C and D: High-power views showing morphological features of giant cells with abundant eosinophilic cytoplasm (C) and mitotic figures (arrow). Addition of toluidine blue (D) reveals darkly staining mast cells (arrows) interspersed among lightly staining giant cells. Original magnifications Ă— 600 (C) and Ă— 400 (D). E and F: Immunoperoxidase staining shows coexpression of neurofilament protein (E) and glial fibrillary acidic protein (F). All sections are from a single patient's tumor specimen. Original magnifications Ă— 200.
Giant cells (also known as balloon cells) are the neuropathological hallmarks of TS. They have prominent nuclei and nucleoli and abundant, glassy, eosinophilic cytoplasm (Fig. 1C and D). The origin of giant cells remains uncertain. Immunohistochemical studies have shown that these cells coexpress proteins normally found in neurons (neurofilament protein, Class III β-tubulin) and in astrocytes (glial fibrillary acidic protein, S100 protein) (Fig. 1E and F). Electron microscopy studies have shown that ultrastructural characteristics of neurons (lamellae of rough endoplasmic reticulum, dense-core granules) and astrocytes (glycogen granules, membrane-bound dense bodies) are found together in individual giant cells. Taken together, these findings support the suggestion that giant cells might originate from mixed glioneuronal precursors.
Subependymal giant cell astrocytoma is the most common brain tumor associated with TS. It is detected in 6% of patients, most often presenting with seizures or hydrocephalus. It usually presents in the first two decades of life, occasionally in the early postnatal period. Rarely has a SEGA been reported in an individual who did not meet the diagnostic criteria of TS. Although this type of tumor is histologically benign and grows slowly, malignant transformation and even massive hemorrhage have been reported.
The cytoarchitecture of SEGAs is indistinguishable from that of SENs, except for the presence of mitotic figures in the tumors. Figure 1 shows the cytological features of a SEGA, including variable cellular packing density (panels A and B), giant cells with interspersed mast cells (panels C and D), and coexpression of neuron-specific and astrocyte-specific intermediate filament proteins (panels E and F). This similarity has fostered the idea that SEGAs arise by transformation of giant cells in preexisting SENs, as reviewed by Gomez. An observation that remains unexplained by this hypothesis is that SEGAs occur exclusively at the foramen of Monro, although nodules are distributed throughout the lateral ventricles.
Historically, intracranial calcification on skull x-ray films was the first radiographic sign of TS. This appearance was due to dystrophic calcification in cortical tubers and SENs. On early pneumoencephalograms, SENs could be seen protruding into the lumen of the lateral ventricle, giving the appearance of molten candle wax ("candle gutterings"). Modern computerized tomography scanning detects brain calcification in 50 to 80% of patients (Fig. 2A). Subependymal nodules are usually densely calcified, where as cortical tubers show variable density, depending on the amount of calcium present. On MR imaging, cortical tubers appear hyperintense on T2-weighted images (Fig. 2B). Subependymal nodules appear slightly more intense than deep gray matter on T1-weighted images. Their signal intensity on T2-weighted MR images varies from patient to patient but is typically hypointense because of the calcification. The appearance of SEGAs on neuroimages closely resembles that of SENs, except that the tumors are larger and they enhance brightly after delivery of intravenous contrast agents (Fig. 2C). In addition, SEGAs are in variably located at the foramen of Monro. Advances in brain imaging have revealed that patients with TS often exhibit developmental anomalies that are not unique to the syndrome, such as agenesis of the corpus callosum, heterotopias, transmantle cortical dysplasia, and schizencephaly. Transmantle cortical dysplasia appears on MR images as radial bands of abnormal signal intensity extending from periventricular to sub cortical regions of the cerebral hemispheres (Fig. 2D). Radial bands sometimes interconnect cortical tubers and SENs. These lesions are typically T1 hyperintense and T2 hypointense in infants and be come T1 hypointense and T2 hyperintense in older children and adults. Radial bands are thought to represent a disturbance in the normal migration of neural progenitor cells from the ventricular germinal matrix to the cerebral cortex during brain development.
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Neuroimages showing the features of TS. A: A computerized tomography scan demonstrating calcified SENs. B: Hyperintense multifocal cortical tubers revealed on T2-weighted MR image. C: Gadolinium-enhanced T1-weighted MR image revealing a SEGA obstructing the right foramen of Monro. D: A fluid-attenuated inversion-recovery MR image showing radial bands (arrows) and multiple cortical tubers.
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Neuroimages showing the features of TS. A: A computerized tomography scan demonstrating calcified SENs. B: Hyperintense multifocal cortical tubers revealed on T2-weighted MR image. C: Gadolinium-enhanced T1-weighted MR image revealing a SEGA obstructing the right foramen of Monro. D: A fluid-attenuated inversion-recovery MR image showing radial bands (arrows) and multiple cortical tubers.
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Neuroimages showing the features of TS. A: A computerized tomography scan demonstrating calcified SENs. B: Hyperintense multifocal cortical tubers revealed on T2-weighted MR image. C: Gadolinium-enhanced T1-weighted MR image revealing a SEGA obstructing the right foramen of Monro. D: A fluid-attenuated inversion-recovery MR image showing radial bands (arrows) and multiple cortical tubers.
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Neuroimages showing the features of TS. A: A computerized tomography scan demonstrating calcified SENs. B: Hyperintense multifocal cortical tubers revealed on T2-weighted MR image. C: Gadolinium-enhanced T1-weighted MR image revealing a SEGA obstructing the right foramen of Monro. D: A fluid-attenuated inversion-recovery MR image showing radial bands (arrows) and multiple cortical tubers.
An extensive body of evidence indicates that TS is caused by loss-of-function mutations in one of two genes, TSC1 located on human chromosome 9q34 or TSC2 located on 16p13. The proteins encoded by TSC1 and TSC2 are called hamartin and tuberin, respectively. A detailed review of the molecular genetics of TS, which contains an analysis of mutations compiled from 446 patients, has been published by Cheadle, et al. In familial cases of TS, the mutation frequency for TSC1 and TSC2 is equal (50% for each gene). In sporadic cases, TSC1 mutations are found in approximately 10 to 15% of patients and TSC2 mutations are found in 70%. In sporadic and familial cases combined, mutations in either TSC1 or TSC2 have been found in 75 to 90% of patients with TS. The absence of mutations in the remaining 10 to 25% of patients most likely reflects limitations in the sensitivity of mutation detection rather than the existence of other genes distinct from TSC1 and TSC2.
Several independent studies have shown close similarities between the phenotypic features associated with TSC1 and TSC2 mutations, suggesting that TS is a disease characterized by locus heterogeneity. Locus heterogeneity predicts that these two genes will encode proteins that function together in the same biochemical pathway. Indeed, hamartin and tuberin, the cellular proteins encoded by TSC1 and TSC2, are now known to interact to suppress the PI3K signal transduction pathway, which will be discussed later. The results of other studies suggest that patients with TSC2 mutations have more disabling neurological impairments than those with TSC1 mutations. For example, a genotype-phenotype correlation in 224 patients showed that TSC1 mutations were associated with lower seizure frequency, milder cognitive impairment, fewer SENs and cortical tubers, less severe kidney and skin disease, and the absence of retinal hamartomas. Patients with TSC2 mutations may be more susceptible to aggressive renal tumors.
Several lines of evidence indicate that TSC1 and TSC2 are tumor suppressor genes. Such genes encode proteins that function normally to inhibit cell growth. According to the Knudson two-hit model, an inherited (germline) mutation in one copy (allele) of a tumor suppressor gene predisposes an individual to tumor formation. A somatic mutation (second hit) that inactivates the remaining normal allele is required to initiate tumor formation. In this model, loss of both allelic copies of a tumor suppressor gene re moves a physiological constraint to cell growth. The second hit is often a deletion of the chromosome region containing the tumor suppressor gene. A molecular signpost for a chromosome deletion is loss of heterozygosity in tumor DNA compared with an individual's normal DNA. Frequent loss of heterozygosity in a particular type of tumor indicates that a tumor suppressor gene important in the genesis of that tumor is present on the missing part of the chromosome.
Loss of heterozygosity for TSC1 and TSC2 has been re ported in a wide variety of hamartomas and tumors resected in patients with TS, including renal angiomyolipomas, cortical tubers, and SEGAs. Approximately 50% of hamartomas have loss of heterozygosity for loci on chromosome 9q34, where TSC2 is located, and 10% have loss of heterozygosity for chromosome 16p13 loci, which are linked to TSC1. The observation that mutations in these genes are usually nonsense, splicing, or frameshift mutations, which encode truncated nonfunctional proteins, is additional evidence that TSC1 and TSC2 are tumor suppressor genes. Furthermore, an immunohistochemical study of nine SEGAs showed that expression of both tuberin and hamartin was absent or barely detectable in eight cases. Interestingly, the tumor cells obtained in one patient with a TSC2 mutation showed no hamartin but abundant tuberin. Taken together, these observations support the concept that hamartomas and other tumors arise in patients with TS when inactivation of either hamartin or tuberin stimulates the growth of susceptible cells.
The proteins hamartin and tuberin, which are encoded by TSC1 and TSC2, bind to one another inside the cytoplasm to form a molecular complex that serves as a gate to control cell growth signals conveyed through the PI3K signal transduction pathway. As reviewed by Cantley, the PI3K pathway is a vital information system that governs many aspects of cell growth. In a review of the literature, Row in sky has reported that the pathway is hyperactive in many types of malignant tumors. Detailed reviews focused on the hamartin/tuberin complex in PI3K signaling have been published by Hay and Sonenberg and by Inoki, et al. One vital function of the PI3K pathway is to transduce cell growth and survival signals conveyed by extracellular growth factors such as IGFs. Salient features of IGF-stimulated PI3K signaling are shown schematically in Fig. 3. When IGFs engage their cognate receptors on the cell surface, the activated tyrosine kinase receptors phosphorylate the cytoplasmic protein IRS-1, thereby creating a molecular docking site for the regulatory subunit of the enzyme PI3K. Consequent activation of the catalytic subunit of PI3K converts the membrane-bound phospholipid PIP2 to PIP3, which recruits the serine/threonine kinase Akt to the plasma membrane, where Akt is phosphorylated and thereby activated catalytically. The tumor suppressor protein PTEN dampens PI3K signaling by dephosphorylating PIP3, thereby preventing attachment of Akt to the plasma membrane.
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Schematic drawing showing the PI3K/mTOR signal transduction pathway. When extracellular growth factors, such as IGF-1, engage and activate their receptors on the cell surface, the receptor tyrosine kinase phosphorylates IRS-1, thereby creating a docking site for the regulatory subunit (p85) of PI3K. Consequent activation of the catalytic subunit (p110) converts membrane-bound PIP2 to PIP3, which recruits Akt to the plasma membrane through the pleckstrin homology domain (PHD) of Akt. The Akt becomes activated by phosphoinositide kinase-dependent kinases (PDKs). Activated Akt phosphorylates TSC2, thereby disrupting its association with TSC1. Dissociation of the TSC1/TSC2 complex derepresses mTOR, thereby stimulating protein synthesis. Nutrient deprivation inhibits protein synthesis by promoting TSC2-mediated repression of mTOR. Activated mTOR imposes feedback inhibition on Akt signaling.
A key downstream component of the PI3K pathway is the cellular protein mTOR. The best-described biochemical function of mTOR is its promotion of mRNA translation and hence stimulation of protein synthesis in the cell. Like Akt, mTOR is a serine/threonine kinase that becomes catalytically activated in response to PI3K signaling. Al though mTOR is a direct phosphorylation substrate for Akt, evidence is mounting that the principal mechanism whereby Akt activates mTOR is not by direct phosphorylation, but rather by disruption of the hamartin/tuberin complex. In the model shown in Fig. 3, the hamartin/tuberin complex functions normally to suppress mTOR activity. Activated Akt phosphorylates tuberin, causing the hamartin/tuberin complex to dissociate. The result is disinhibition of mTOR and consequent stimulation of mRNA translation, protein synthesis, and cell cycle progression. Although Akt and mTOR are functionally coupled in the PI3K pathway, each of these proteins transduces cell signals through a unique set of downstream effector molecules.
In addition to relaying growth factor signals to the nucleus of the cell, mTOR functions as a nutrient sensor during cell metabolism. Under conditions of nutrient deprivation, intracellular levels of ATP and amino acids fall. In response, mTOR activity declines. This compensatory mechanism makes sense for cells because it enables them to shut down their energy-demanding processes, like protein synthesis, during nutritionally hard times. The biochemical re actions that couple intracellular ATP levels with mTOR activity are not fully understood, but tuberin clearly plays a role. When cells are in low-energy states, the enzyme adenosine mono phosphate-activated protein kinase phosphorylates tuberin, and this phosphorylated form of tuberin protects cells from apoptotic death induced by energy de privation. Thus, ha martin and tuberin, by regulating mTOR, integrate two of the most important signals governing cell growth: growth factors and nutrients.
Considering the fact that hamartin and tuberin apply the brakes in a signal transduction pathway that is frequently activated in many types of malignant tumors, it is surprising that lesions in patients with TS rarely become malignant. Mouse models of TS provide an explanation. Mouse embryos in which either the Tsc1 or the Tsc2 gene is completely knocked out fail to develop beyond midgestation be cause they suffer hypoplasia of the liver and enlargement of the heart. Mice that are heterozygous defective for Tsc1 or Tsc2 develop normally, but they are predisposed to renal adenomas, which progress at low frequency to malignant cancers. These mice also develop hepatic hemangiomas and peripheral angiosarcomas. Interestingly, brain lesions, which are so common in cases of TS in humans, do not occur in these mouse models.
When Tsc2 mice are crossed with mice that are heterozygous defective for the Pten tumor suppressor gene, the resultant compound heterozygotes (Tsc2 Pten mice) show an earlier onset and higher incidence of hepatic hemangiomas and peripheral angiosarcomas. Analysis of signaling molecules specifically regulated by Akt and mTOR shows that, in hepatic hemangiomas, signaling down stream of Akt is attenuated in the benign tumors found in Tsc2 mice but is enhanced in the more aggressive tumors present in Tsc2 Pten mice. This suggests that loss of Tsc2 expression creates an inhibitory feedback loop, in which mTOR or one of its downstream effectors suppresses Akt signaling (Fig. 3). The Pten deficiency can overcome this inhibition by enabling PI3K signaling to increase cellular levels of activated Akt. Extrapolating these findings to TS in humans provides an explanation for the fact that malignant tumors rarely occur, although hamartomas, which result from aberrant cell growth control, are abundant. Tuberin deficiency simultaneously stimulates mTOR-mediated protein synthesis and inhibits Akt-mediated cell survival and proliferation (Fig. 3). The combined effect is to perturb cell growth control just enough to generate hamartomas but not sufficiently to induce malignant transformation.
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