INTRODUCTION

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Keywords: lymphangiomyomatosis; tuberous sclerosis; genes; suppressor; tumor; loss of heterozygosity; chromosomes; human; pair 16

Lymphangiomyomatosis (LAM) is a rare disease that affects almost exclusively women (1). LAM consists of a diffuse proliferation of smooth muscle cells around lymphatic vessels, blood vessels, and airways. Retroperitoneal and pelvic lymph node involvement can also occur (2). The symptoms of LAM include dyspnea, cough, and pneumothorax (3). The average age at diagnosis is approximately 30 yr. Lung transplantation appears to be the only effective option for patients with end-stage disease (4, 5), although recurrences after transplantation can occur (1).

LAM can occur as an isolated disorder, referred to here as sporadic LAM, or in association with tuberous sclerosis complex (TSC), referred to here as TSC-LAM. TSC is an autosomal dominant disorder characterized by seizures, mental retardation, autism, and tumors of the brain, heart, and kidney. These tumors include cerebral cortical tubers, subependymal giant cell astrocytomas, retinal hamartomas, cardiac rhabdomyomas, renal angiomyolipomas, and renal cell carcinomas. Germline mutations in two tumor suppressor genes, TSC1 on chromosome 9q34 (6) and TSC2 on chromosome 16p13 (7), cause TSC. Among patients with TSC, LAM is the third most frequent cause of TSC-related death, after renal disease and brain tumors (8).

We recently found somatic TSC2 mutations in angiomyolipomas and the pulmonary smooth muscle cells from four patients with sporadic LAM (9), with loss of heterozygosity (LOH) inactivating the wild-type TSC2 allele. In each case, the same mutation was found in the angiomyolipoma and LAM cells, but not in normal lung or normal kidney. This suggested the possibility of a highly unusual disease mechanism: the migration of abnormal smooth muscle cells with a common genetic origin between the lung and kidney.

We have previously shown that different tumors from a single patient with TSC have differing extents of LOH (10, 11). This is consistent with the Knudson tumor suppressor gene model (12), which predicts that each angiomyolipoma arises from a genetic event inactivating the wild-type TSC2 allele. We reasoned that if angiomyolipomas and LAM arise separately with different genetic events inactivating the wild-type TSC2 allele, we might detect differences in their LOH patterns. If angiomyolipoma and LAM cells have a common origin, the LOH patterns should be identical. Therefore, we compared the LOH region on chromosome 16 between angiomyolipoma and pulmonary LAM in two patients with sporadic LAM using multiple microsatellite markers spanning the chromosome. We also analyzed laser capture microdissected pulmonary LAM cells from a patient with TSC-LAM for LOH in the TSC2 region of chromosome 16.

	METHODS

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This study was approved by the institutional review board of Fox Chase Cancer Center and was performed with the informed consent of the participants.

DNA Extraction and Laser Capture Microdissection

DNA was extracted from paraffin-embedded tissue specimens as described previously (11). For the pulmonary LAM and normal lung samples, we used laser capture microdissection (PixCell II; Arcturus Engineering, Mountain View, CA). To identify the abnormal smooth muscle cells, adjacent tissue sections were stained with muscle-specific actin (BioGenex, San Ramon, CA) and hematoxylin and eosin. The hematoxylin-eosin slide was used for the microdissection. Regions containing primarily normal lung were identified morphologically. Approximately 500 nuclei of normal lung and LAM cells were captured. Because only two or three reactions could be performed per microdissection, several separate microdissections were performed for each patient. The DNA was extracted by overnight incubation in 10 µl of extraction buffer (5% Tween 20, 2 mg/ml proteinase K, 0.5 M Tris-HCl, pH 8.9, 20 mM ethylenediaminetetraacetic acid [EDTA], and 10 mM NaCl).

LOH Analyses

We used a panel of microsatellite markers spanning chromosome 16: D16S475, D16S525, Kg8, D16S291, D16S663, D16S423, D16S418, D16S287, D16S749, D16S769, D16S766, and D16S2624 (Research Genetics, Huntsville, AL). The cytogenetic locations of these markers are shown on the Web Repository. A 2.5-µl aliquot of DNA was used in a 10-µl polymerase chain reaction (PCR) containing 10% glycerol. The PCR amplification consisted of 95° C for 5 min, 40 cycles of 94° C for 30 s, 55° C for 30 s, 72° C for 45 s, and a final extension of 72° C for 10 min. PCR was performed with radioactive phosphorus-labeled deoxyguanosine triphosphate ([³²P]dGTP) in the reaction mix. TaqStart antibody (Clontech, Palo Alto, CA) at a final concentration of 0.056 µM was used in PCR reaction to enhance the specificity. The PCR products were resolved by denaturing 8 M urea polyacrylamide gel electrophoresis (Gibco, Grand Island, NY) and visualized by autoradiography. All results were repeated at least twice for confirmation.

	RESULTS

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TSC-LAM

Patient 603 has a history of bilateral pneumothoraces. Lung biopsy showed smooth muscle proliferation and cystic changes consistent with LAM. Magnetic resonance imaging of her brain showed a lesion consistent with tuberous sclerosis, and an abdominal computed tomographic (CT) scan showed multiple, bilateral renal lesions consistent with angiomyolipomas. These lesions have not been biopsied or removed. The patient has no family history of TSC, and she has no children. In a prior study (13), we failed to identify a germline mutation in either TSC1 or TSC2 in her peripheral blood lymphocytes using single-strand conformation polymorphism analysis (SSCP). SSCP has a sensitivity of approximately 60% for the detection of TSC gene mutations in patients with known TSC (14).

DNA prepared from laser capture microdissected pulmonary LAM cells was tested for LOH in the chromosome 16p region. LOH was detected in the pulmonary LAM cells using the marker D16S475 (Figure 1). Unfortunately, the other markers (D16S525, Kg8, D16S291, D16S663, D16S423, D16S418, and D16S749) were all homozygous or had closely spaced alleles unsuitable for LOH analysis. Limited tissue availability prevented us from testing other markers.

View larger version (47K): [in this window] [in a new window]   Figure 1.   Examples of LOH analyses. For each panel, the patient number and the marker are indicated. Patient 603 has TSC-LAM, and Patients 487 and 490 have sporadic LAM. The tissues studied included lymphoblastoid cells (LB), microdissected normal lung (NL), microdissected pulmonary LAM (L), normal kidney (NK), and angiomyolipoma (A). The location of the upper band representing each of the two alleles in normal tissues is indicated with a line. The "lost" allele is indicated with an arrow. Some residual signal from the lost allele is evident in the microdissected LAM specimens and to a lesser degree in the angiomyolipomas, reflecting the presence of contaminating normal cells. Retention of heterozygosity is seen at the marker D16S2624 for Patient 487, with both alleles present in normal kidney, angiomyolipoma, normal lung, and LAM.

Sporadic LAM

We previously reported TSC2 LOH in the angiomyolipomas (15) and microdissected pulmonary LAM cells (9) of Patients 487 and 490 with sporadic LAM. These LOH studies were performed using four markers (D16S525, Kg8, D16S291, and D16S283) within 600 kb of TSC2. Both patients also had inactivating TSC2 mutations in the remaining copy of TSC2 in their angiomyolipoma and pulmonary LAM cells. Patient 487 had a nonsense mutation G1096T in exon 10 and Patient 490 had a 4-bp deletion in exon 18 (9).

In this study, we analyzed nine additional microsatellite markers in the angiomyolipoma, normal kidney, microdissected pulmonary LAM cells, and microdissected normal lung from Patient 487, and five additional markers in tissues from Patient 490. Limited tissue availability prevented us from testing all nine markers in pulmonary LAM cells from Patient 490.

Representative LOH results for Patient 487 are shown in Figure 1. For Patient 487, four markers from this study (D16S663, D16S423, D16S418, and D16S749) and one marker from the previous study (Kg8) showed LOH in both the angiomyolipoma and LAM cells. Three markers from this study (D16S475, D16S287, and D16S769) were homozygous. Two markers from this study (D16S766 and D16S2624) had retained heterozygosity. The comparison of LOH results in angiomyolipoma and pulmonary LAM from Patient 487 is summarized in Figure 2.

View larger version (38K): [in this window] [in a new window]   Figure 2.   Chromosome 16 LOH results for Patients 487 and 490. Microsatellite markers used in the LOH analyses are listed at the left. The TSC2 gene location at 16p13.3 is indicated. Columns from left to right represent normal kidney and normal lung (N ), angiomyolipoma cells (A), and microdissected LAM cells (L). Dotted boxes represent homozygous markers. Gray boxes represent heterozygous markers. Black boxes indicate that LOH was detected. Open boxes represent markers that were not tested.

Representative LOH results for Patient 490 are shown in Figure 1. Four markers from this study (D16S475, D16S663, D16S418, and D16S749) and one marker from the previous study (D16S525) showed LOH in both angiomyolipoma and LAM cells. One marker from this study (D16S423) was homozygous. One marker from this study (D16S2624) showed retained heterozygosity. The comparison of LOH results in angiomyolipoma and pulmonary LAM from Patient 490 is summarized in Figure 2.

	DISCUSSION

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In previous work, we identified somatic TSC2 mutations in angiomyolipomas and the pulmonary smooth muscle cells from four patients with sporadic LAM (9). We also found chromosome 16 LOH in the angiomyolipomas (15) and microdissected pulmonary LAM cells (9) from patients with sporadic LAM, consistent with the two-hit tumor suppressor gene model (12). Because we found the same mutations in the angiomyolipoma and LAM cells, we hypothesized that pulmonary LAM could arise from the migration of smooth muscle cells from an angiomyolipoma to the lung.

In this study, we first asked whether the two-hit model also applies to TSC-associated LAM. We then compared the LOH region on chromosome 16 between angiomyolipoma and pulmonary LAM cells from two patients with sporadic LAM to determine whether these cells appear to have a common genetic origin.

We found LOH in the TSC2 region of chromosome 16p13 in microdissected pulmonary LAM cells from a female patient with TSC. This indicates that TSC-LAM, like sporadic LAM, fits the two-hit model. It also underscores the similarities between TSC-LAM and sporadic LAM at the immunohistochemical, ultrastructural, and now at the genetic levels.

To our knowledge, there is only one prior report of a patient with TSC-LAM in whom LOH was sought in pulmonary tissue (16). In that study, LOH was not detected. There are at least two possible reasons why we detected LOH in our study and the previous study did not. First, LOH is detected in only approximately 60% of TSC tumors. This suggests that many tumors have other types of genetic events inactivating the wild-type allele that are not detected in LOH analyses. For example, missense changes or intragenic deletions are not detected in an LOH analysis. Second, the authors of the previous report did not use microdissection. We have found that we can detect the LOH event only in the abnormal smooth muscle cells (as identified by muscle-specific actin immunoreactivity) and that laser capture microdissection facilitates the isolation of these cells.

For the second part of this study, we focused on two patients with sporadic LAM in whom we previously detected LOH in both angiomyolipoma and pulmonary LAM cells. Our goal was to determine whether any differences in the LOH pattern between angiomyolipoma and LAM could be detected. Previously we proposed two possible models for sporadic LAM: low-level somatic mosaicism for cells with a "first-hit" mutation, or migration of cells with both a first and second mutation between the lung and the kidney. We reasoned that if patients with sporadic LAM have a low level of mosaicism for a somatic "first-hit" mutation in TSC2, the second-hit events would arise independently in the lung and kidney. In this case, the angiomyolipoma and pulmonary LAM cells would not have exactly the same region of chromosome 16 loss. Alternatively, the cells could sustain a second hit such as LOH and then migrate from the angiomyolipoma to the lung, or from the lung to the kidney. In this case, the LOH pattern should be identical between angiomyolipoma and pulmonary LAM cells.

We had previously analyzed four markers in the TSC2 region of chromosome 16p13 (9) for LOH in tissue from Patients 487 and 490. At each heterozygous marker, the angiomyolipoma and pulmonary LAM cells were concordant for LOH. Here, we report for the first time the analysis of additional markers on chromosome 16 extending across the chromosome. For each marker, the pattern was concordant between the angiomyolipoma and the pulmonary LAM cells. LOH was seen in both the angiomyolipoma and the pulmonary LAM at four markers in Patient 487 and at four markers in Patient 490. Retention of heterozygosity was seen at two markers in patient 487 and at one marker in patient 490.

Our findings are consistent with a model in which LAM cells and angiomyolipoma cells have a common genetic origin. Previous data from our group (10, 11) and from other laboratories (17) clearly demonstrate that different TSC tumors have different second-hit mutations, and that these differences can often be detected using LOH analyses. For example, we previously analyzed two angiomyolipomas from a patient with TSC. Both tumors had LOH at the marker Kg8 but one had retained heterozygosity at D16S525 (10). In another study, we compared six angiomyolipomas from a patient with TSC. Four had LOH in the TSC1 region of chromosome 9q34. Among the four tumors with LOH, three different regions of LOH could be identified using four heterozygous markers, consistent with these individual tumors having separate genetic origins (11).

If a model in which LAM cells and angiomyolipoma cells have a common genetic origin proves to be correct, it will be of great importance to determine the biologic mechanism through which this occurs. One possibility is that the abnormal smooth muscle cells migrate from the angiomyolipoma to the lung, or from the lung to the kidney. There are at least two other diseases in which benign smooth muscle cells appear to metastasize: benign metastasizing leiomyoma (18) and disseminated peritoneal leiomyomatosis (19). It is not unusual to find metastatic foci of angiomyolipoma cells in perirenal lymph nodes (20), suggesting that angiomyolipoma cells are capable of migrating via the lymphatics.

The mechanism of LAM pathogenesis could involve activation of the small guanosine triphosphatase Rho factor (GTPase Rho). Recently the TSC1 gene product, hamartin, was found to regulate cell adhesion by activating Rho (21). Rho activation is believed to play a critical role in cancer cell motility, invasion, and metastasis, based on data from a number of different experimental systems (22). It is not yet known whether the TSC2 gene product, tuberin, also activates Rho, but hamartin and tuberin appear to physically interact and therefore are likely to function in the same cellular pathways (26, 27).

Proving or disproving the hypothesis that the pathogenesis of LAM involves the migration of abnormal smooth muscle cells from one organ to another may be challenging. Ultimately, it may require the development of animal models to fully understand the pathogenesis of LAM. To date, pathology consistent with angiomyolipomas or LAM has not been observed in the Eker rat model of TSC2 (28) or in either of two mouse models of TSC2 (29, 30). However, the Eker rat does develop uterine leiomyomas that appear to have important similarities to angiomyolipoma and LAM cells, including estrogen receptor expression (31).

In summary, we report here, for the first time, the identification of TSC2 LOH in microdissected pulmonary LAM cells from a patient with TSC-LAM. We also compared the LOH pattern between the angiomyolipoma and pulmonary LAM cells from two patients with sporadic LAM using markers spanning chromosome 16. At each heterozygous marker, the angiomyolipoma and pulmonary LAM cells were concordant for either loss or retention of heterozygosity. These results are consistent with a model in which LAM cells and angiomyolipoma cells have a common genetic origin.