INCIDENCE AND DEMOGRAPHIC FEATURES

The reported prevalence of lymphangioleiomyomatosis is around one per million in the United Kingdom,4 France,5and the United States, although the true prevalence is likely to be greater. The disease is well described in Asia6 although data on prevalence are not available.

Lymphangioleiomyomatosis is exclusively confined to women, the mean age of onset being 34 years.1 ,2 ,6-8 Presentation after the menopause is very unusual, occurring in only eight of 186 patients in the larger series,1 ,2 ,4 ,6-8 six of whom were taking oestrogen containing hormone replacement therapy. This fits with evidence suggesting that oestrogen administration9 and pregnancy10-12 may accelerate disease progression whereas oophorectomy and progesterone2 ,4 may reduce it (see below).

PATHOLOGICAL FEATURES

The lung in lymphangioleiomyomatosis contains numerous cysts, ranging in diameter from millimetres to centimetres,1 and these are responsible for the pneumothoraces and the striking appearance of blebs over the lung surface (fig 1). Infiltration of lung and pleural lymphatics causes obliteration and dilatation of the lymphatic channels leading to lymph stasis, chyloptysis, and septal lines on the chest radiograph. The thoracic duct is often thickened and formed of multiple narrow channels leading to chylous pleural effusions and ascites. Pulmonary vascular occlusion can cause pulmonary haemorrhage, haemoptysis, and haemosiderosis.1

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

(A) Thoracotomy showing abnormal lung surrounded by a chylous collection. (B) Close up of the lung showing the characteristic appearance of multiple blebs.

Microscopically the lungs are characterised by cystic air spaces and a nodular proliferation of abnormal smooth muscle cells (LAM cells). In early lesions LAM cells accumulate in the alveolar walls, collagen and elastic fibres are partially degraded, and oedema, haemorrhage and haemosiderin laden macrophages are seen around the alveoli.13 ,14 With more advanced disease the nodules are larger and associated with compact collagen bundles whereas oedema, haemorrhage, and macrophages are less prominent.

The thin walled cysts are lined by flattened epithelial and ciliated bronchiolar epithelial cells14 and are thought to be formed by the amalgamation of damaged alveoli, probably as a result of uncontrolled proteolytic activity.13 ,14 The nodules surround and may jut into the cysts, alveoli, and bronchioles causing thickening of the small airway walls, often in an irregular fashion (fig 2). LAM cells appear to be of smooth muscle origin since they stain for vimentin, desmin, and α-smooth muscle actin,15-17 and myofilaments are seen on electron microscopy. The cells also display features that are not typical of muscle cells—namely, multiple indented round nuclei, a prominent endoplasmic reticulum, microfilament bundles with dense bodies, numerous electron dense membrane bound granules13 ,18—and they characteristically stain with the monoclonal antibody HMB 45.19 ,20 There appear to be three forms of LAM cell according to their microscopic appearance, large spindle shaped cells, smaller cells with little cytoplasm, and epithelioid cells.14 Interestingly, the three cell types also have differing immunophenotypes which suggests that they have different functions.13 ,14 Receptors for oestrogen and/or progesterone are found on cells from roughly half the patients.21-24 The significance of these findings is uncertain and is discussed below.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Photomicrographs showing nodular proliferation of abnormal smooth muscle and cysts in (A) a lung biopsy specimen stained with haematoxylin and eosin and (B) brown HMB 45 positive LAM cells lining a cystic space.

Angiomyolipomas are rare mesenchymal tumours containing smooth muscle, fat and blood vessels, which also contain HMB 45 positive smooth muscle cells.

CLINICAL FEATURES

Patients with lymphangioleiomyomatosis present most commonly with dyspnoea (59%), pneumothorax (49%), and cough (39%) according to the larger series.1 ,2 ,5 ,6 ,25 Chest pain (22%), chylous pleural effusions (13%), haemoptysis (18%), and wheeze are less common. Chyloptysis, presenting as white, sticky sputum, may occur7 and, following pleurodesis, patients may complain of occasional gurgling in their chest (possibly due to ruptured cysts). Extrapulmonary features include lymph node masses, cystic soft tissue masses,26 ,27 and chylous ascites in up to a third of patients.1 ,25 Uterine fibroids (leiomyomas)8and renal angiomyolipomas28 ,29 are also seen; angiomyolipomas are often asymptomatic although bleeding may occur (see below).

The natural history of lymphangioleiomyomatosis is of progressive airflow obstruction leading to respiratory failure and cor pulmonale.1 The rate of progression is highly variable between patients, ranging from a rapid decline over a few years1 to a more indolent disease over two or three decades.2 ,30 In a review of 47 patients with lymphangioleiomyomatosis in the UK the mean fall in forced expiratory volume in one second (FEV₁) was 118 ml/year although there was marked variability between patients.4 Two thirds of women will develop a pneumothorax and these were recurrent in more than two thirds of our patients. Chylous pleural effusions occur in a fifth of patients.5

Estimates of life expectancy in lymphangioleiomyomatosis are still relatively uncertain as previous figures were based on series from tertiary referral centres and the advent of CT scanning may enable the diagnosis to be made earlier. The most recent series reports a 10 year survival of 79%5 but some caution is needed since case ascertainment will inevitably favour those with a better prognosis.

Lung function tests in patients with lymphangioleiomyomatosis show a variety of abnormalities with airflow obstruction and impaired gas transfer the predominant features. Restrictive lung defects are sometimes seen but usually in combination with airflow obstruction and often as the result of pleural effusions, pleurectomy, or thoracotomy.1 ,6 ,31 ,32 Lung function is occasionally normal at presentation6 ,32 but disease progression is associated with increased airflow obstruction and impaired gas transfer.25

DIAGNOSIS

Because lymphangioleiomyomatosis often presents with dyspnoea and airflow obstruction, the diagnosis is often missed initially. The mean interval between onset of symptoms and diagnosis was over four years in our series (range 0–25 years) and the picture is similar elsewhere.2 The diagnosis should be suspected in women when dyspnoea or asthma is associated with a pneumothorax, haemoptysis or abnormal chest radiograph, when emphysema has been diagnosed without a history of smoking or α₁-antitrypsin deficiency, or when interstitial lung disease occurs with airflow obstruction.

Open lung biopsy has been the gold standard for the diagnosis of lymphangioleiomyomatosis. The sensitivity and specificity of histological analysis has improved considerably with the use of the monoclonal antibody HMB 45 which, in the lung, will only stain LAM cells19 ,20 thus excluding conditions which may mimic lymphangioleiomyomatosis such as metastatic endometrial sarcoma.33 Open lung biopsy can determine the hormone receptor status of LAM cells and whether smooth muscle proliferation or cystic change predominates but, as the clinical significance of these findings is uncertain, they do not alone justify taking open lung biopsy specimens. Transbronchial biopsy specimens in conjunction with HMB 45 staining can be sufficient to make a diagnosis of lymphangioleiomyomatosis.20

The plain radiograph may be normal initially but, as the disease progresses, reticular shadowing and cystic changes develop whilst lung volumes are either preserved or increased (fig 3).31 ,34The association of large lungs and diffuse shadowing on the plain radiograph may also be seen in Langerhans’ cell histiocytosis, sarcoidosis, and extrinsic allergic alveolitis. The appearances on high resolution computerised tomographic (HRCT) scanning are characteristic and striking with thin walled cysts 2–20 mm or more in diameter distributed throughout the lung fields (fig 3).31 ,34Other features include occasional hilar and mediastinal nodes and alveolar shadowing, which may represent haemorrhage, in up to 60% of scans.6 ,25 ,34 The main radiological differential diagnosis is Langerhans’ cell histiocytosis which, unlike lymphangioleiomyomatosis, usually has a nodular appearance at least in the early stages and it spares the costophrenic angles.35

• Download figure
• Open in new tab
• Download powerpoint

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

(A) Chest radiograph showing a reticular pattern which appears most prominent at the bases. (B) High resolution CT scan showing the classical findings of multiple thin walled cysts evenly distributed throughout the lung fields; the intervening parenchyma appears normal.

Because the CT findings in lymphangioleiomyomatosis are so distinctive, many physicians do not necessarily resort to open lung biopsy to make the diagnosis. This strategy is probably reasonable if the CT appearance is classical and the history is fully consistent with lymphangioleiomyomatosis; the finding of renal angiomyolipomas or a chylous effusion obviously strengthens the diagnosis. In practice, lung tissue often becomes available when patients undergo surgery for pneumothorax or chylous effusion.

MANAGEMENT

RELATIONSHIP BETWEEN LYMPHANGIOLEIOMYOMATOSIS AND THE TUBEROUS SCLEROSIS COMPLEX (TSC)

Lymphangioleiomyomatosis and angiomyolipomas can occur as part of TSC and understanding the differences between lymphangioleiomyomatosis and TSC may help to elucidate the aetiology of lymphangioleiomyomatosis. Although the link between the two diseases has been recognised for some years,1 ,28 ,29 ,63 the idea that TSC genes may be involved in lymphangioleiomyomatosis has only been explored more recently.68 ,69 In TSC there is a germ line mutation in one of two genes (TSC-1 or TSC-2).70-72 A second somatic mutation (or hit) termed loss of heterozygosity results in loss of the gene product in that cell (fig 4).73 As the TSC genes are tumour suppressors74 this allows cellular proliferation and the formation of a hamartoma; in TSC multiple second hits (mutations in the normal allele) result in multiple hamartomas.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4

Schematic representation of how loss of heterozygosity for TCS-2 might occur in lymphangioleiomyomatosis. When the cell has one mutant allelle (black) normal protein is synthesised by the normal allelle (white). A second sequential somatic mutation results in loss of heterozygosity for that allelle and loss of protein function. Since TSC-2 is a tumour suppressor gene cellular proliferation results. In tuberous sclerosis the initial mutation is in the germ line: in lymphangioleiomyomatosis the initial mutation is unlikely to be in the germ line and probably represents somatic mosaicism.

An important advance in the genetics of lymphangioleiomyomatosis was made this year by Smolarek et al.69 They found loss of heterozygosity for TSC-2 (but not TSC-1) in renal angiomyolipomas from seven of 13 patients with lymphangioleiomyomatosis but with no cutaneous or neurological features of TSC, and in the lymph nodes of a further patient. Lung tissue was not examined, probably because the diffuse nature of the lung disease makes it difficult to separate the DNA from LAM cells and normal lung tissue to enable loss of heterozygosity to be identified. Loss of heterozygosity for TSC-2 had been shown previously in sporadic angiomyolipomas (without lymphangioleiomyomatosis or tuberous sclerosis),75 but the relatively high incidence of TSC-2 abnormalities in the study by Smolarek et al 69 and the fact that they also occurred in lymph nodes suggest that the findings are relevant to lymphangioleiomyomatosis. Since a germ line mutation of TSC-2 is unlikely in lymphangioleiomyomatosis, these patients may be mosaics—that is, they may only carry the TSC-2 mutation in a small population of cells or may have sequential somatic mutations of the TSC-2 gene.

CELLULAR ROLE OF THE TUBEROUS SCLEROSIS-2 GENE PRODUCT TUBERIN AND RELATED PROTEINS

The TSC-2 gene product tuberin is constitutively expressed in a large number of tissue and cell types and is therefore likely to perform a function of basic cellular importance. Tuberin is a 180 kDa protein which acts as a tumour suppressor73; it has guanine triphosphate (GTP) activating protein (GAP) activity for the GTP binding proteins rap 176 and rab 5.77 GTP binding proteins regulate cellular pathways and are active or inactive according to whether GDP or GTP is bound. GAPs such as rap 1 GAP and tuberin facilitate the hydrolysis (inactivation) of GTP attached to the GTP binding protein (fig 5A). Recent studies are beginning to shed light on the cellular role of tuberin in inhibiting rap 1, a modulator of the ras pathway, an important pathway linking cell surface tyrosine kinase receptors and the production of transcription factors involved in cell proliferation (fig 5B). Activation of tyrosine kinase receptors (by growth factors) results in activation of ras by the phosphorylation of intermediate proteins. This induces activation of raf-1 and thence a protein kinase cascade via mitogen activated protein kinases resulting in the upregulation of transcription factors and cellular proliferation. Rap 1 binds many of the same regulators and effectors as ras78 and, according to cell type, can either inhibit or stimulate proliferation: it reverses the transformation of NIH3T3 cells by the oncogene vKi ras79 but induces DNA synthesis when injected into Swiss 3T3 cells.80 Loss of tuberin may therefore cause cellular proliferation through one or more of the following mechanisms: (1) in cells where rap 1 promotes mitogenesis loss of tuberin would lead to activation of rap and thence proliferation; (2) in cells where rap 1 antagonises ras (is anti-mitogenic), loss of tuberin binding to rap may allow rap to associate with its other effectors to promote mitogenesis; (3) tuberin may have functions other than its rap GAP activity including GAP activity for other proteins or as an effector for non-GAP functions in these or other pathways.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5

(A) Regulation of guanylyl triphosphate (GTP) binding proteins. GTP binding proteins act as molecular switches being active when bound to GTP and inactive when bound to guanylyl diphosphate (GDP). GTP hydrolysis to the inactive form is promoted by specific GTP activating proteins (GAPs) including rap 1 GAP and tuberin. The active form is promoted by antagonistic guanine dissociation factors (GDF). GAPs themselves may also be GTP binding proteins. (B) Simplified representation of how the tyrosine kinase receptor (TKR)/ras/raf/protein kinase cascade leads to the production of transcription factors that are important in cellular proliferation. The figure shows the possible interactions of tuberin and rap 1 with unproven associations shown shaded. Binding of the TKR ligand activates protein phosphorylation via intermediate proteins to activate ras and thence raf 1. Further phosphorylation events occur downstream via mitogen activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK) resulting in the upregulation of nuclear transcription factors and proliferation. Tuberin inhibits rap 1 which in some cells inhibits the ras pathway whereas in others it promotes mitogenesis. — denotes inhibition.

Evidence linking defects in the ras pathway to lymphangioleiomyomatosis is at present circumstantial. Depletion of tuberin protein by antisense oligonucleotides allowed serum depleted fibroblasts to enter the S phase of the cell cycle unlike tuberin replete cells. The response was similar to cells maximally stimulated by serum,81suggesting that cells depleted of tuberin may proliferate in the absence of mitogenic stimuli. In the Eker rat, an animal model with a germ line truncating mutation of TSC-2 used to study tuberous sclerosis, homozygous mutants die in utero whilst heterozygotes develop renal and uterine tumours. If wild type TSC-2 is introduced into these tumour cells their ability to proliferate is reduced.82 It has been argued that the GAP activity of tuberin for rap 1 is weak,76 but there seems little doubt that the tumour suppressor activity of tuberin is due to its rap 1 GAP activity since the region homologous with rap 1 GAP showed similar tumour suppressor activity to the full length protein.82 There are parallels between this hypothesis and neurofibromatosis (NF) where the NF 1 gene product neurofibromin has GAP activity for ras.83 NF tumour cells express very little neurofibromin and their ras proteins appear to be constitutively activated as judged by GTP binding.83

The other known function of tuberin is as a GAP for rab 5, one of the rab proteins that regulate intracellular transport. Rab 5 is the rate limiting step in the docking and fusion process of the endocytic pathway84 and there is evidence to suggest that tuberin may inactivate rab 5 in vivo, thereby reducing endocytosis. Immune complexes of both recombinant and native tuberin have rab 5 GAP activity. Rab 5 GAP activity is low in tuberin deficient cells and increases following the introduction of tuberin as does the rate of fluid phase endocytosis.77 Whether tuberin is a more important GAP for rap 1 or rab 5 in LAM cells and which of these two functions is more relevant to lymphangioleiomyomatosis is uncertain. We need to know more about the activities of rap 1 and rab 5 in LAM and normal smooth muscle cells and the relative GTPase activity of tuberin for these two proteins.

TSC-1 was sequenced in 1997.64 The predicted protein, named hamartin, also appears to have tumour suppressor activity since most of the genetic abnormalities described in hamartomas of TSC patients with TSC-1 mutations have been truncating mutations which are likely to cause loss of protein function.64 Hamartin co-localises with tuberin in cells and the two proteins interact via coiled coil domains85 so it is possible that a tuberin/hamartin complex is required for normal cellular function. This could explain the virtually identical phenotypes seen in patients with TSC irrespective of genotype. Loss of heterozygosity for TSC-1 is seen less frequently in TSC hamartomas and has not been described in lymphangioleiomyomatosis,69although it might be expected to give rise to lymphangioleiomyomatosis in the same way as loss of heterozygosity for tuberin.

ROLE OF OESTROGEN AND PROGESTERONE IN LYMPHANGIOLEIOMYOMATOSIS

Hormonal factors appear to play a part in both the initiation and progression of lymphangioleiomyomatosis as evidenced by the female predilection for the disease, the finding of hormone receptors on some LAM cells, and the suggestion that exogenous oestrogens9and pregnancy may exacerbate the disease whilst progesterone treatment may reduce its progression. Furthermore, although there is no sex difference in TSC, the 3% of patients with TSC who have lymphangioleiomyomatosis are predominantly, if not exclusively, women.3 ,86 The mean age of onset for TSC in these patients was 16 years whereas the onset of lung disease was 33 years, similar to that seen in isolated lymphangioleiomyomatosis.3 These differences suggest that factors in addition to an absence of tuberin are required for the development of lymphangioleiomyomatosis and these may be hormonal.

How sex hormones might affect the development and progression of lymphangioleiomyomatosis is speculative. Oestrogen may amplify the effect of a mutant protein by downregulating tuberin or possibly other proteins with GAP activity which would otherwise compensate for a defect in tuberin activity. Differential expression of such proteins would explain the predilection of these lesions for certain tissues. Some LAM cells express oestrogen and/or progesterone receptors which may be important in cell growth. LAM cells which express oestrogen receptors also express the protein Bcl-2 which acts as a suppressor of apoptosis.87 Increasing the ratio of anti-apototic proteins such as Bcl-2 to its pro-apototic homologue Bax would reduce apoptosis of LAM cells and may represent a way in which oestrogen potentiates lymphangioleiomyomatosis. The biological significance of oestrogen and progesterone receptors and why they are not uniformly expressed on LAM cells needs to be explained, however.

ROLE OF MELANOMA PROTEINS EXPRESSED IN LAM CELLS

The monoclonal antibody HMB 45 is used by pathologists to diagnose malignant melanoma; the discovery that it stains LAM cells has led to considerable interest in its target proteins and their role in LAM cells.88 HMB 45 binds to two 10 kDa glycoproteins (Pmel 17 and GP100) which are expressed by malignant melanoma and other cell lines of melanoma origin. HMB 45 positivity and other ultrastructural features suggest a developmental relationship between lymphangioleiomyomatosis, angiomyolipoma, and the rare clear cell lung tumour (which can also be associated with TSC and lymphangioleiomyomatosis).18 ,89-92 This relationship is not shared by other hamartomas in TSC, however,93 which suggests that they have a different aetiology or that lymphangioleiomyomatosis and angiomyolipoma require an additional factor for their development. The Pmel 17 gene product controls the synthesis of eumelanin94 which is localised to pre-melanosomes in melanoma cells and structurally similar electron dense membrane bound vesicles are seen in LAM cells. HMB 45 negative LAM cells stain for proliferating cell nuclear antigen, a marker of proliferation, whereas HMB 45 positive cells do not,95which suggests that they may not be actively proliferating and that different populations of LAM cells have different functions. Whether expression of the HMB 45 ligand is important in the pathogenesis of lymphangioleiomyomatosis or a bystander phenomenon is uncertain.

ABERRANT EXPRESSION OF GROWTH AND TRANSCRIPTION FACTORS

Proliferation of normal cells may be enhanced by growth factors—whether autocrine, paracrine or circulating—and by increased or aberrant receptor expression. The diffuse and widespread nature of the smooth muscle proliferation seen in the lung in lymphangioleiomyomatosis and the recurrence of disease in the donor lungs of transplant recipients, including the apparent transformation of male donor cells,50-52 suggests that circulating factors may be important. Normal airway smooth muscle cells are capable of producing a large number of growth factors and other mitogens96 and a number of abnormalities in growth and transcription factors have been described in LAM cells in the last two years (at present only in abstract form). These include: (1) increased expression of basic fibroblast growth factor97 and platelet derived growth factor β-chain and receptors98in biopsy specimens from patients with lymphangioleiomyomatosis; (2) increased production of angiotensin II in LAM cells99 and increased circulating levels of angiotensin converting enzyme in patients. This is of interest since angiotensin II upregulates transcription factors that cause hypertrophy of normal airway smooth muscle cells.100

High mobility group protein I-C (HMGI-C) was expressed in lung biopsy specimens of four patients with lymphangioleiomyomatosis but not in control subjects.101 The HMG proteins are a family of chromatin associated proteins that regulate gene expression in undifferentiated embryonic cells102 ,103 and they are frequently aberrant or overexpressed in uterine leiomyomas104 and pulmonary hamartomas.105Overexpression of aberrant HMGI-C transcripts in LAM cells may permit uncontrolled cellular proliferation.

Some of these phenomena could be secondary to uncontrolled cellular proliferation resulting from a defect in TSC-2 but where does the TSC-2 hypothesis fit in with the recurrent and widespread smooth muscle proliferation seen in donor lungs after transplantation? One possibility is that clonal expansion of one cell type secreting mitogenic factors leads to recruitment of a second reactive cell population.