Appendix 3
Additional pathology details.
Pathologic criteria for diagnosis of LAM
Although the pathologic features of LAM are characteristic and diagnostic, the diagnosis is often delayed.
LAM is a rare disease and general pathologists may never have seen a case, and therefore face problems of
recognizing the unique features of LAM. However specialist lung pathologists may make a firm diagnosis
without the help of ancillary techniques such as immunohistochemistry.
Two lesions characterize LAM: cysts and a multifocal nodular proliferation of immature smooth muscle and
perivascular epithelioid cells (LAM cells)[1-3]. Both lesions are found together in variable percentages: the
findings may be inconspicuous in early disease. Epithelioid and immature looking smooth muscle cells form
the nodular lesions characteristic of LAM. A few cells, but also aggregates of more than 20 cells can form
the nodules. While perivascular epithelioid cells and spindle cells are present in LAM, intermediate forms
with a combined muscular and epithelioid phenotype can be encountered. Thus it seems appropriate to
consider them as a morpho-phenotypical modulation of the same cell, probably arising from a common
precursor[4, 5]. Sporadic and tuberous sclerosis complex (TSC)-associated LAM is morphologically and
phenotypically indistinguishable.
The sensitivity and specificity of the pathologic changes seen in LAM have not been determined. As a
consequence the correct diagnosis relies on the experience of the pathologist. Where a typical proliferation of
immature smooth muscle cells and epithelioid cells outside the normal muscular structures occur, associated
with cyst formation, routine H&E staining in combination with adequate clinical and radiological
information is sufficient to make the diagnosis in most cases. Immunohistochemistry for smooth muscle
actin, desmin, and HMB45 is an important adjunct to diagnosis. Again, the specificity and sensitivity of
these markers has not been defined. Smooth muscle actin will stain all muscular cells, desmin will stain the
more mature smooth muscle cells, and HMB45 will stain strongly the epithelioid LAM cells, but also some
of the immature spindle cells. Not all LAM cells are positive for HMB45 the proportion of positive cells can
vary from 17% - 67%[5-8]. HMB45 is a highly sensitive marker in LAM, but in early disease only a few
scattered cells are present and may be overlooked unless there is a strong clinical suspicion of LAM. Given
the presence of an immature smooth muscle and epithelioid cell proliferation at an unusual structural position
within the lung, HMB45 positivity in a proportion of these cells confirms the diagnosis of LAM. In rare
cases, when HMB45 is completely negative but the characteristic lesions are present, the diagnosis of LAM
can be established[6, 7]. In such cases a correlation with clinical and high-resolution CT scan is essential to
increase the confidence level of the diagnosis. In about half of the cases oestrogen and/or progesterone
receptor can be detected by immunohistochemistry[9, 10]. This is not specific to LAM and other
proliferations of smooth muscle such as benign metastasizing leiomyoma or metastatic leiomyosarcoma can
express these antigens.
Differential diagnosis in lung biopsy tissue
Muscular proliferations can occur in emphysema[11] but in contrast to LAM the proliferation is not nodular,
there are neither perivascular epithelioid cell proliferations nor lymphatic cysts. A similar smooth muscle
proliferation is seen in some variants of pulmonary hypertension. It is usually associated with focal chronic
hypoxia[12]. Interstitial pneumonias, especially usual interstitial pneumonia might enter the differential
diagnosis. Usual interstitial pneumonia is characterized by nodular proliferations of myofibroblasts
(fibroblastic foci) as well as by focal fibrosis, scarring and cysts [13]. In other nodular muscular
proliferations such as in emphysema, hypertrophic bronchitis / bronchiolitis, some variants of pulmonary
hypertension, and others, the smooth muscle cells are always HMB45 negative. Metastasis of malignant
melanoma, peripheral nerve sheath tumours, tumours of smooth muscle, Schwann cell or perivascular
epithelioid cell origin can positively stain with HMB45, but have a different morphologic appearance.
Metastasis of stromal sarcoma of the uterus might mimic LAM, but can be ruled out by a negative reaction
for HMB45[14].
1
Where transbronchial biopsy tissue is available a diagnosis can be made in cases where the muscular
proliferation and cystic changes are present. A focal proliferation of LAM cells is also very helpful but the
lesions can be easily missed due to limited sampling. HMB45 is especially important for examination of
transbronchial biopsies[6].
Extra-pulmonary involvement
If lymph nodes are involved, a lymph node biopsy from the retroperitoneum or mediastinum may be
diagnostic of LAM. In severe cases the thoracic duct can be involved. Rarely, other organs such as the serosa
of the uterus or fallopian tubes can be involved[7, 15-17]. Lymphatics alone will not be helpful in assessing
the diagnosis; there are rare diseases with lymphangiectasis and localized as well as systemic lymphatic
proliferation. Some of these might also present with chylothorax. Pulmonary lymphangiomatosis can pose a
problem, as well as Gorham-Stout syndrome, which is a systemic lymphangiomatosis. The lymphatic
dilation and proliferation in Gorham-Stout syndrome can involve mediastinal lymphatics and look similar to
LAM. There is also some muscular proliferation in Gorham-Stout syndrome. In addition there is a rare
lymphangioleiomyoma of the thoracic duct, which can mimic LAM [18, 19]. The above implies that where
lymphatic tissue is sampled the characteristic immature smooth muscle component must convincingly be
present to make a clear diagnosis of LAM.
The diagnosis of angiomyolipoma can be suspected by CT or ultrasound; the histologic diagnosis of
angiomyolipoma is straightforward, due to the mixture of proliferating small and medium sized blood
vessels, perivascular epithelioid cells, smooth muscle and fat cells and can often be made on frozen section.
For a long time, classic renal cell carcinoma has been considered a feature of kidney pathology in TSC.
Recent findings have shown that some cases diagnosed as carcinoma were, in fact, a carcinoma-like variant
of angiomyolipoma. At the moment renal cell carcinoma is not considered a feature of kidney in tuberous
sclerosis complex (TSC), and patients with TSC are not at higher risk of developing renal cell carcinoma.
Recommendation
Cells
Structure
Evidence level
Diagnostic Criteria
Immature smooth muscle
cells (A), perivascular
epithelioid cells (B),
increase and dilation of
lymphatics in alveolar walls
(C)
HMB45: perivascular
epithelioid cells and part of
smooth muscle cells (F)
Smooth muscle actin: (G)
in smooth muscle cells
Desmin (H): in smooth
muscle cells
Oestrogen and progesterone
receptor
Cysts related to LAM cell
infiltration (D),
emphysematous changes
(E)
A= high probability*
B= high probability *
C= low probability *
D= high probability*
E= low probability*
Immunohistochemistry
F= high probability*
G= low probability*
H= medium
probability*
Table. a4.1 Histological and immunohistochemical parameters for the diagnosis of pulmonary LAM.
* Probability can only be estimated, since there are not enough cases for a meaningful statistical evaluation of the
different features of LAM. If A+B+D are present the diagnosis can be made even without immunohistochemistry. If
one of the major criteria (A, B, D) is present the addition of F is diagnostic too. All other criteria have either low or
medium probability; the diagnosis of LAM can be suspected but not proven.
Molecular pathology
LAM cells are characterised by dysregulation of the mammalian target of rapamycin (mTOR) pathway as the
result of mutations in the TSC1/2 genes[20]. This leads to a complex perivascular epithelioid cell (PEC)
phenotype capable of uncontrolled growth, metastatic behaviour and the expression of hormone receptors
and growth factors including vascular endothelial growth factor (VEGF). The mTOR pathway has several
key functions including the relaying of growth factor signals to advance the cell cycle, the integration of
2
nutrient signals with the cell cycle, protein synthesis and the regulation of apoptosis. Constitutive activation
of mTOR and its downstream targets is the result of inactivating mutations in either the TSC-1, or more
commonly TSC-2 gene [21-23]. These genes code for hamartin and tuberin respectively which form an
intracellular heterodimer to repress the pivotal cellular kinase mTOR via the small GTPase rheb (ras
homologue enriched in brain)[24]. In those with TSC there is a pre-existing germline mutation in TSC-1 or 2 and a second mutation (hit) at the same locus resulting in loss of heterozygosity[25]. In those with sporadic
LAM the same molecular defect is thought to result from sequential somatic mutations in TSC-1/2.
LAM may arise through a unique mechanism with circulating LAM cells akin to ‘benign metastases’
targeting the lungs, kidneys and lymphatics[26]. This unusual mechanism was discovered after the
observation that LAM cells in the lungs, kidneys and lymphatics carried identical genetic mutations
suggesting that they had arisen from the same source[27]. In support of this idea, LAM cells of donor
(female) origin have been found in the (male) graft of lung transplant patient who developed recurrent
LAM[28]. In addition, circulating cells with loss of heterozygosity for TSC-2 (LAM cells) have been
isolated in the blood and urine of patients with LAM[29]. The mechanisms targeting circulating LAM cells
to the lungs, kidneys and lymphatics are not yet understood.
LAM cells, in common with other hamartomatous lesions including angiomyolipoma, and clear cell tumour
of the lung express melanoma related antigens including GP100 (the target of the HMB45 antibody) in
intracytoplasmic structures similar to pre-melanosomes This is a feature of a group of entities termed
perivascular epithelioid cells, the normal cellular counterpart of which has not been identified[30]. In
common with other perivascular epithelioid cell lesions LAM cells also have cytoplasmic oestrogen and
progesterone receptors[31]. Although of uncertain significance it is likely that these receptors confer the
female specificity and oestrogen responsiveness of the disease, possibly by an interaction with the
Akt/mTOR pathway and tuberin[32]. Activation of oestrogen receptors in LAM derived cells being linked
with increased cell growth and possibly, via an association with the anti-apoptotic protein BCL-2, may
confer enhanced cell survival[9]. Other anti-apoptotic mechanisms may also exist including increased
expression of the protein survivin[33].
The mechanism of lung parenchymal destruction in LAM is uncertain although increased expression of the
extra-cellular matrix degrading enzymes, matrix metalloproteinases (MMP) -1, -2 and -14 have been
observed in the lungs[34]. MMP-2 was particularly strongly expressed and in lung biopsies was associated
with both MMP-14, its activating enzyme, and disrupted collagen fibres in the walls of LAM cysts. Increased
expression of elastase has also been reported in lung tissue from women with LAM[35].
Other hamartoma syndromes are characterised by defects in the Pi3 OH-kinase / Akt / mTOR pathway,
including retinoblastoma, Cowden’s syndrome and Peutz-Jehgers syndrome[36]. In common with these
entities TSC and LAM cells have enhanced stabilisation of the transcription factor hypoxia inducible factor
and proteins under its transcriptional control, including VEGF are overexpressed in these diseases[36].
Consistent with this observation, raised VEGF-D levels are seen in the plasma of LAM patients[37].
References
1. Carrington CB, Cugell DW, Gaensler EA, Marks A, Redding RA, Schaaf JT, Tomasian A.
Lymphangioleiomyomatosis. Physiologic-pathologic-radiologic correlations. Am Rev Respir Dis 1977:
116(6): 977-995.
2. Matsui K, Beasley MB, Nelson WK, Barnes PM, Bechtle J, Falk R, Ferrans VJ, Moss J, Travis WD.
Prognostic significance of pulmonary lymphangioleiomyomatosis histologic score. Am J Surg Pathol
2001: 25(4): 479-484.
3. Corrin B, Liebow A, Friedman P. Pulmonary lymphangiomyomatosis. A review. Am J Pathol 1975: 79:
348-382.
4. Krymskaya VP. Smooth muscle like cells in pulmonary lymphangioleiomyomatosis. Proc Am Thorac Soc
2008: 5(1): 119-126.
5. Zhe X, Schuger L. Combined smooth muscle and melanocytic differentiation in
lymphangioleiomyomatosis. J Histochem Cytochem 2004: 52(12): 1537-1542.
3
6. Bonetti F, Chiodera PL, Pea M, Martignoni G, Bosi F, Zamboni G, Mariuzzi GM. Transbronchial biopsy
in lymphangiomyomatosis of the lung. HMB45 for diagnosis. Am J Surg Pathol 1993: 17(11): 10921102.
7. Chu SC, Horiba K, Usuki J, Avila NA, Chen CC, Travis WD, Ferrans VJ, Moss J. Comprehensive
evaluation of 35 patients with lymphangioleiomyomatosis. Chest 1999: 115(4): 1041-1052.
8. Matsumoto Y, Horiba K, Usuki J, Chu SC, Ferrans VJ, Moss J. Markers of cell proliferation and
expression of melanosomal antigen in lymphangioleiomyomatosis. Am J Respir Cell Mol Biol 1999:
21(3): 327-336.
9. Matsui K, Takeda K, Yu ZX, Valencia J, Travis WD, Moss J, Ferrans VJ. Downregulation of estrogen
and progesterone receptors in the abnormal smooth muscle cells in pulmonary
lymphangioleiomyomatosis following therapy. An immunohistochemical study. Am J Respir Crit Care
Med 2000: 161(3 Pt 1): 1002-1009.
10. Glassberg MK, Elliot SJ, Fritz J, Catanuto P, Potier M, Donahue R, Stetler-Stevenson W, Karl M.
Activation of the Estrogen Receptor contributes to the Progression of Pulmonary
Lymphangioleiomyomatosis via MMP-induced Cell Invasiveness. J Clin Endocrinol Metab 2008.
11. Glasser SW, Detmer EA, Ikegami M, Na CL, Stahlman MT, Whitsett JA. Pneumonitis and emphysema in
sp-C gene targeted mice. J Biol Chem 2003: 278(16): 14291-14298.
12. Pietra GG, Capron F, Stewart S, Leone O, Humbert M, Robbins IM, Reid LM, Tuder RM. Pathologic
assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol 2004: 43(12 Suppl S): 25S32S.
13. Flaherty KR, Travis WD, Colby TV, Toews GB, Kazerooni EA, Gross BH, Jain A, Strawderman RL,
Flint A, Lynch JP, Martinez FJ. Histopathologic variability in usual and nonspecific interstitial
pneumonias. Am J Respir Crit Care Med 2001: 164(9): 1722-1727.
14. Itoh T, Mochizuki M, Kumazaki S, Ishihara T, Fukayama M. Cystic pulmonary metastases of
endometrial stromal sarcoma of the uterus, mimicking lymphangiomyomatosis: a case report with
immunohistochemistry of HMB45. Pathol Int 1997: 47(10): 725-729.
15. Sullivan EJ. Lymphangioleiomyomatosis: a review. Chest 1998: 114(6): 1689-1703.
16. Matsui K, Tatsuguchi A, Valencia J, Yu Z, Bechtle J, Beasley MB, Avila N, Travis WD, Moss J, Ferrans
VJ. Extrapulmonary lymphangioleiomyomatosis (LAM): clinicopathologic features in 22 cases. Hum
Pathol 2000: 31(10): 1242-1248.
17. Goh SG, Ho JM, Chuah KL, Tan PH, Poh WT, Riddell RH. Leiomyomatosis-like
lymphangioleiomyomatosis of the colon in a female with tuberous sclerosis. Mod Pathol 2001: 14(11):
1141-1146.
18. Tazelaar HD, Kerr D, Yousem SA, Saldana MJ, Langston C, Colby TV. Diffuse pulmonary
lymphangiomatosis. Hum Pathol 1993: 24(12): 1313-1322.
19. Moller G, Priemel M, Amling M, Werner M, Kuhlmey AS, Delling G. The Gorham-Stout syndrome
(Gorham's massive osteolysis). A report of six cases with histopathological findings. J Bone Joint Surg Br
1999: 81(3): 501-506.
20. Goncharova EA, Goncharov DA, Eszterhas A, Hunter DS, Glassberg MK, Yeung RS, Walker CL,
Noonan D, Kwiatkowski DJ, Chou MM, Panettieri RA, Jr., Krymskaya VP. Tuberin Regulates p70 S6
Kinase Activation and Ribosomal Protein S6 Phosphorylation. A role for the TSC2 tumor suppressor
gene in pulmonary lymphangioleiomyomatosis (LAM). J Biol Chem 2002: 277(34): 30958-30967.
21. Sato T, Seyama K, Kumasaka T, Fujii H, Setoguchi Y, Shirai T, Tomino Y, Hino O, Fukuchi Y. A patient
with TSC1 germline mutation whose clinical phenotype was limited to lymphangioleiomyomatosis. J
Intern Med 2004: 256(2): 166-173.
22. Sato T, Seyama K, Fujii H, Maruyama H, Setoguchi Y, Iwakami S, Fukuchi Y, Hino O. Mutation
analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis.
Journal of Human Genetics 2002: 47(1): 20-28.
23. McManus EJ, Alessi DR. TSC1-TSC2: a complex tale of PKB-mediated S6K regulation. Nature Cell
Biology 2002: 4(9): E214-E216.
24. Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct target of TSC2 GAP activity and regulates
mTOR signaling. Genes & Development 2003: 17(15): 1829-1834.
25. Sepp T, Yates JR, Green AJ. Loss of heterozygosity in tuberous sclerosis hamartomas. Journal of
Medical Genetics 1996: 33(11): 962-964.
26. Henske EP. Metastasis of benign tumor cells in tuberous sclerosis complex. Genes, Chromosomes &
Cancer 2003: 38(4): 376-381.
4
27. Carsillo T, Astrinidis A, Henske EP. Mutations in the tuberous sclerosis complex gene TSC2 are a cause
of sporadic pulmonary lymphangioleiomyomatosis. Proc Natl Acad Sci U S A 2000: 97(11): 6085-6090.
28. Karbowniczek M, Astrinidis A, Balsara BR, Testa JR, Lium JH, Colby TV, McCormack FX, Henske EP.
Recurrent Lymphangiomyomatosis after Transplantation: Genetic Analyses Reveal a Metastatic
Mechanism. Am J Respir Crit Care Med 2003: 167(7): 976-982.
29. Crooks DM, Pacheco-Rodriguez G, DeCastro RM, McCoy JP, Jr., Wang J-a, Kumaki F, Darling T, Moss
J. Molecular and genetic analysis of disseminated neoplastic cells in lymphangioleiomyomatosis. PNAS
2004: 0407971101.
30. Bonetti F, Chiodera P. Lymphangioleiomyomatosis and tuberous sclerosis: where is the border? Eur
Respir J 1996: 9(3): 399-401.
31. Brentani MM, Carvalho CR, Saldiva PH, Pacheco MM, Oshima CT. Steroid receptors in pulmonary
lymphangiomyomatosis. Chest 1984: 85(1): 96-99.
32. Finlay GA, York B, Karas RH, Fanburg BL, Zhang H, Kwiatkowski DJ, Noonan DJ. Estrogen-induced
Smooth Muscle Cell Growth Is Regulated by Tuberin and Associated with Altered Activation of Plateletderived Growth Factor Receptor-{beta} and ERK-1/2. J Biol Chem 2004: 279(22): 23114-23122.
33. Carelli S, Lesma E, Paratore S, Grande V, Zadra G, Bosari S, Di Giulio A, Gorio A. Survivin expression
in tuberous sclerosis complex cells. . Molecular medicine: 13(3-4): 166-177.
34. Matsui K, Takeda K, Yu ZX, Travis WD, Moss J, Ferrans VJ. Role for activation of matrix
metalloproteinases in the pathogenesis of pulmonary lymphangioleiomyomatosis. Arch Pathol Lab Med
2000: 124(2): 267-275.
35. Fukuda Y, Kawamoto M, Yamamoto A, Ishizaki M, Basset F, Masugi Y. Role of elastic fiber degradation
in emphysema-like lesions of pulmonary lymphangiomyomatosis. Human Pathology 1991: 22: 727-728.
36. Brugarolas J, Kaelin WG, Jr. Dysregulation of HIF and VEGF is a unifying feature of the familial
hamartoma syndromes. Cancer Cell 2004: 6(1): 7-10.
37. Seyama K, Kumasaka T, Souma S, Sato T, Kurihara M, Mitani K, Tominaga S, Fukuchi Y. Vascular
Endothelial Growth Factor-D Is Increased in Serum of Patients with Lymphangioleiomyomatosis.
Lymphatic Research and Biology 2006: 4: 143-152.
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