American Journal of Respiratory and Critical Care Medicine

Lymphangiomyomatosis (LAM) is characterized by the proliferation of abnormal smooth muscle cells and cystic degeneration of the lung. LAM affects almost exclusively young women. Although lung transplantation provides effective therapy for end-stage LAM, there are reports of LAM recurrence after lung transplantation. Whether these recurrent LAM cells arise from the patient or the lung transplant donor is an area of controversy. We used microsatellite marker fingerprinting and TSC2 gene mutational analysis to study a patient with recurrent LAM after single-lung transplantation. The DNA microsatellite marker pattern indicated the presence of patient-derived LAM cells in the allograft. A somatic one base pair deletion in exon 18 of the TSC2 gene was identified in pulmonary and lymph node LAM cells before transplantation. The same mutation was in the recurrent LAM, demonstrating that the recurrent LAM was derived from the patient. Fluorescence in situ hybridization revealed that cells immunoreactive with the monoclonal antibody HMB-45 did not contain a Y chromosome. These data indicate that histologically benign LAM cells can migrate or metastasize in vivo to the transplanted lung. In addition, the patient had no evidence of a renal angiomyolipoma at autopsy and therefore demonstrated for the first time that somatic TSC2 mutations cause LAM in patients without angiomyolipomas.

Lymphangiomyomatosis (LAM), which affects almost exclusively young women (1, 2), is characterized by widespread pulmonary proliferation of abnormal smooth muscle cells. Renal angiomyolipomas occur in 50% of sporadic LAM patients (3). LAM can occur as an isolated disorder (sporadic LAM) or in women with tuberous sclerosis complex (TSC). TSC is a tumor suppressor gene syndrome in which the manifestations include seizures, mental retardation, autism, and tumors of brain, kidney, heart, and skin. The incidence of radiographic evidence of LAM among women with TSC is 26–39% (46). In prior work, we found that angiomyolipoma cells and pulmonary LAM cells from sporadic LAM patients contain inactivating mutations in the TSC2 gene (7). These mutations were not present in normal cells, indicating that they arose somatically. The presence of the same TSC2 mutation and loss of heterozygosity pattern (8) in LAM and angiomyolipoma cells led to the hypothesis that pulmonary LAM results from the metastatic spread of angiomyolipoma cells.

There are three documented cases of LAM recurrence after lung transplantation (911). In all three cases, the donor lungs were from men. In two cases, using in situ hybridization with Y chromosome probes, the foci of recurrent LAM were shown to contain Y chromosomes (10, 11). These findings suggested that a circulating growth factor induced the LAM cell phenotype.

To determine definitively the genetic origin of recurrent LAM cells after lung transplantation, we used laser capture microdissection and examined recurrent LAM cells genetically. Analysis of microsatellite markers revealed that foci of recurrent LAM in the allograft contain patient-derived cells and that the same TSC2 gene mutation present in the native LAM cells was also found in the recurrent LAM cells. This demonstrates that the recurrent LAM cells are derived from the patient's original LAM cells.

Laser Capture Microdissection and DNA Extraction

Paraffin sections were immunostained with muscle-specific actin and HMB-45 (both from BioGenex, San Ramon, CA). Laser capture microdissection (PixCell II; Arcturus Engineering, Mountain View, CA) was used to isolate cells (7). DNA was extracted by overnight incubation in 30 μl of extraction buffer (0.5% Tween 20, 0.2 mg/ml of proteinase K, 50 μM of Tris-HCl [pH 8.9], 2 mM of ethylenediaminetetraacetic acid, and 1 mM of NaCl).

Microdissected bronchial epithelial cells within the native left lung and the allograft lung were used to prepare normal DNA from the patient and donor, respectively. LAM cells were microdissected from the diagnostic lung biopsy specimen (2 years before transplantation), the native lung autopsy specimen (Figures 1A and 1B)

, four mediastinal lymph nodes removed during the diagnostic lung biopsy (Figures 1C and 1D), and the allograft (Figures 1E–1H).

Microsatellite Fingerprinting and Loss of Heterozygosity Analyses

Microsatellite markers on chromosome 1p (D1S1661), chromosome 9q (D9S1198), and chromosome 16p (D16S287, D16S291, D16S418) were used to compare the allele pattern of the donor, the patient, and the recurrent LAM. A 2.5-μl aliquot of the DNA solution was used in a 10-μl polymerase chain reaction (12). Polymerase chain reaction was performed with 32P-dGTP in the reaction mix. The polymerase chain reaction products were resolved by denaturating 8M-urea polyacrylamide gel electrophoresis and visualized by autoradiography. All results were repeated for confirmation. For loss of heterozygosity analyses, microsatellite markers near TSC2 on chromosome 16p13 were used (Research Genetics, Huntsville, AL).

Single-strand Conformation Polymorphism Analysis

Single-strand conformation polymorphism analysis (SSCP) was used to screen for mutations in each of 41 exons of TSC2 gene (7, 13). A single round of amplification using 35 cycles was performed with 32P-dGTP in the reaction mix. The polymerase chain reaction products were run on Mutation Detection Enhancement (MDE) gels (FMC Bioproducts, Rockland, ME). All reactions were repeated for confirmation.

DNA Sequencing

DNA was amplified and sequenced in both forward and reverse directions. The exon 18 variant band was also separately excised from the gel and was reamplified and sequenced.

Fluorescence In Situ Hybridization and Coimmunofluorescence Analysis

The spectrum orange labeled α satellite (CEP Y) DNA probe (Vysis, Inc., Downers Grove, IL), containing centromere sequences specific to chromosome Y, was hybridized to 5-μm paraffin sections according to the Spectra Vysion Assay Protocol (Vysis, Inc.). For coimmunofluorescence, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 minutes at room temperature. Nonspecific background was eliminated by incubating the tissue with normal goat serum (BioGenex, San Ramon, CA) for 10 minutes at room temperature. The sections were then incubated in a humidified chamber with mouse monoclonal antibodies against either muscle-specific actin or HMB-45 antigen and then rinsed and incubated with Cy2-conjugated donkey antimouse antibody (Jackson Immunoresearch, West Grove, PA), washed, and counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Fluorescent signals were captured using a Zeiss Axiophot epifluorescence microscope equipped with a cooled charge-coupled device camera (Photometrics, Tuscon, AZ).

Clinical Features

The patient was a 44-year-old woman who underwent single lung transplantation for LAM, which had been previously treated by oophorectomy and pleurodesis. Postoperatively, she was immunosuppressed with cyclosporine, azathioprine, and prednisone. She died of Aspergillus pneumonia 22 months later. There was no evidence of a renal angiomyolipoma before transplantation or at autopsy. The right lung allograft showed multifocal involvement by small foci of recurrent LAM cells (9). Tissue available for this study consisted of a diagnostic lung biopsy performed 2 years before lung transplantation, four mediastinal lymph nodes containing LAM removed during the biopsy, and autopsy specimens from both the native (left) lung and the allograft (right) lung. The recurrent LAM cells were immunoreactive with the HMB-45 antibody (Figures 1G and 1H).

Recurrent LAM Contains Patient-derived Cells

We used microsatellite markers on chromosome 1 (D1S1661), chromosome 9 (D9S1998), and chromosome 16 (D16S287 and D16S418) to compare the microsatellite pattern of the donor, the patient, and the recurrent LAM cells. At the marker D1S1661, the donor was homozygous for a lower allele, and the patient was heterozygous, with the same lower allele as the donor (Figure 2A)

. The native LAM showed the patient's alleles, as expected. The recurrent LAM also showed the upper allele, indicating the presence of patient-derived DNA. At the marker D9S1198, the patient was homozygous for a lower allele, whereas the donor was heterozygous for two higher alleles (Figure 2B). A smaller amount of the patient allele was also present in the donor DNA, which was microdissected from the allograft lung. This indicates the presence of a small number of host-derived cells (possibly inflammatory) within the allograft. The native LAM (from the left lung) showed the patient's alleles, as expected, and the recurrent LAM cells also showed a predominance of the patient's alleles. At the marker D16S418, the patient and donor were both heterozygous, with one shared allele (Figure 2C). The native LAM showed the patient-derived alleles, as expected, and the recurrent LAM also showed the presence of patient-derived DNA.

Loss of Heterozygosity Analysis

Loss of heterozygosity was detected using the chromosome 16 markers D16S418 (Figure 2C) and D16S291 (Figure 2D) in the native pulmonary LAM cells. Loss of heterozygosity at the marker D16S291 was detected in the native pulmonary LAM and also in LAM cells microdissected from four separate mediastinal lymph nodes that were removed from the patient at the time of the initial diagnosis of LAM (Figure 2D).

Presence of a Somatic TSC2 Exon 18 Mutation in Native Pulmonary and Lymph Node LAM Cells

DNA was prepared from microdissected LAM cells from the patient's native LAM (left lung), and SSCP was used to screen all 41 exons of the TSC2 gene for mutations. A variant band in exon 18 of the TSC2 gene was found in native pulmonary LAM cells and in LAM cells from four separate mediastinal lymph nodes (Figure 3A)

. This variant band was not present in the patient normal lung DNA, and sequencing of exon 18 from the normal lung showed wild-type sequence (Figure 3B), indicating that the mutation was somatic rather than germline. Sequencing of the microdissected LAM cell DNA revealed a one base pair deletion beginning at base pair 2069 (Figure 3C), which is predicted to result in premature protein truncation at amino acid 697. Sequencing of the variant band cut from the SSCP gel confirmed this deletion (Figure 3D). DNA from mediastinal lymph node LAM cells also contained the mutation (Figures 3E and 3F).

Presence of the Exon 18 Mutation in the Recurrent LAM Cells

The exon 18 variant band was also present in microdissected recurrent LAM cells, but not in donor normal lung (Figure 4A)

. DNA sequencing confirmed that the mutation was not present in the donor normal lung (Figure 4B). In contrast, DNA sequencing of exon 18 from the recurrent LAM cells showed the one base pair deletion at position 2069, resulting in overlapping peaks at subsequent bases (Figure 4C). Sequencing of the SSCP variant band from the recurrent LAM cells confirmed this deletion (Figure 4D). Loss of heterozygosity at the marker D16S291 was also seen in the recurrent LAM cells.

The Y Chromosome Is Not Present in Recurrent LAM Cells

Y-chromosome fluorescence in situ hybridization was performed in conjunction with immunostaining using muscle-specific actin and the HMB-45 antibodies. The Y chromosome was not found in recurrent LAM cells expressing HMB-45 antigen (Figure 5A)

or muscle-specific actin (Figure 5B). As expected, Y-chromosome fluorescence was present in endothelial cells (Figures 5A and 5C) and bronchial epithelial cells (Figure 5D) within the allograft lung. As a positive control, normal male peripheral blood lymphocytes were hybridized and showed a single Y-chromosome signal (data not shown).

The literature contains at least three reports of recurrent LAM after lung transplantation (911, 14). Earlier analyses of two cases of recurrent LAM demonstrated that the foci of recurrent LAM contained Y chromosomes (10, 11). This led to the conclusion that recurrent LAM after lung transplantation arises from donor cells. More recently, however, somatic inactivating mutations in the TSC2 gene were identified in pulmonary LAM and renal angiomyolipomas from sporadic LAM patients (7, 15). These mutations led to the hypothesis that sporadic LAM results from the migration of benign smooth muscle cells from the angiomyolipoma to the lung.

In this study, we examined the genetic basis of recurrent LAM using laser capture microdissection and two genetic approaches: microsatellite marker fingerprinting and TSC2 mutational analysis. The pattern of microsatellite alleles revealed that cells of patient origin were present within foci of recurrent LAM. We next screened DNA from native pulmonary LAM for mutations in all 41 exons of the TSC2 gene. A one base pair frame-shifting deletion in exon 18 was identified. Morphologically normal bronchial epithelial cells from this patient did not contain the mutation. This is consistent with previous studies in which patients with sporadic LAM were found to have somatic TSC2 mutations (7, 15). Four separate mediastinal lymph nodes also showed the exon 18 mutation and had a loss of heterozygosity in the TSC2 gene region. The recurrent LAM cells contained the same SSCP variant band and one base pair deletion as the native pulmonary LAM and the four lymph node LAM specimens. To validate these genetic findings further, we performed Y-chromosome fluorescence in situ hybridization on sections immunostained for either muscle-specific actin or HMB-45. Y chromosomes were present in bronchial epithelial cells and endothelial cells within the allograft lung, as expected, but were not present in LAM cells identified using either muscle-specific actin or HMB-45.

Our data demonstrate, for the first time, that LAM cells migrate or metastasize in vivo, despite their histologically benign features. Our data contrast with two previous reports of recurrent LAM in which Y-chromosome in situ hybridization appeared to show that the recurrent LAM cells were of donor origin (9, 10). Since these earlier reports, it has become clear that regions of pulmonary LAM are not homogeneous. We previously found that LAM cells, which immunostain positively with muscle specific actin, are tightly intermingled with other cells that are not immunoreactive with muscle specific actin (7). Others have found that LAM is frequently accompanied by reactive hyperplasia of type II pneumocytes (16). We speculate, therefore, that the Y chromosomes seen within foci of recurrent LAM in previous studies (10, 11) were within donor-derived endothelial cells or reactive pneumocytes. It has been previously shown that endothelial cells, as well as bronchial and alveolar epithelial cells, are of donor origin after lung transplantation (17). We cannot exclude the possibility that LAM has multiple causes and that the previously reported cases are biologically different than the case studied here. Fluorescence in situ hybridization analyses combined with HMB-45 immunostaining and TSC2 mutational detection on laser-captured material could be used to re-evaluate the earlier reports. Alternative explanations should also be considered, including stem cell fusion (18) and microchimerism (19).

The patient in our study had no evidence of renal angiomyolipomas either during her life or at autopsy. Approximately 60% of sporadic LAM patients have angiomyolipomas (3). To our knowledge, this patient represents the first time that a TSC1 or TSC2 mutation has been sought in a LAM patient in whom the absence of angiomyolipomas has been documented. We and others have proposed that cells with mutational inactivation of the TSC1 or TSC2 gene migrate from the smooth muscle component of renal angiomyolipomas to the lung, resulting in LAM (7, 8, 15). The inactivation of TSC2 in the LAM cells from this patient, who lacks an angiomyolipoma, challenges this model and raises the possibility that LAM cells can arise in other sites. If LAM or angiomyolipoma cells enter the circulation, it is surprising that they are present primarily in axial lymph nodes, lung, and kidney, rather than other sites.

A model in which LAM cells migrate or metastasize to the lung challenges the boundary between benign and malignant diseases. There are other examples of histologically benign diseases in which cells appear to metastasize, including benign metastasizing leiomyoma, and disseminated peritoneal leiomyomatosis (20). If LAM cells containing TSC2 gene mutations have the potential to migrate in vivo, it may indicate that the TSC genes have functional roles related to cellular metastasis. TSC2 encodes tuberin, a 200-kD protein that has a domain of homology to rap1GTPase activating protein (GAP) near the carboxy terminus. Tuberin has been shown to possess GAP activity for rap1A (21) and rab5 (22) and to function in multiple pathways in mammalian cells: vesicular trafficking (22), cell cycle regulation (2327), steroid hormone function (28), and signaling via ribosomal protein S6, which regulates protein synthesis and cell growth (29, 30). Hamartin, the protein product of the TSC1 gene, was recently found to activate the small GTPase Rho and to regulate focal adhesion and stress fiber formation via an interaction with the ezrin–radixin–moesin family of cytoskeletal proteins (31). The significance of these cellular pathways to the clinical manifestations of LAM remains an active area of investigation.

In conclusion, we found that LAM cells in this patient migrated or metastasized to the allograft lung after transplantation, despite their histologically benign features. Further studies will be required to determine whether this pathogenic mechanism applies to other cases of LAM, including cases in which TSC2 mutations are not detected. Our data appear to place LAM in a small group of diseases, all of which affect women, resulting from the metastasis of histologically benign smooth muscle cells. The biology of LAM may have broad significance in elucidating the molecular basis of cellular migration and/or metastasis.

The authors are grateful to Dr. Alfonso Bellacosa for critical review of the manuscript and to the Fox Chase Cancer Center Histopathology and Sequencing Facilities for technical assistance.

No part of this research was funded by tobacco industry sources. This study was approved by the Institutional Review Board of Fox Chase Cancer Center.

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Correspondence and requests for reprints should be addressed to Elizabeth Petri Henske, M.D., Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. E-mail:


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