INTRODUCTION

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Pulmonary lymphangioleiomyomatosis (LAM) is an uncommon disease characterized by patchy proliferation of specialized smooth muscle cells. The modified smooth muscle cells are immunophenotypically distinct in that they express a melanogenesis-associated antigen recognized by HMB45 and also frequently express estrogen and progesterone receptor proteins (1, 2). LAM occurs most frequently as a sporadic lesion in women of child-bearing age (3). A small percentage of affected patients have tuberous sclerosis complex (TSC). Micronodular pneumocyte hyperplasia (MNPH), a circumscribed proliferation of type 2 pneumocytes, occurs sporadically in association with LAM or TSC (4). MNPH occurs more commonly in women but can also affect men (4). LAM, occurring in isolation, in combination with MNPH, or in association with TSC has never been unequivocally documented in a man. We describe LAM, associated with MNPH and TSC, in a phenotypic and genotypic man.

	CASE REPORT

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A 39-yr-old African American man was seen at an emergency room after an epileptic seizure. He had a history of recurrent seizures, schizophrenia, and mild mental retardation. He also had a history of lung disease of undetermined nature. A unilateral nephrectomy had been performed for a solitary mass 7 cm in greatest dimension. He had been a cigarette smoker for many years. He denied any fever, chills, or chest pain but described exertional dyspnea and intermittent hemoptysis. His mother died of cancer and his father died of an unknown cause. There was no known history of TSC in the family, which included 13 siblings.

On initial examination, he was lethargic, afebrile, and coughing up blood. He was a phenotypically normal male with normally developed external genitalia, normal sexual hair and body fat distribution, and no gynecomastia. His skin showed typical facial angiofibromas. Auscultation of his lungs revealed diffuse bilateral inspiratory crackles. Arterial oxygen saturation measured by pulse oximetry was 83% on room air. Arterial blood gas study on room air showed Pa_O2 40, Pa_CO2 34, and pH 7.41. Chest radiograph showed diffuse interstitial infiltrates. Echocardiography showed normal-sized cardiac chambers and function. Spirometry was performed but the results were not interpretable because of poor cooperation. He was treated with supplemental oxygen, intravenous antibiotics, and olanzapine, an antipsychotic agent. Over the following 7 d of hospitalization his hemoptysis subsided and dyspnea improved. His arterial oxygen saturation on room air increased to 91% at rest and his chest radiographs showed partial clearing of infiltrates.

The patient was seen 9 mo later for further evaluation of persistent lung infiltrates. He described mild exertional dyspnea but denied hemoptysis. Auscultation of his chest again revealed bilateral inspiratory crackles. High-resolution computed tomography (HRCT) of the chest (Figure 1) showed diffuse cystic changes and raised the possibility of pulmonary Langerhans' cell histiocytosis (eosinophilic granuloma). Three video-assisted thoracoscopic biopsies of the left lower and upper lobes were obtained. The postoperative course was unremarkable. He received no specific treatment for his lung disease and was doing well with minimal exertional dyspnea 3 mo after surgery.

View larger version (141K): [in this window] [in a new window]   Figure 1.   HRCT scan of the chest showing multiple thin-walled cysts typical of LAM.

	METHODS

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Histology and Immunohistochemistry

Routine hematoxylin-eosin-stained sections of the lung biopsy and previously resected renal mass were reviewed. Immunohistochemical stains for HMB45 (1:100; Dako, Carpintera, CA) were performed on a Ventana ES autostainer (Ventana, Tucson, AZ) using a standard streptavidin-biotin technique. Stains for estrogen receptor (1:150; Immunotech, Hialeah, FL) and progesterone receptor (prediluted; Ventana) proteins were performed on a Biotek autostainer (Ventana) using a standard avidin-biotin complex (ABC) method. Immunohistochemical stains for hamartin and tuberin were performed using an ABC technique as previously described by Plank and coworkers (8, 9).

Polymerase Chain Reaction (PCR) for Loss of Heterozygosity (LOH)

DNA was extracted from unstained paraffin-embedded sections of the patient's previously resected renal angiomyolipoma and amplified using a PCR technique for chromosome 16p13 microsatellite markers D16S291 as described elsewhere (10). LOH was determined by visual comparison of autoradiographic signals obtained from amplification of angiomyolipoma and normal kidney DNA.

Interphase Fluorescence In Situ Hybridization (FISH)

Five-micron sections from the lung biopsy, comprising mainly normal lung parenchyma, were placed on probe plus slides for interphase FISH. Each slide was heated for 1 to 2 h at 90° C, deparaffinized in two changes of xylene, treated with 100% ethanol for 5 min, and air-dried. Slides were microwaved in 0.1 M citric acid (pH 6.0), rinsed in 2 × saline sodium citrate (SSC) (17.53 g sodium chloride and 0.4 g sodium citrate in 1 L of distilled water), and treated with pepsin (0.05 g/100 ml of 0.01 N HCl) for 20 to 40 min at 37° C. Each slide was then rinsed, examined under phase-contrast microscopy, and dehydrated in 70%, 85%, and 100% ethanol. Centromere-specific, direct labeled probes for 18, X, and Y in aqua, green, and orange (Vysis, Downers Grove, IL) were used.

The premixed probes were placed on each slide, coverslipped and sealed with rubber cement, placed in HYBrite (Vysis, Downers Grove, IL), and hybridized overnight at 37° C. The slides were washed in 2× SSC/0.1 Nonidet P-40 (NP-40) at 70° C for 2 min, rinsed in 2× SSC/ 0.1% NP-40 at room temperature, counterstained with DAPI (4',6'- diamino-2 phenylindole dihydrochloride), and coverslipped.

	RESULTS

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The major finding in the lung biopsy was LAM. At low magnification, multiple cystic spaces were associated with focal interstitial thickening and hemosiderin pigment (Figure 2A). At higher magnification, the areas of interstitial thickening showed spindle cells with centrally located nuclei, inconspicuous nucleoli, and tapering cytoplasmic processes (Figure 2B). The cells were arranged in ill-defined fascicles with no distinct architectural pattern. Circumscribed proliferations of pneumocytes typical of MNPH were also present and formed multiple microscopic nodules. The microscopic nodules were composed of cytologically bland cuboidal epithelial cells arranged in a linear fashion along thickened alveolar septa. The abnormal pneumocyte proliferations had a sharp interface with surrounding lung tissue and were discrete from the lesions of LAM. Review of the previously resected renal mass showed an angiomyolipoma.

View larger version (112K): [in this window] [in a new window]   View larger version (137K): [in this window] [in a new window]   Figure 2.   (A) Low-magnification photomicrograph showing LAM (hematoxylin-eosin stain). A cystic space shows focal thickening proliferation of spindle smooth muscle cells. Original magnification: ×40. (B) Higher magnification photomicrograph showing short bundles of smooth muscle cells. The cells in this field are epithelioid and have vesicular nuclei with visible nucleoli. Original magnification: ×100.

Paraffin section immunostains of the lung biopsy showed strong nuclear immunoreactivity for both estrogen and progesterone receptor proteins in more than half the spindle cells of LAM. Cytoplasmic immunoreactivity was also focally present for HMB45 (Figure 3). Stains performed on the renal angiomyolipoma also showed focal immunoreactivity for HMB45. Immunohistochemical stains showed loss of tuberin (TSC2) in both the angiomyolipoma and in the LAM. Hamartin (TSC1) immunoreactivity was present in the angiomyolipoma and the LAM.

View larger version (149K): [in this window] [in a new window]   Figure 3.   Photomicrograph showing immunoreactivity for HMB45 in the smooth muscle cells of LAM. Original magnification: ×100.

LOH in the TSC2 region of chromosome 16p13 was demonstrated in the angiomyolipoma using the marker D16S291. No LOH was seen in the TSC1 region of chromosome 9q34 using the markers D9S1199. Results were repeated at least twice for confirmation. Ninety-nine of 100 cells analyzed by FISH showed two signals each for chromosome 18 and one each for X and Y chromosomes (Figure 4).

View larger version (86K): [in this window] [in a new window]   Figure 4.   High-magnification fluorescence photomicrograph illustrating a single cell with two signals for chromosome 18 (aqua), one for X (green), and one for Y (red ), consistent with a normal male sex chromosome pattern.

	DISCUSSION

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Our report is the first to confirm the existence of LAM in a phenotypic and genotypic male. A recent review underscores the views of many in affirming that "LAM occurs exclusively in women" (11). The registry of patients established by the LAM Foundation, which includes over 300 patients, has no confirmed examples of LAM in a male patient (Francis X. McCormack, M.D., personal communication). A review of the English-language literature reveals only six previous reports of LAM occurring in men, with or without evidence of associated TSC. One of these published patients was subsequently identified as female (12), and another had only mediastinal disease without evidence of lung involvement (13). One had histologic findings atypical for LAM and likely suffered from some other unrelated condition (14), and two had no histologic confirmation of the diagnosis (15). This leaves only a single previously reported example of LAM in a male patient (16). This same patient was excluded from a subsequent review of Korean patients with pulmonary LAM after review of his biopsy material (17).

Interphase FISH analysis performed on our patient's lung biopsy confirmed a normal XY genotype. The positive X and Y chromosome signals in 99 of 100 cells virtually eliminates the possibility of an XX/XY chimerism (18). Furthermore, XX/XY chimeras rarely present as phenotypically normal men (19). Syndromes of androgen insensitivity (testicular feminization) are associated with a 46, XY karyotype; however, some degree of feminization or abnormalities of external genitalia are typical, even in incomplete forms of the syndrome, which were not present in our patient (20).

In our patient, LAM occurred in the setting of TSC. The combination of LAM, renal angiomyolipoma, and facial angiofibromas is diagnostic of TSC (21). LAM is a rare pulmonary manifestation of TSC, occurring in 1 to 3% of patients (22). These patients are interesting in that they are invariably women, usually of reproductive age, and commonly develop respiratory symptoms typical of those seen in patients with sporadic LAM. Our patient also had MNPH, an unusual epithelial proliferation originally described in the lungs of a 38-yr-old woman with TSC and LAM (5). Muir and coworkers reported a series of 14 patients with MNPH, including nearly all previously reported examples (4). Ten (71.4%) of their patients, all women, had associated LAM and seven of these had other manifestations of TSC. Two additional patients had TSC without evidence of LAM, including one man (24 yr of age). Two patients had MNPH as an isolated finding without TSC or LAM, and one of these was also a man (57 yr of age). Our report is the first to describe the combination of LAM and MNPH in a man.

Observation of LAM in a male raises questions about the role of estrogen and progesterone in the pathogenesis of LAM. Therapeutic strategies have targeted modulation of estrogen and progesterone levels, and have included oophorectomy, progesterone, and tamoxifen in various combinations. Indeed a trial of medroxyprogesterone acetate is the currently recommended treatment of choice (23, 24). Lung transplantation has also been effective in selected patients. Although the proliferating smooth muscle cells in our patient were immunoreactive for both estrogen and progesterone receptor proteins, he received no specific treatment and was stable after short-term follow-up.