Lymphangioleiomyomatosis Lesions Research Articles

Cross talk between LAM cells and fibroblasts may influence alveolar epithelial cell behavior in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-specific cystic lung disease in which tuberous sclerosis complex 2 (TSC2)-deficient LAM cells, LAM-associated fibroblasts (LAFs), and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial (AT2) cells. We hypothesized that the behavior of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in the patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared with parenchymal AT2 cells, demonstrated by increased Ki67 expression. Furthermore, nodular AT2 cells express proteins associated with epithelial activation in other disease states including matrix metalloproteinase 7, and fibroblast growth factor 7 (FGF7). In vitro, LAF-conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair, and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, a potential mediator of fibroblast-epithelial cross talk, in a mechanistic target of rapamycin (mTOR)-dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behavior. Fibroblast-derived FGF7 may contribute to the cross talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

Isolation and characterisation of lymphatic endothelial cells from lung tissues affected by lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare pulmonary disease characterised by the proliferation of smooth muscle-like cells (LAM cells), and an abundance of lymphatic vessels in LAM lesions. Studies reported that vascular endothelial growth factor-D (VEGF-D) secreted by LAM cells contributes to LAM-associated lymphangiogenesis, however, the precise mechanisms of lymphangiogenesis and characteristics of lymphatic endothelial cells (LECs) in LAM lesions have not yet been elucidated. In this study, human primary-cultured LECs were obtained both from LAM-affected lung tissues (LAM-LECs) and normal lung tissues (control LECs) using fluorescence-activated cell sorting (FACS). We found that LAM-LECs had significantly higher ability of proliferation and migration compared to control LECs. VEGF-D significantly promoted migration of LECs but not proliferation of LECs in vitro. cDNA microarray and FACS analysis revealed the expression of vascular endothelial growth factor receptor (VEGFR)-3 and integrin α9 were elevated in LAM-LECs. Inhibition of VEGFR-3 suppressed proliferation and migration of LECs, and blockade of integrin α9 reduced VEGF-D-induced migration of LECs. Our data uncovered the distinct features of LAM-associated LECs, increased proliferation and migration, which may be due to higher expression of VEGFR-3 and integrin α9. Furthermore, we also found VEGF-D/VEGFR-3 and VEGF-D/ integrin α9 signaling play an important role in LAM-associated lymphangiogenesis.

Efficacy and Tolerability of Sirolimus in Lymphangioleiomyomatosis Patients after Lung Transplantation: Single-Center Experience in Japan

<h3>Purpose</h3> Lung transplantation (LTx) is an effective treatment for patients with end-stage pulmonary lymphangioleiomyomatosis (LAM). However, native lung-related complications, such as chylothorax, chylous sputum, and native lung hyperinflation, can develop after a single LTx. LAM lesion recurrence in transplanted lungs and abdominal LAM lesions may also occur post-LTx. Further, while sirolimus therapy can be used to treat these complications, its efficacy and tolerability in patients with LAM after LTx remain unclear. This study aimed to evaluate the effect of sirolimus on post-LTx LAM complications. <h3>Methods</h3> We retrospectively reviewed the records of LTx recipients who received sirolimus for treating post-LTx LAM complications from March 2000 to December 2019. <h3>Results</h3> Of the 40 patients who underwent LTx for LAM treatment, eight underwent sirolimus treatment for post-LTx LAM complications. Two patients exhibited chylous sputum; two, chylous ascites; one, chylothorax; one, native lung hyperinflation; one, LAM recurrence in the transplanted lung; and one, angiomyolipoma. Patients' immunosuppressive regimen consisting of tacrolimus (TAC), mycophenolate mofetil (MMF), and steroids was not changed, but the target trough level of TAC was reduced with the baseline levels of TAC and sirolimus being 5 ng/mL each. Sirolimus was discontinued for one patient because nausea occurred soon after the first dose. LAM lesions improved or were suppressed in six patients (Figure 1). The forced expiratory volume at 1 s post-sirolimus administration was stable (Figure 2). Acute rejection and infection were not observed, but one patient underwent a second LTx on the contralateral side due to chronic lung allograft dysfunction. <h3>Conclusion</h3> Providing sirolimus immunosuppressive therapy after LTx for LAM treatment can be effective in managing post-LTx LAM complications and may be acceptable when combined with TAC, MMF, and steroids.

Adoptive T-Cell Transfer to Treat Lymphangioleiomyomatosis.

Patients with lymphangioleiomyomatosis (LAM) develop pulmonary cysts associated with neoplastic, smooth muscle-like cells that feature neuroendocrine cell markers. The disease preferentially affects premenopausal women. Existing therapeutics do not cure LAM. As gp100 is a diagnostic marker expressed by LAM lesions, we proposed to target this immunogenic glycoprotein using TCR transgenic T cells. To reproduce the genetic mutations underlying LAM, we cultured Tsc2-/- kidney tumor cells from aged Tsc2 heterozygous mice and generated a stable gp100-expressing cell line by lentiviral transduction. T cells were isolated from major histocompatibility complex-matched TCR transgenic pmel-1 mice to measure cytotoxicity in vitro, and 80% cytotoxicity was observed within 48 hours. Antigen-specific cytotoxicity was likewise observed using pmel-1 TCR-transduced mouse T cells, suggesting that transgenic T cells may likewise be useful to treat LAM in vivo. On intravenous injection, slow-growing gp100+ LAM-like cells formed lung nodules that were readily detectable in severe combined immunodeficient/beige mice. Adoptive transfer of gp100-reactive but not wild-type T cells into mice significantly shrunk established lung tumors, even in the absence of anti-PD-1 therapy. These results demonstrate the treatment potential of adoptively transferred T cells to eliminate pulmonary lesions in LAM.

SAT-141 Glycoprotein NMB (GPNMB) Is Pro-Tumorigenic in TSC2-Null Cancer Cells and Is a Potential Drug Target and Biomarker for Lymphangioleiomyomatosis (LAM)

Lymphangioleiomyomatosis (LAM) is an estrogen-sensitive lung disease found almost exclusively in women that is characterized by hyperproliferation of smooth muscle cells forming small tumors, or LAM lesions throughout the lungs of patients. Growth of these tumors leads to progressive loss of pulmonary function, and sometimes subsequent lung transplantation. LAM tumor cells contain mutations in either the TSC1 or TSC2 genes, leading to activation of the mTORC1 pathway. In fact, mTOR inhibitors such as sirolimus are commonly used to treat LAM; however, these drugs are not always effective and have significant side effects, suggesting the need for new therapeutic targets. Interestingly, another important feature of LAM cells is that they express melanocytic markers that are normally found in melanocytes or melanoma cells. From RNASeq analysis of a mouse model for LAM that we designed, we discovered significant upregulation of the melanocytic marker Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB), a type I transmembrane protein. GPNMB was not only highly expressed in our mouse model (a uterine specific TSC2-null mouse), it was also expressed in TSC2-null cell lines, and human LAM patient lung samples. In our hands, knocking down GPNMB expression by siRNA directed against GPNMB mRNA decreased migration and proliferation in TSC2-null cells. Additionally, we found that GPNMB’s large ectodomain is shed by TSC2-null cells and can be detected in the blood of human patients with LAM. Finally, MMP 2 and 9 can be secreted as a result of ectodomain shedding and its interaction with integrins. Accordingly, we did indeed see a decrease in MMP 2/9 expression in TSC2-null cells with reduced GPNMB expression from treatment with siRNA directed against GPNMB mRNA. Overall, our results demonstrate the potential importance of GPNMB in LAM tumor progression, and suggest that GPNMB may be a possible LAM biomarker and target for its treatment.

Uterine precursor lesions in patients with incidental nodal lymphangioleiomyomatosis: A report of 4 cases

Uterine sections from 6 patients with incidental nodal lymphangioleiomyomatosis (LAM) were examined for LAM lesions by screening these sections with cathepsin K immunohistochemistry (IHC) stains. The hysterectomy specimens were all concurrent with the lymph node dissections in which the nodal LAM was discovered. In 4 of 6 patients microscopic lesions of pre-LAM were identified and confirmed by IHC staining for HMB-45 and beta-catenin. All lesions were grossly inapparent and also inapparent by routine hematoxylin and eosin stains. Three variants of pre-LAM lesions were identified. None of the pre-LAM lesions had an associated lymphatic proliferation. It is proposed that these pre-LAM lesions gave rise to the incidental nodal LAM lesions. Furthermore, it is suggested that the absence of an associated lymphatic proliferation associated with these lesions may be a factor in the attenuated potential for spread and the only rare association of these nodal lesions with pulmonary LAM.

Inhibition of Growth of TSC2-Null Cells by a PI3K/mTOR Inhibitor but Not by a Selective MNK1/2 Inhibitor.

Lymphangioleiomyomatosis (LAM) is a rare metastatic cystic lung disease due to a mutation in a TSC tumor suppressor, resulting in hyperactive mTOR growth pathways. Sirolimus (rapamycin), an allosteric mTORC1 inhibitor, is a therapeutic option for women with LAM but it only maintains lung volume during treatment and does not provide benefit for all LAM patients. The two major mTORC1 protein synthesis pathways are via S6K/S6 or 4E-BP/eIF4E activation. We aimed to investigate rapamycin in combination with compounds that target associated growth pathways, with the potential to be additive to rapamycin. In this study we demonstrated that rapamycin, at a clinically tolerable concentration (10 nM), inhibited the phosphorylation of S6, but not the critical eIF4E releasing Thr 37/46 phosphorylation sites of 4E-BP1 in TSC2-deficient LAM-derived cells. We also characterized the abundant protein expression of peIF4E within LAM lesions. A selective MNK1/2 inhibitor eFT508 inhibited the phosphorylation of eIF4E but did not reduce TSC2-null cell growth. In contrast, a PI3K/mTOR inhibitor omipalisib blocked the phosphorylation of Akt and both S6K/S6 and 4E-BP/eIF4E branches, and additively decreased the growth of TSC2-null cells with rapamycin. Omipalisib, or another inhibitor of both major mTORC1 growth pathways and pAkt, might provide therapeutic options for TSC2-deficient cancers including, but not limited to, LAM.

Identification of the Lymphangioleiomyomatosis Cell and Its Uterine Origin

Lymphangioleiomyomatosis (LAM) is a metastasizing neoplasm of reproductive age women that causes cystic lung remodeling and progressive respiratory failure. The source of LAM cells that invade the lung and the reasons that LAM targets women have remained elusive. We employed single cell and single nuclei RNA sequencing on LAM lesions within explanted LAM lungs, known to contain smooth muscle like cells bearing mTOR activating mutations in TSC1 or TSC2, and identified a unique population of cells that were readily distinguished from those of endogenous lung cells. LAM CORE cells shared closest transcriptomic similarity to normal uterus and neural crest. Immunofluorescence microscopy demonstrated the expression of LAMCORE cell signature genes within LAM lesions in both lung and uterus. Serum aptamer proteomics and ELISA identified biomarkers predicted to be secreted by LAM CORE cells. Single cell transcriptomics strongly supports a uterine neural crest origin of LAM CORE cells; providing insights into disease pathogenesis and informing future treatment strategies for LAM.

Rapalog resistance is associated with mesenchymal-type changes in Tsc2-null cells

Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) are caused by inactivating mutations in TSC1 or TSC2, leading to mTORC1 hyperactivation. The mTORC1 inhibitors rapamycin and analogs (rapalogs) are approved for treating of TSC and LAM. Due to their cytostatic and not cytocidal action, discontinuation of treatment leads to tumor regrowth and decline in pulmonary function. Therefore, life-long rapalog treatment is proposed for the control of TSC and LAM lesions, which increases the chances for the development of acquired drug resistance. Understanding the signaling perturbations leading to rapalog resistance is critical for the development of better therapeutic strategies. We developed the first Tsc2-null rapamycin-resistant cell line, ELT3-245, which is highly tumorigenic in mice, and refractory to rapamycin treatment. In vitro ELT3-245 cells exhibit enhanced anchorage-independent cell survival, resistance to anoikis, and loss of epithelial markers. A key alteration in ELT3-245 is increased β-catenin signaling. We propose that a subset of cells in TSC and LAM lesions have additional signaling aberrations, thus possess the potential to become resistant to rapalogs. Alternatively, when challenged with rapalogs TSC-null cells are reprogrammed to express mesenchymal-like markers. These signaling changes could be further exploited to induce clinically-relevant long-term remissions.

Rapamycin-independent IGF2 expression in Tsc2-null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells.

Lymphangioleiomyomatosis (LAM) is a rare, almost exclusively female lung disease linked to inactivating mutations in tuberous sclerosis complex 2 (TSC2), a tumor suppressor gene that controls cell metabolic state and growth via regulation of the mechanistic target of rapamycin (mTORC1) signaling. mTORC1 is frequently activated in human cancers and, although the mTORC1 inhibitor rapamycin has a cytostatic effect, it is, in general, unable to elicit a robust curative effect or tumor regression. Using RNA-Seq, we identified (1) Insulin-like Growth Factor (IGF2) as one of the genes with the highest fold-change difference between human TSC2-null and TSC2-expressing angiomyolipoma cells from a patient with LAM, and (2) the mouse IGF2 homolog Igf2, as a top-ranking gene according to fold change between Tsc2-/- and Tsc2+/+ mouse embryo fibroblasts (MEFs). We extended transcript-level findings to protein level, observing increased Igf2 protein expression and Igf2 secretion by Tsc2-/- MEFs. Increased Igf2 expression was not due to epigenetic imprinting, but was partially mediated through the Stat3 pathway and was completely insensitive to rapamycin treatment. An siRNA-mediated decrease of Igf2 resulted in decreased Stat3 phosphorylation, suggesting presence of an autocrine Igf2/Stat3 amplification cycle in Tsc2-/- MEFs. In human pulmonary LAM lesions and metastatic cell clusters, high levels of IGF2 were associated with mTORC1 activation. In addition, treatment of three primary IGF2-expressing LAM lung cell lines with rapamycin did not result in IGF2 level changes. Thus, targeting of IGF2 signaling may be of therapeutic value to LAM patients, particularly those who are unresponsive to rapamycin.

Urokinase-type plasminogen activator (uPA) is critical for progression of tuberous sclerosis complex 2 (TSC2)-deficient tumors

Lymphangioleiomyomatosis (LAM) is a fatal lung disease associated with germline or somatic inactivating mutations in tuberous sclerosis complex genes (TSC1 or TSC2). LAM is characterized by neoplastic growth of smooth muscle-α-actin-positive cells that destroy lung parenchyma and by the formation of benign renal neoplasms called angiolipomas. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin slows progression of these diseases but is not curative and associated with notable toxicity at clinically effective doses, highlighting the need for better understanding LAM's molecular etiology. We report here that LAM lesions and angiomyolipomas overexpress urokinase-type plasminogen activator (uPA). Tsc1-/- and Tsc2-/- mouse embryonic fibroblasts expressed higher uPA levels than their WT counterparts, resulting from the TSC inactivation. Inhibition of uPA expression in Tsc2-null cells reduced the growth and invasiveness and increased susceptibility to apoptosis. However, rapamycin further increased uPA expression in TSC2-null tumor cells and immortalized TSC2-null angiomyolipoma cells, but not in cells with intact TSC. Induction of glucocorticoid receptor signaling or forkhead box (FOXO) 1/3 inhibition abolished the rapamycin-induced uPA expression in TSC-compromised cells. Moreover, rapamycin-enhanced migration of TSC2-null cells was inhibited by the uPA inhibitor UK122, dexamethasone, and a FOXO inhibitor. uPA-knock-out mice developed fewer and smaller TSC2-null lung tumors, and introduction of uPA shRNA in tumor cells or amiloride-induced uPA inhibition reduced tumorigenesis in vivo These findings suggest that interference with the uPA-dependent pathway, when used along with rapamycin, might attenuate LAM progression and potentially other TSC-related disorders.

NK cell activating receptor ligand expression in lymphangioleiomyomatosis is associated with lung function decline.

Lymphangioleiomyomatosis (LAM) is a rare lung disease of women that leads to progressive cyst formation and accelerated loss of pulmonary function. Neoplastic smooth muscle cells from an unknown source metastasize to the lung and drive destructive remodeling. Given the role of NK cells in immune surveillance, we postulated that NK cell activating receptors and their cognate ligands are involved in LAM pathogenesis. We found that ligands for the NKG2D activating receptor UL-16 binding protein 2 (ULBP2) and ULBP3 are localized in cystic LAM lesions and pulmonary nodules. We found elevated soluble serum ULBP2 (mean = 575 pg/ml ± 142) in 50 of 100 subjects and ULBP3 in 30 of 100 (mean = 8,300 pg/ml ± 1,515) subjects. LAM patients had fewer circulating NKG2D+ NK cells and decreased NKG2D surface expression. Lung function decline was associated with soluble NKG2D ligand (sNKG2DL) detection. The greatest rate of decline forced expiratory volume in 1 second (FEV1, -124 ± 30 ml/year) in the 48 months after enrollment (NHLBI LAM Registry) occurred in patients expressing both ULBP2 and ULBP3, whereas patients with undetectable sNKG2DL levels had the lowest rate of FEV1 decline (-32.7 ± 10 ml/year). These data suggest a role for NK cells, sNKG2DL, and the innate immune system in LAM pathogenesis.

Isolation of individual cellular components from lung tissues of patients with lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease entailing cystic destruction of the lungs and progressive respiratory failure. LAM lungs are histologically characterized by the proliferation of smooth muscle-like cells (LAM cells) and an abundance of lymphatic vessels. To elucidate the pathophysiological processes of LAM, cell-type-specific analyses are required. However, no method exists for isolating the individual types of cells in LAM lesions. Therefore, we established a fluorescence-activated cell sorting (FACS)-based method for the direct isolation of LAM cells and other various cellular components from LAM-affected lung tissue. We obtained LAM-affected lung tissue from resections or transplant recipients and prepared single-cell suspensions. FACS, immunohistochemical, and molecular analysis were used cooperatively to isolate HMB45-positive LAM cells with tuberous sclerosis complex (TSC) 2 loss of heterozygosity (LOH). Using a combination of antibodies against an epithelial cell adhesion molecule (EpCAM) and podoplanin, we fractionated CD45-negative lung cells into three groups: lymphatic endothelial cells (LEC) (EpCAM(-)/podoplanin(hi) subset), alveolar type II cells (EpCAM(hi)/podoplanin(-) subset), and mesenchymal cells (EpCAM(-)/podoplanin(-/low) subset). During subsequent analysis of HMB45 expression, as a LAM-specific marker, we clearly identified LAM cells in the mesenchymal cell population. We then discovered that CD90(+)/CD34(-) cells in the mesenchymal cell population are not only positive for HBM45 but also had TSC2 LOH. These isolated cells were viable and subsequently amenable to cell culture. This method enables us to isolate LAM cells and other cellular components, including LAM-associated LEC, from LAM-affected lung tissues, providing new research opportunities in this field.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

The neural crest lineage as a driver of disease heterogeneity in Tuberous Sclerosis Complex and Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease, best characterized by the formation of proliferative nodules that express smooth muscle and melanocytic antigens within the lung parenchyma, leading to progressive destruction of lung tissue and function. The pathological basis of LAM is associated with Tuberous Sclerosis Complex (TSC), a multi-system disorder marked by low-grade tumors in the brain, kidneys, heart, eyes, lung and skin, arising from inherited or spontaneous germ-line mutations in either of the TSC1 or TSC2 genes. LAM can develop either in a patient with TSC (TSC-LAM) or spontaneously (S-LAM), and it is clear that the majority of LAM lesions of both forms are characterized by an inactivating mutation in either TSC1 or TSC2, as in TSC. Despite this genetic commonality, there is considerable heterogeneity in the tumor spectrum of TSC and LAM patients, the basis for which is currently unknown. There is extensive clinical evidence to suggest that the cell of origin for LAM, as well as many of the TSC-associated tumors, is a neural crest cell, a highly migratory cell type with extensive multi-lineage potential. Here we explore the hypothesis that the types of tumors that develop and the tissues that are affected in TSC and LAM are dictated by the developmental timing of TSC gene mutations, which determines the identities of the affected cell types and the size of downstream populations that acquire a mutation. We further discuss the evidence to support a neural crest origin for LAM and TSC tumors, and propose approaches for generating humanized models of TSC and LAM that will allow cell of origin theories to be experimentally tested. Identifying the cell of origin and developing appropriate humanized models is necessary to truly understand LAM and TSC pathology and to establish effective and long-lasting therapeutic approaches for these patients.

Exonic Mutations of TSC2/TSC1 Are Common but Not Seen in All Sporadic Pulmonary Lymphangioleiomyomatosis

Exonic Mutations of <i>TSC2/TSC1</i> Are Common but Not Seen in All Sporadic Pulmonary Lymphangioleiomyomatosis

Prevalence of Uterine and Adnexal Involvement in Pulmonary Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM), a systemic disorder affecting almost exclusively young women, is characterized by the abnormal proliferation of smooth muscle-like cells (LAM cells). LAM can occur either in association with the tuberous sclerosis complex (TSC) (TSC-LAM) or without TSC (sporadic LAM). Recent studies have demonstrated that LAM is a neoplasm arising from constitutive activation of the mammalian target of rapamycin signaling pathway dysregulated by a functional loss of TSC genes, but the primary organ of origin remains unclear. Therefore, we performed histologic and immunohistologic analyses of gynecologic organs in 20 patients, half with and the other half without pulmonary LAM, to determine how often LAM involves the uterus. The results showed that 9 of 10 (90%) patients with pulmonary LAM had uterine LAM lesions. In contrast, no patients without pulmonary LAM had so. All uterine LAM lesions were accompanied by LAM lesions in retroperitoneal or pelvic lymph nodes and LAM cell clusters, each enveloped by a monolayer of vascular endothelial growth factor receptor-3-positive lymphatic endothelial cells. Furthermore, when we compared uterine lesions of TSC-LAM with those of sporadic LAM, proliferation of HMB45-positive epithelioid-shaped LAM cells and infiltrates with a tongue-like growth pattern was more prominent in the former, whereas the extent of lymphangiogenesis within the myometrium was greater in the latter. These results indicate that uterine involvement is a common manifestation of LAM, and, possibly, that the uterus or an adjacent locale in the retroperitoneum or pelvic cavity is the primary site of origin of LAM.

Effects of prolactin on TSC2-null Eker rat cells and in pulmonary lymphangioleiomyomatosis.

Lymphangioleiomyomatosis, a cystic lung disease of women, is characterized by proliferation of smooth muscle-like lymphangioleiomyomatosis cells, which possess mutations in the tuberous sclerosis complex genes, TSC1/TSC2. Growth factors involved in lymphangioleiomyomatosis cell proliferation are unknown. Prolactin, an important reproductive hormone in women, is known to promote cell proliferation and survival in other tissues. To determine the role of prolactin in signaling and proliferation in lymphangioleiomyomatosis. Prolactin levels in the sera of patients with lymphangioleiomyomatosis were correlated with clinical status. Components of prolactin signal transduction pathways were assessed in lymphangioleiomyomatosis lesions from human lung explants by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Prolactin effects on proliferation and signaling were quantified in tuberin-deficient and tuberin-expressing rat cells in vitro. Higher prolactin levels in the sera of patients with lymphangioleiomyomatosis were associated with a faster rate of decline in FEV(1) and an increased history of pneumothorax (P < 0.01). Higher levels of prolactin and prolactin receptor mRNA and immunoreactivity were found in lymphangioleiomyomatosis lesions when compared with vascular smooth muscle cells in the same region of tissue. This was accompanied by evidence of activation of signal transducer and activator of transcription-1 (STAT1), STAT3, p44/42, and p38 mitogen-activated protein kinase. Tsc2(-/-) Eker rat embryonic fibroblasts expressed more prolactin receptor than did Tsc2(+/+) cells, and responded to prolactin with increased proliferation and activation of the same signaling pathways seen in vivo. Prolactin may be an important growth factor in the pathogenesis of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis: A Disease Involving the Lymphatic System

Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease in which abnormal smooth muscle-like cells (LAM cells) proliferate in the lungs and along the axial lymphatic systems, including the lymph nodes and thoracic ducts. LAM cells are transformed due to loss-of-function type mutations of either the TSC1 or TSC2 tumor suppressor genes. The pathological features include the proliferation of benign-looking LAM cells and the existence of abundant lymphatic vessels that are associated with clinical conditions such as chyle leakage. LAM cells produce potent lymphangiogenic growth factors (VEGF-C and VEGF-D) and the lymphatic vessel density within LAM lesions correlates with the histologic severity of LAM. The serum VEGF-D level increases in LAM, especially in patients with lymphatic involvement. LAM cell clusters (LCCs), which are postulated pathologically to be generated by lymphangiogenesis-mediated fragmentation and subsequent shedding into the lymphatic circulation, are observed in both chylous effusion and LAM-associated lymphatics within LAM tissue specimens. The identification of LCCs in chylous effusion together with the characteristic clinical manifestations can therefore be an alternative for a lung biopsy if LAM patients are complicated with chylous effusion. LAM appears to be a disease involving a dysfunction of the lymphatic system and a fascinating model of tumor dissemination that is exclusively lymphangitic. LAM-associated lymphangiogenesis that mediates the shedding of LCCs seems to play a central role in the dissemination of LAM cells and progression in LAM and it may also be a potential therapeutic target as well as the dysregulated mTOR signaling pathway.

Utility of [18F]2-Fluoro-2-Deoxyglucose-PET in Sporadic and Tuberous Sclerosis-Associated Lymphangioleiomyomatosis

Mutations in tuberous sclerosis complex (TSC) genes are associated with dysregulated mammalian target of rapamycin (mTOR)/Akt signaling and unusual neoplasms called perivascular epithelioid cell tumors (PEComas), including angiomyolipomas (AMLs) and lymphangioleiomyomatosis (LAM). Tools that quantify metabolic activity and total body burden of AML and LAM cells would be valuable for the assessment of disease progression and the response to therapy in patients with TSC and LAM. Our hypothesis was that constitutive activation of mTOR in LAM and AML cells would result in increased glucose uptake of [(18)F]2-fluoro-2-deoxyglucose (FDG) on PET scanning, as has been suggested by a single prior case report. After institutional review board approval, FDG-PET scanning was performed in six LAM patients. Six additional LAM patients underwent FDG-PET scanning for clinical evaluation of suspected malignancy. Pleural uptake related to prior therapy was identified in four individuals with a remote history of talc pleurodesis. Focal increased uptake was observed in a supraclavicular lymph node in a patient with Hodgkin lymphoma and in a lung nodule in a patient with a biopsy-documented primary lung adenocarcinoma. In one TSC-LAM patient with a biopsy-documented malignant uterine PEComa, robust uptake was noted in metastatic nodules in the lung but not in the LAM-involved lung parenchyma or the patient's massive abdominal lymphangioleiomyomas. No abnormal uptake was identified in the AMLs or LAM lesions in any patients. This pilot study suggests that FDG-PET scans are negative in patients with benign PEComas and therefore are not likely to be useful for estimating the burden of disease in patients with TSC or LAM, but that FDG-PET scans can be used to identify or exclude other neoplasms in these patients.