American Journal ofpathology, Vol. 151, No. 6, December 1997 Copyright ) American Society for Investigative Pathology

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1 American Journal ofpathology, Vol. 151, No. 6, December 1997 Copyright ) American Society for Investigative Pathology Loss of Tuberin in Both Subependymal Giant Cell Astrocytomas and Angiomyolipomas Supports a Two-Hit Model for the Pathogenesis of Tuberous Sclerosis Tumors Elizabeth Petri Henske,* Lisa L. Wessner,* Jeffrey Golden,t Bernd W. Scheithauer,* Alexander 0. Vortmeyer, Zhengping Zhuang, Andres J. P. Klein-Szanto,11 David J. Kwiatkowski,11 and Raymond S. Yeung** From the Departments ofmedical Oncology,* Pathology,9' and Surgical Oncology,** Fox Chase Cancer Center, Philadelphia, Pennsylvania; Department ofneuropathology,t Children's Hospital ofphiladelphia, Philadelphia, Pennsylvania; Department of Pathology,t Mayo Clinic, Rochester, Minnesota; Department of Pathology,5 National Cancer Institute, Bethesda, Maryland; and Division of Experimental Medicine,11 Brigham and Women's Hospital, Boston, Massachusetts Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and tumors of skin, brain, heart, and kidney. In this study, we focused on two of the most frequent tumors in TSC patients, renal angiomyolipomas and subependymal giant cell astrocytomas (SE- GAs). Two questions were addressed. First, is loss of tuberin, the product of the TSC2 gene, seen in both renal and central nervous system tumors from TSC patients? Second, when loss of tuberin occurs, does it affect each of the cell types seen in these tumors? We used a loss of heterozygosity approach to identify tumors from TSC2 patients. We found loss of tuberin immunostalning in the spindle and epithelioid cells but not in the giant cells of six TSC2 SEGAs. We also found loss of tuberin immunostaning in all three cell types (smooth muscle, fat, and vessels) of six TSC2 anglomyolipomas. Chromosome 16pl3 loss of heterozygosity occurred in both spindle and epithelioid cells of a SEGA and in smooth muscle and fat but not the vessels of two anglomyolipomas. These results support a two-hit tumor suppressor model for the pathogenesis of SEGAs and angiomyolipomas. The vascular elements of angiomyolipomas and the giant cells of SEGAs may be reactive rather than neoplastic. (AmJ Pathol 1997, 151: ) Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and the development of tumors in multiple organs including brain, heart, kidney, and skin. Most tumors in TSC, with the exception of renal cancers,1 are benign, including renal angiomyolipomas, facial angiofibromas, cardiac rhabdomyomas, and subependymal giant cell astrocytomas (SEGAs). SEGAs typically arise in the subventricular region of the brain, most commonly in the lateral ventricle, and can cause hydrocephalus and increased intracranial pressure.2'3 Symptomatic SEGAs occur in 6% of TSC patients,4 but radiographic evidence of subependymal nodules, the precursor of SEGAs, is found in 88% of TSC patients.5 Both SEGAs and angiomyolipomas are characterized by cellular pleomorphism. Histologically, SEGAs are composed of three cell types: spindle, intermediate-sized polygonal or epithelioid, and giant.6 Immunohistochemistry and immuno-electron microscopy have demonstrated that cells with the same morphology can express both glial and neuron-associated epitopes, suggesting that the cells giving rise to SEGAs are capable of divergent differentiation.7 At autopsy, angiomyolipomas are found in 67% of TSC kidneys.6 They also occur in the liver and other organs. Angiomyolipomas are vascular tumors consisting of blood vessels, smooth muscle, and adipose tissue. The blood vessels typically have thick, abnormally formed walls with disruption or even absence of the internal elastic laminae as well as replacement of the vascular smooth muscle by dense fibrous connective tissue.9 Genetic linkage analysis of families that segregate TSC indicates that there are two genes that cause this disease: TSC1 on chromosome 9q34 and TSC2 on chromosome 16p13.10 Approximately half of TSC families demonstrate linkage to TSC1 and half to TSC2. Tuberin, the product of the TSC2 gene11 is widely expressed in nor- Supported by grants from the National Institutes of Health, CA61889 (R. S. Yeung), and the National Tuberous Sclerosis Association (R. S. Yeung and E. P. Henske). Accepted for publication September 11, Address reprint requests to Dr. Elizabeth Petri Henske, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA henske@tuberous.fccc.edu. R S. Yeung's present address is the Department of Surgery, University of Washington School of Medicine, Seattle, WA

2 1640 Henske et al mal tissues12-15 and has GTPase activating (GAP) activity for rapl16 and rab5.17 Both rapl and rab5 are members of the ras superfamily of small GTP-binding proteins. The TSC1 gene on chromosome 9q34 was recently identified.18 Its function is unknown. A number of findings indicate that the tuberous sclerosis genes are tumor suppressor genes. Loss of heterozygosity (LOH) on chromosome 16p1319,20 and 9q3421,22 has been demonstrated in TSC lesions, consistent with tumor suppressor functions for the TSC1 and TSC2 genes. Germline TSC2 gene mutations are predicted to cause loss of functional tuberin.11 The Eker rat model of autosomal dominant renal cancer provides direct evidence that the TSC2 gene is a tumor suppressor gene. Eker rats develop renal cancer23 as well as other tumors including subependymal giant cell hamartomas.24 A germline mutation in the rat homologue of TSC2 has been identified in the Eker rat,2526 and loss of wild-type TSC2 occurs in the renal cancers.2728 Cell proliferation and the tumorigenicity of Eker renal carcinoma cells are suppressed by transfection of either wild-type TSC2 or the C-terminal region of TSC2 including the GAP domain.29 To determine whether the two-hit tumor suppressor model applies to both central nervous system (CNS) and renal tumors in tuberous sclerosis, and to determine which cells are affected by the second hit, we studied SEGAs and angiomyolipomas from five TSC2 patients and Eker rat subependymal hamartomas. In SEGAs we found an absence of tuberin in both spindle and epithelioid cells but not in giant cells. In angiomyolipomas, the loss of tuberin was seen in all three cell types. Analysis of microdissected tissue was consistent with a two-hit inactivation of TSC2 in both spindle and epithelioid cells in SEGAs and in smooth muscle and fat (but not vessels) in angiomyolipomas. These results demonstrate that TSC2 tumors, despite their cellular pleomorphism, share a common molecular pathogenesis. Materials and Methods Patients and Tumors All samples were from patients with a clinical diagnosis of tuberous sclerosis using the criteria of Gomez.30 Patient 312 was the only patient from whom multiple affected and unaffected family members were available for genetic linkage analysis. This analysis was consistent with linkage of TSC to chromosome 16p13 and not to chromosome 9q34 (results not shown). Loss of Heterozygosity Analysis Loss of heterozygosity in the TSC2 region of chromosome 16p was used to establish that the patients in this study had TSC2 disease. Patient 32 had three angiomyolipomas, which all showed chromosome 16p13 LOH but not chromosome 9q34 LOH.20 Patient 97 had 11 angiomyolipomas (eight of which showed chromosome 16p13 LOH and none of which showed chromosome 9q34 LOH) and Tuberin Immunostaining of SEGAs and Angiomyolipomas four SEGAs (none of which showed LOH on either 9q34 or 16p1331). Patient 104 had one SEGA that demonstrated LOH on chromosome 16p13 with retained heterozygosity on chromosome 9q34.31 Patient 309 had two renal angiomyolipomas, one of which showed LOH on chromosome 16p13, and both tumors retained heterozygosity on chromosome 9q34. For microdissection of the SEGA from patient 104, unstained sections of paraffin embedded tissue were aligned with hematoxylin and eosin (H&E) stained sections. Tissue from the giant cell and spindle cell regions was removed separately using an 18-gauge needle. For microdissection of the angiomyolipomas from patient 97, sections were lightly stained with H&E. Entire vascular structures including endothelial and perivascular cells were isolated. Microdissection was performed while the section was visualized under the 1Ox objective of an Olympus microscope. DNA was extracted from the paraffin-embedded tissue in 50 mmol/l KCI, 10 mmol/l Tris (ph 8.3), 1.5 mmol/l MgC12, 100,ug/ml bovine serum albumin, 0.45% Tween 20, 0.45% Nonidet P-40, and 100,ug/ml proteinase K at 500C for 18 hours. Proteinase K was heat inactivated at 1000C for 10 minutes. Control DNA for each patient was obtained from histologically normal tissue. One gl of aliquot was used in a 10-,ul polymerase chain reaction (PCR). Amplification was performed using primers for the chromosome 16p13 microsatellite markers kg8 and D16S28332 in the presence of [32P]dGTP, as previously described.31 PCR products were resolved by denaturing 8 mol/l urea-polyacrylamide gel electrophoresis and visualized by autoradiography. Five-micron paraffin sections were deparaffinized, rehydrated, and processed for antigen retrieval by boiling in 6 mol/l urea in a microwave oven (750 W) at a low setting for 3 minutes. Endogenous peroxidase was blocked using 3% H202 in phosphate-buffered saline. Sections were incubated with C20, an affinity-purified polyclonal antibody to the C-terminal polypeptide of tuberin (1:100, Santa Cruz Biotechnology, CA) or L3, an IgG purified polyclonal antibody to a glutathione S-transferase-tuberin fusion protein,17 for 2 hours at room temperature. L3 was used for preliminary studies only as we found that nonspecific staining was greater with L3 than with C20 (see results section). Negative controls were incubated with phosphate-buffered saline. The slides were washed, exposed to secondary biotinylated goat anti-rabbit antibody, and incubated in ABC reagent (avidin and biotinylated horseradish peroxidase complex, Vector Laboratories, Burlingame, CA). Signals were detected using diaminobenzidine tetrahydrochloride as chromogen substrate. Sections were lightly counterstained with Gill's hematoxylin and mounted in Permount (Fisher Scientific, Fair Lawn, NJ). For blocking experiments, the antigen (C20 or N19 peptide, Santa Cruz Biotechnology)

3 Loss of Tuberin in Tuberous Sclerosis 2 Tumors 1641 IP HeLa E4 E8 al3 afhit + ptsc2*_ IgG_o ~+ Figure 1. The tuberin C20 antibody (Santa Cruz) specifically recognizes the 190-kd TSC2 product. Endogenous tuberin was immunoprecipitated by anti- L3"7 from whole cell lysates and detected by immunoblotting analysis using the C20 antibody in human cervical carcinoma cells (HeLa) and in TSC2 +/+ embryonic fibroblasts (E4). The specific band representing tuberin is not present in immunoprecipitate from an unrelated antibody, anti-fhit, nor in embryonic fibroblasts derived from a homozygous TSC2 -/- Eker rat (E8). was preincubated with the antiserum for 1 hour at room temperature before incubation with the tissues. Results Characterization of the Tuberin Antibody We used L3, a previously characterized antibody to a glutathione S-transferase-fusion product of tuberin,'7 to determine the specificity of the commercially available affinitypurified antibody, C20. Whole cell lysates from human derived HeLa cells and rat derived fibroblasts with (E4) or without (E8) endogenous tuberin were immunoprecipitated with anti-l3 or an unrelated antibody (anti-fhit), followed by immunoblotting using the C20 antibody. The C20 antibody recognized a specific 190-kd band corresponding to tuberin in HeLa and E4 cells but not in E8 cells that lack tuberin (Figure 1). For immunohistochemistry, the IgG-purified anti-l3 preparation revealed greater nonspecific staining than the C20 antibody, although both reagents showed similar patterns of cytoplasmic staining (data not shown). We therefore used the C20 antibody for this study. To establish the specificity of C20 reactivity in paraffin-embedded tissues, we demonstrated that cytoplasmic staining in renal tubular epithelium can be blocked by preincubation of C20 with its antigen but cannot be blocked by a peptide from the N terminus of TSC2, N19 (Figure 2). The pattern of tuberin reactivity in the normal kidney using C20 was identical to that previously reported.13 4,40 Immunostaining of Human Angiomyolipomas and SEGAs Paraffin-embedded sections from six angiomyolipomas were immunostained with the C20 tuberin antibody. Three were from patient 97 and one each was from patients 32, 312, and 309. Only angiomyolipomas with both tumor and normal tissue present in the same section were used to provide an internal positive control for the tuberin immunostaining. We found loss of tuberin in smooth muscle, fat, and vessels in all six angiomyolipomas (Figure 3A). The expected expression of tuberin in normal tissues was seen on each slide (Figure 3B).13 On one section from patient 97, the non-neoplastic renal tissue contained six microscopic foci of smooth muscle proliferation, all of which showed absent tuberin immunostaining. Eleven human SEGAs were immunostained. Six were from patients with TSC2 disease (four from patient 97, one from patient 104, and one from patient 309), and five were from patients of unknown TSC1 versus TSC2 status. Positive tuberin immunostaining in the ependymal lining cells (Figure 4D), which has been previously reported,14'15 was seen in all samples, providing an internal control for the level of staining. Tuberin staining was absent in the spindle and epithelioid cells and was faintly present in the giant cells of all six TSC2 SEGAs (Figure 4, B and C) and in four of the five SEGAs of unknown TSC1 versus TSC2 status. One SEGA of unknown TSC status showed prominent immunostaining in all three cell types (Figure 4E). Immunostaining of Eker Rat Subependymal Hamartomas Rat and human subependymal tumors have similar spindle and epithelioid components, although the rat tumors do not have giant cells.24 We examined the pattern of tuberin expression in Eker rat subependymal hamartomas by immunohistochemistry. Consistent with the findings in human SEGAs described above, the majority of the cellular components (spindle and epithelioid) within the rat subependymal hamartomas stained negatively for tuberin (Figure 5), as compared with adjacent normal neurons and ependymal cells. Microdissection and Genetic Analysis of Human Tumors We screened 10 angiomyolipomas in which chromosome 16p13 LOH had been previously detected and identified two with discrete regions of vascular, smooth muscle, and adipose tissue that were suitable for microdissection. One was from patient 97 (tumor 97-T17) and one was from patient 32 (tumor 32-3T). PCR analysis of the microdissected samples using the microsatellite marker kg8 showed clear loss of heterozygosity in the smooth muscle and fat components, as shown in Figure 6A. A faint signal from the lost allele was seen, which we and others have

4 1642 Henske et al SE-4.. t' A Negative control X B atub (C20) C C20 peptide t : D atub(c20) + C20 peptide i.: E atub(c20) + N19 peptide U.. r, Figure 2. Specific immunohistochemical staining of the tuberin C20 antibody in normal rat kidney. Paraffin-embedded tissue sections were incubated in the absence (A) or presence (B) of C20 antibody. Specific immunoreactivity is restricted to the tubular epithelium of the kidney. The immunoreactivity is not present with the C20 peptide alone (C) and is blocked when the C20 antibody is preincubated with its immunogen (D) but not blocked with the N19 peptide (E). found in most TSC tumors analyzed for LOH using a PCR-based method.31'33 Although clear evidence of LOH was not seen in the vascular component of the angiomyolipomas (Figure 6A), there was an imbalance of alleles with the upper allele approximately twofold less intense than the lower allele. This allelic imbalance may be from smooth muscle cells with LOH that were intermingled with the vessels or may indicate that some components of the vessels have LOH, whereas other components do not. The one SEGA with chromosome 16p13 LOH had two histologically distinct regions. One region had approximately 95% spindle cells, and another smaller region had approximately 90% epithelioid cells. The epithelioid region contained approximately 5% giant cells and 5% spindle cells. The spindle and epithelioid regions of this tumor were microdissected and separately analyzed for LOH. Loss of heterozygosity at the chromosome 16p13 markers kg8 (Figure 6B) and D16S283 were seen in both the spindle cell region and the epithelioid cell region with a nearly identical balance of intensity between the retained and the lost alleles, demonstrating that the second hit exists in both the spindle and the epithelioid cell populations. Discussion We undertook this study to address two specific questions. First, does the pathogenesis of TSC2-associated CNS tumors involve two hits in TSC2 with loss of functional tuberin? Second, in tumors such as SEGAs and angiomyolipomas that exhibit cellular pleomorphism, does the loss of tuberin occur in all cell types?

5 Loss of Tuberin in Tuberous Sclerosis 2 Tumors 1643 ~~M.~ A)~~~~~. BaT 20 Figure 3. Loss of tuberin immunostaining in a TSC2 angiomyolipoma. Paraffin-embedded tissue sections from patient 97 were immunostained with the C20 antibody. No tuberin immunostaining was detected in the vascular (large arrow), fat (arrowheads), or smooth muscle elements of the angiomyolipoma (A). Renal tubules from a region of normal kidney on the same slide stained positively (B). These questions were raised in part by our recent finding of a difference in the frequency of loss of heterozygosity in tuberous sclerosis CNS tumors compared with renal tumors.31 LOH was detected in 60% of renal angiomyolipomas but in only 4% of CNS tumors (0 of 11 cortical tubers and only 1 of 14 SEGAs).31 This led to the hypothesis that a somatic second hit is not required for the pathogenesis of CNS tumors in TSC.34 An alternative explanation for the low incidence of LOH in SEGAs is that SEGAs consist of a mixture of cells, only some of which have sustained a second hit. A third possibility is that second hits in the CNS are frequently small deletions or point mutations that are not detectable using an LOH analysis. It is not known whether loss of the TSC1 gene product affects the levels of tuberin or whether loss of tuberin affects the levels of the TSC1 gene product. Therefore, to use the results of immunostaining to develop a model for the role of tuberin in tumor pathogenesis it is essential to know whether the tumors being studied are from individuals with TSC2 (chromosome 16p13) or TSC1 (chromosome 9q34) mutations. However, the distinction between TSC1 and TSC2 disease can be extremely difficult to make. No clinical differences between TSC1 and TSC2 mutations have yet been identified. Establishment of TSC1 versus TSC2 status by genetic linkage analysis is often impossible as 60% of TSC cases represent apparent new mutations in individuals with no family history of the disease. Finally, although the TSC2 gene has been identified, its size makes mutational analysis difficult. The lack of a TSC2 mutation could mean either that the mutation was not detected or that the patient has TSC1 disease. Preliminary mutational studies of all 41 exons of TSC2 using single strand conformation polymorphism analysis have detected variant bands in only 21 of 90 TSC patients.35 In our study, we determined the TSC1 versus TSC2 status of four patients using a loss of heterozygosity approach. Individuals with tumors in which chromosome 16p13 LOH was detected were considered to have tuberous sclerosis due to a TSC2 mutation. Our decision to use this method to identify TSC2 tumors for this study was based on the LOH analyses of 158 benign TSC tumors by three groups.19'31'33'36 These studies clearly show that LOH in TSC tumors is specific, occurring on either chromosome 9q34 or 16p13. No tumors with LOH at both chromosomal loci have been ever reported by any group. The specificity of this method is also supported by the fact that when multiple tumors from a single patient have LOH, it is always for the same chromosomal haplotype.20'31'33 When the family is large enough to establish genetic linkage to either chromosome 16 or 9, it has been possible to confirm that the wild-type copy of the TSC1 or TSC2 gene is lost.22 The sensitivity of this method is greater when multiple tumors are analyzed. Sixty percent of renal angiomyolipomas demonstrate LOH at either the TSC1 or TSC2 chromosomal region, and 85% of individuals with at least three angiomyolipomas have LOH detected in at least one tumor.31 Therefore we estimate that the sensitivity of this method is 60% if one angiomyolipoma is analyzed and 85% if three or more angiomyolipomas are analyzed. We studied tuberin expression in SEGAs and angiomyolipomas from five patients with TSC2 disease. Loss of tuberin immunoreactivity was seen in smooth muscle, fat, and vessels of all six angiomyolipomas from these patients. Using microdissection, regions of two angiomyolipomas consisting primarily of smooth muscle, fat, and vessels were analyzed for LOH. Both the smooth muscle and the fat showed LOH, whereas the vascular component did not. These results suggest that the vascular elements of angiomyolipomas are reactive and not neoplastic. We found this result surprising as tuberin immunostaining was absent in the vascular component of the tumor (Figure 3A), whereas high levels of tuberin expression were seen in the vascular smooth muscle of normal kidney in this (results not shown) and another13 study. It is possible that some components of the vessels are

6 1644 Henske et al neoplastic (for example the endothelial cells), whereas other parts (for example the vascular smooth muscle) are reactive, or that some vessels within an angiomyolipoma are neoplastic and others are reactive, or that these dysplastic cells simply do not express high levels of tuberin despite having an intact copy of the TSC2 gene. Additional studies will be required to address the origin of the vascular elements of angiomyolipomas. All six SEGAs from TSC2 patients showed loss of tuberin immunostaining in spindle cells and epithelioid cells. Faint reactivity was seen in giant cells. The giant cell staining was more intense than staining in spindle and epithelioid cells from the tumor, but less intense than the staining of normal neurons or ependymal cells. Tuberin immunoreactivity was also lost in both spindle cells and epithelioid cells from Eker rat subependymal hamar- Figure 4. Absence of tuberin immunostaining in a TSC2 SEGA. Paraffinembedded tissue from patient 104 was stained with H&E (A). Giant cells (large arrow), epithelioid cells (arrowhead), and spindle cells (small arrow) are indicated. Tuberin immunostaining was absent in epithelioid cells (B) and spindle cells (C) and faintly present in giant cells (B). Positive immunostaining in ependymal lining cells, an example of which is shown in D, was used as an internal control of tuberin immunostaining. One SEGA, from patient 106, showed positive immunostaining in all three cell types (E). "ti '-`~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.P"4t. :'A ^,2 ei A H&E B-E atub (C20)

7 Loss of Tuberin in Tuberous Sclerosis 2 Tumors A,B ttub (C20) C,D H &E Figure 5. Absence of tuberin immunostaining in subependymal hamartomas of the Eker rat. Low magnification view of an Eker subependymal hamartoma immunostained with the C20 antibody (A) shows little or no staining compared with adjacent structures. Higher magnification of a second tumor shows loss of tuberin in both the spindle and epithelioid components of the lesion and positively stained neurons in the adjacent normal tissue (B). Corresponding H&E-stained sections are shown (C and D). tomas. Giant cells have not been found in Eker rat subependymal tumors. Microdissection followed by PCR analysis demonstrated that the spindle and the epithelioid regions have chromosome 16p13 LOH. The neuronal versus glial origin of subependymal giant cell astrocytomas has been debated for decades4'37-41 with some cells appearing to express both neuronal and glial markers.7'42 Our results strongly support a two-hit model for the pathogenesis of SEGAs and suggest that the precursor cell that sustained the second hit may have the potential for differentiation along both neuronal and glial pathways. In addition to the SEGAs from TSC2 patients, we also immunostained five SEGAs from patients with unknown TSC1 versus TSC2 status. Four showed loss of tuberin immunostaining with a similar pattern to the TSC2 SEGAs. One SEGA showed strong immunoreactivity for tuberin in all three cell types (Figure 4E). Two previous studies14'43 have assessed tuberin levels in SEGAs. One study43 found loss of tuberin expression in three SEGAs by Western blot analysis. The second study14 found moderate levels of tuberin immunoreactivity in SEGAs using immunohistochemistry. These two studies, along with our data, highlight the difficulty in interpreting tuberin expression studies when the TSC1 versus TSC2 status is unknown. The one SEGA in our study with detectable tuberin expression and the SEGAs with moderate tuberin expression in the previous study14 are consistent with either TSC1 gene mutations or TSC2 gene mutation that preserve tuberin immunoreactivity. Similarly, SEGAs with loss of tuberin expression may have inactivating TSC2 gene mutations, or loss of the TSC1 gene product could secondarily affect tuberin expression. In conclusion, we report the first immunostaining analysis of TSC lesions in which the TSC1 versus TSC2 status of the patients was specifically ascertained. We found that both human and rat TSC2 subependymal tumors have loss of tuberin immunostaining in spindle and epithelioid cells and that chromosome 16p13 LOH can affect both spindle and epithelioid cells. Additional analysis using single-cell microdissection will be required to determine whether the giant cells of human SEGAs have sustained a second hit. We also found loss of tuberin expression in all three components (fat, smooth muscle,

8 1646 Henske et al B 97 - T T _ S5-= smooth muscle V= vessels _ je _ Fw~~~~~~F=fat K- normal kidney S V F K S V F K N E S N. normal brin E= epitheliold region S= spindle cell region Figure 6. Loss of heterozygosity in different cellular components of angiomyolipomas (A) and a subependymal giant cell astrocytoma (B). In A, microdissection was used to selectively procure regions consisting primarily of smooth muscle (S), vessels (V), and fat (F) from angiomyolipomas from patient 97 (97-T17) and 32 (32-3T). DNA was isolated and amplified using the marker kg8. Two alleles, each of which is represented by a doublet, are seen in normal kidney (K). The upper band of each allele is indicated with an arrow. By chance, both patients have the same two kg8 alleles. Decreased intensity of the upper allele relative to the lower is seen in smooth muscle (S) and fat (F) from both tumors, indicating loss of heterozygosity. Loss is not seen in the samples consisting primarily of vessels (V). In B, microdissection was used to separate a region of spindle cells (S) from a region of primarily epithelioid and giant cells (E) from a SEGA from patient 104. Two alleles, each represented by a doublet, are seen in normal DNA (N) from this patient after PCR amplification using the marker kg8. Loss of the lower allele is seen in both the epithelioid and the spindle components of the tumor. and vessels) of angiomyolipomas, but only the smooth muscle and fat showed LOH. Angiomyolipoma vessels, therefore, do not appear to be part of the neoplastic process. This study demonstrates that cells with a second hit in TSC2 can undergo divergent differentiation into spindle cells and epithelioid cells in SEGAs and into fat and smooth muscle in angiomyolipomas, indicating that tuberin may play a role in the control of cellular differentiation. Acknowledgments We thank William A. Petri, Sr. and Dr. Alfred Knudson for critical review of the manuscript, the Harvard Brain Tissue Resource Center for providing tissue for this study, and Catherine Renner and Fang Jin for expert technical assistance. References 1. Bjornsson J, Short MP, Kwiatkowski DJ, Henske EP: Tuberous sclerosis-associated renal cell carcinoma: clinical, pathologic, and genetic features. Am J Pathol 1996, 149: Buki A, Horvath Z, Kover F, Veto F, Doczi T: Occlusive hydrocephalus complicating tuberous sclerosis - report of two cases. Eur J Neurol 1996, 3: Dirocco C, lannelli A, Marchese E: On the treatment of subependymal giant cell astrocytomas and associated hydrocephalus in tuberous sclerosis. Pediatr Neurosurg 1995, 23: Shepherd CWv, Scheithauer BW, Gomez MR. Altermatt HJ, Katzmann JA: Subependymal giant cell astrocytoma: a clinical, pathological, and flow cytometric study. Neurosurgery 1991, 28: Shepherd CW, Gomez MR, Lie JT: Causes of death in patients with tuberous sclerosis. 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