Abstract
Rationale: The three previously reported cases of conclusively documented pulmonary lymphangioleiomyomatosis (LAM) in men were associated with definite or probable tuberous sclerosis complex (TSC).
Objectives: To describe an unequivocal case of pulmonary LAM occurring in a man with no clinical or genotypic evidence of TSC.
Methods: At high-resolution computed tomography, a 37-year-old phenotypically and karyotypically normal man with left pneumothorax and massive pulmonary collapse had widespread thin-walled cysts throughout both lungs. Histological diagnosis of LAM was performed on biopsy material, and immunohistochemically confirmed with the HMB-45 monoclonal antibody.
Measurements and Main Results: Remarkably, the HMB-45–positive cells lining the cysts also showed strong reactivity for estrogen and progesterone receptor proteins. TSC was clinically excluded, and TSC1 and TSC2 germline mutations were not detected at DNA analysis.
Conclusions: This article indicates that occurrence of LAM may be possible in a chromosomally normal man unaffected by TSC. On diagnostic grounds, the possibility of LAM should be borne in mind when diffuse cystic lung disease occurs in a man, even in the absence of signs of TSC.
Pulmonary lymphangioleiomyomatosis (LAM) is a rare interstitial disease of uncertain etiology, which is almost exclusively confined to women and is frequently associated with tuberous sclerosis complex (TSC) (1–5). Histologically, LAM is characterized by proliferation of peculiar smooth muscle cells (LAM cells) that show reactivity to the human melanoma black (HMB)–45 monoclonal antibody (6) in the bronchioles, pulmonary veins, and lymphatic vessels. A systematic review of the literature found reports of only nine men bearing a possible, but unproven, diagnosis of pulmonary LAM between 1939 and 1997 (7). A current search of PubMed and Embase identified three subsequent reports of pulmonary LAM in men, all with either definite (8, 9) or probable (10) TSC. Here, we describe an unequivocal case of pulmonary LAM occurring in a man with no evidence at DNA analysis of TSC1 or TSC2 gene mutations.
In December 2003, a 37-year-old phenotypically normal man was urgently admitted to our hospital due to left pneumothorax and massive pulmonary collapse. The patient, who was a company manager and father of a normal child, denied having ever smoked and reported no personal history of serious illness or hospitalization, seizures, mechanical ventilation, or prematurity. Family anamnesis did not disclose possible cases of LAM, TSC, emphysema, pneumothorax, renal cancer, seizures, or mental retardation. Thoracic drainage was performed and the patient was discharged after 10 days. One month later, a high-resolution computed tomography (CT) scan revealed widespread thin-walled cysts (0.5–1.5 cm in diameter) throughout both lungs, showing the characteristic features of pulmonary LAM (Figure 1). Serological α1-antitrypsin values were normal. Clinical signs and laboratory tests for Sjögren's syndrome were negative. Brain and abdominal CT scans were negative for cerebral calcifications and angiomyolipomas; no subtle anomalies were apparent. Abdominal echography confirmed existing diagnoses of a 3-cm renal cyst (6 Hounsfield units at CT) and a 3-cm hepatic hemangioma in the VII segment (hyperechogenic with a clear border). Video-assisted thoracoscopic biopsies were taken at the lingula and apex of the left lung, and a pleural abrasion was performed. Histological examination and immunostaining (see Figure 2) confirmed a final diagnosis of LAM, with characteristic cytoplasmic reactivity to the HMB-45 monoclonal antibody in LAM cells lining the cysts. Remarkably, the same cells showed strong nuclear reactivity for estrogen and progesterone receptor proteins.
Figure 1. High-resolution computed tomography scan showing widespread thin-walled, oval cysts in a background of normal parenchyma.
[More] [Minimize]
Figure 2. Histological and immunohistochemical findings at biopsy. (A) The cyst wall is composed of vessels and smooth muscle cells (lymphangioleiomyomatosis [LAM] cells) interspersed with occasional inflammatory elements (hematoxylin and eosin; original magnification, ×10). LAM cells in the cyst walls show (B) cytoplasm immunoreactivity for the HMB-45 monoclonal antibody (original magnification, ×60), and nuclear reactivity for the (C) estrogen receptor and (D) progesterone receptor antibodies (original magnification, both ×40).
Regarding gender status, estrogen, progesterone, estrone, and testosterone values were all within the normal range. Physically, the patient had normal developmental features, including two descending testicles and male pattern frontal balding and hair distribution. Karyotyping demonstrated that the patient had a normal male complement of chromosomes: conventional GTG- and QF-banding (band resolution, 550) of metaphase chromosomes obtained from phytohemagglutinin (PHA)-stimulated blood lymphocyte cultures showed a normal 46,XY karyotype in 100 examined metaphases, thereby excluding the possibility of chimerisms.
Clinical signs of TSC were systematically investigated. An expert dermatologist was unable to find signs of TSC, such as angiofibromas, subungual fibromas, hypopigmented macules (a Wood's lamp was used), or confetti lesions. An ophthalmologist was also unable to find signs of TSC. Mutation analysis on DNA from cyst wall tissue and peripheral blood lymphocytes was performed to search for TSC1 or TSC2 mutations.
Of note, in February 2004, a decision was made based on the clinicians' judgment (and in concordance with the patient) to use hormonal manipulation therapy, a treatment option that has been tried in women using various pharmacological strategies (5) (we administered leuprorelin, a gonadotropin releasing hormone [GnRH]-antagonist analog, initially associated with letrozole, an aromatase inhibitor). At the time of writing (December 2006) the patient feels in normal health and appears to be clinically and functionally stable.
Routine hematoxylin-and-eosin–stained sections of the lung biopsy were examined. Immunohistochemical stains for HMB-45 (1:30; BioGenex, San Ramon, CA), estrogen receptor (1:50, Clone 1D5; Dako, Glostrup, Denmark) and progesterone receptor (1:50, Clone PgR 636; Dako), smooth muscle actin (1:40, Clone HHF35; BioGenex), and desmin (1:200 Clone D33; Dako) were performed on a Dako Autostainer (Dako, Carpintera, CA) using a standard avidin–biotin complex (Dako, Glostrup, Denmark) method.
Cyst walls containing LAM cells were identified on four serial 4-μm sections from paraffin blocks and were mechanically microdissected under an inverted microscope to avoid contamination from normal cells. Both on cyst wall tissue and peripheral blood lymphocytes, DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) before mutation analysis.
All 23 exons of TSC1 (including the first two transcribed but untranslated exons), the 41 coding exons of TSC2 (including short flanking segments containing splicing signals), and the promoters of both genes were screened by denaturing high-performance liquid chromatography (DHPLC) on a WAVE DNA fragment analysis system (Transgenomics, Crewe, UK). Polymerase chain reaction (PCR) products were automatically loaded on a DNasep column (Transgenomics) and eluted with a linear acetonitrile gradient in a 0.1 M triethylamine acetate buffer (pH 7) at a constant flow rate of 1.5 ml/minute. Samples were analyzed at two temperatures (recommended temperature [RTm] and RTm + 2°C) according to DHPLCMelt software (http://insertion.stanford.edu/melt.html; accessed December 15, 2005).
Large deletions in the TSC1 and TSC2 genes were screened using multiple ligation-dependent probe amplification (MLPA) (11). MLPA was performed according to the protocols suggested by the TSC1 (version 1) and TSC2 (version 3) probe kits from MRC-Holland (Amsterdam, The Netherlands).The TSC1 MLPA kit contains 16 probes specific for exons 1–7, 10, 12–13, 15, 17–18 20–21, and 23. The TSC2 MLPA kit contains 31 probes for the exons 1–5, 7–11, 13–22, 24–26, 28–30, 33, 35–37, and 41, and 1 probe for the last exon of the adjacent PKD1 gene (exon 46).
LAM cells showed strong nuclear immunoreactivity for estrogen and progesterone receptor monoclonal antibodies, and cytoplasm staining for monoclonal antibodies against HMB-45 and smooth muscle actin (Figure 2).
No TSC1 or TSC2 mutation gene was detected on DNA from the cyst wall and from peripheral blood lymphocytes (in our experience, both DHPLC and MLPA have detection rates of about 75–80%). We were able to confirm (in blood cells) the heterozygosity observed at a polymorphic site within TSC1 (2829 C/T on exon 22), thus excluding loss of heterozygosity for this gene.
To our knowledge, this is the first report of definite diagnosis of pulmonary LAM in a karyotypically normal man, apparently not affected by TSC (in addition to the negative expert clinical evaluation, TSC1 and TSC2 germline mutations were not detected). The present case suggests that TSC is not a necessary condition for onset of LAM in men. The only finding in our karyotypically normal man that could provide a pathogenetic clue was the presence of estrogen and progesterone receptor proteins in the LAM cells. This is a common finding in women affected by LAM, and a similar observation was recorded in one of the three cases previously reported in men (8) (the LAM cells of one of the two other men showed only mild positivity for progesterone receptors [10]).
The negative results at DNA analysis require some comment. In the lung tissue, we did not identify any mutation in the TSC1 or TSC2 genes. For TSC1, loss of heterozygosity was excluded in exon 22, where a polymorphism was found in both blood and lung tissue (no polymorphism was detected in any exon of TSC2). Regarding sensitivity, reported detection rates range from 37 to 83%, with the best results deriving from DHPLC, long-range PCR, and quantitative PCR (12). Our current detection rate using MLPA, DHPLC, and sequencing is 75 to 80%. A plausible explanation for the minority (15–20%) of TSC cases who test negative is the existence of a mutation in regulatory regions of the genes, or in a deep intron not investigated by DHPLC. Because mechanical microdissection was used in the present case, it also cannot be ruled out that the presence of normal tissue might have masked mutations in the LAM cells (although we believe this is unlikely in view of the extreme scarcity of normal tissue in the specimens used).
Knowledge that LAM may occur in a chromosomally normal man in the absence of TSC could be relevant for research into the etiology and pathogenesis of this rare disease. On diagnostic grounds, the possibility of LAM should be borne in mind when diffuse cystic lung disease occurs in a man, even in the absence of signs of TSC.
| 1. | Ryu JH, Moss J, Beck GJ, Lee JC, Brown KK, Chapman JT, Finlay GA, Olson EJ, Ruoss SJ, Maurer JR, et al.; NHLBI LAM Registry Group. The NHLBI lymphangioleiomyomatosis registry: characteristics of 230 patients at enrollment. Am J Respir Crit Care Med 2006;173:105–111. |
| 2. | Cohen MM, Pollock-BarZiv S, Johnson SR. Emerging clinical picture of lymphangioleiomyomatosis. Thorax 2005;60:875–879. |
| 3. | Finlay G. The LAM cell: what is it, where does it come from, and why does it grow? Am J Physiol Lung Cell Mol Physiol 2004;28:L694–L700. |
| 4. | Kelly J, Moss J. Lymphangioleiomyomatosis. Am J Med Sci 2001;321:17–25. |
| 5. | Johnson S. Rare diseases. 1. Lymphangioleiomyomatosis: clinical features, management and basic mechanisms. Thorax 1999;54:254–264. |
| 6. | Chan JK, Tsang WY, Pau MY, Tang MC, Pang SW, Fletcher CD. Lymphangiomyomatosis and angiomyolipoma: closely related entities characterized by hamartomatous proliferation of HMB-45-positive smooth muscle. Histopathology 1993;22:445–455. |
| 7. | Hancock E, Osborne J. Lymphangioleiomyomatosis: a review of the literature. Respir Med 2002;96:1–6. |
| 8. | Aubry MC, Myers JL, Ryu JH, Henske EP, Logginidou H, Jalal SM, Tazelaar HD. Pulmonary lymphangioleiomyomatosis in a man. Am J Respir Crit Care Med 2000;162:749–752. |
| 9. | Miyake M, Tateishi U, Maeda T, Kusumoto M, Satake M, Arai Y, Sugimura K. Pulmonary lymphangioleiomyomatosis in a male patient with tuberous sclerosis complex. Radiat Med 2005;23:525–527. |
| 10. | Kim NR, Chung MP, Park CK, Lee KS, Han J. Pulmonary lymphangioleiomyomatosis and multiple hepatic angiomyolipomas in a man. Pathol Int 2003;53:231–235. |
| 11. | Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002;30:e57. |
| 12. | Dabora SL, Jozwiak S, Franz DN, Roberts PS, Nieto A, Chung J, Choy YS, Reeve MP, Thiele E, Egelhoff JC, et al. Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am J Hum Genet 2001;68:64–80. |
