HistopathoZogH 1991, 18, 347-354
AWNIS
Micronodular hyperplasia of type I1 pneumocytes-a lung lesion associated with tuberous sclerosis
030901679100063E
new
H.H.POPPER, F.M. JUETTNER-SMOLLE* & M.G.PONGRATZt Institute of Pathology, *Department of Thoracic and Hyperbaric Surgery, University of Graz and ?Department of Pulmology, General Hospital of Graz, Austria Date of submission 6 November 1990 Accepted for publication 20 November 1990
POPPER H . H . , JUETTNER-SMOLLE F.M. & PONGRATZ M . G .
(1991) Histopathology 18, 347-354
Micronodular hyperplasia of type II pneumocytes-a tuberous sclerosis
new lung lesion associated with
Open lung biopsy in a 38-year-old female with Pringle-Bourneville syndrome and recurrent pneumothorax revealed a micronodular pneumocyte I1 hyperplasia, a new entity probably associated with the tuberous sclerosis syndrome. The lesion caused an obstruction of the alveolar lymphatic vessels and alveolar ducts, resulting in an emphysema-like picture. This cystic dilation of alveoli and draining lymphatics followed by rupture caused the recurrent pneumothorax. The epithelial pneumocytic nature of the lesion was confirmed by immunohistochemistry and electronmicroscopy. Keywords: tuberous sclerosis, Pringle-Bourneville syndrome, micronodular pneumocytic hyperplasia, pneumothorax
Introduction Tuberous sclerosis is a very rare hereditary autosomal dominant disease with an incidence of approx. 1: 15 0001,a high but incomplete penetrance and great variability in expression, even within familie~l-~. It is characterized by both dysplastic development and heterotopia of the ectodermal cells of the central nervous system. Clinically, it is often accompanied by other developmental abnormalities, l i e sebaceous adenoma and fibromas of the skin, fibrovascular subungual nodules, renal tumours5 (angiomyolipofibromas),mental retardation and epilepsy6.Pulmonary involvement, such as lymphangioleiomyomatosis,fibrosis and hamartomas. is rarely seen6-8. There exist some reports concerning lymphangioleiomyomatosis,but no description of a micronodular hyperplasia of pneumocytes.
Case report A 38-year-old female was admitted to the department of pneumology for severe chest pain due to recurrent rightAddress for correspondence:Dr H.H.Popper, Institute of Pathology, University of Graz. School of Medicine, Auenbruggerplatz 25, A-8036 Graz. Austria.
sided pneumothorax. Pringle-Bourneville syndrome with prominent cutaneous manifestations and bilateral cystonephrosis had been diagnosed in her first year of life. The history revealed a needle biopsy of the left kidney at the age of 20 and an episode of severe left-sided chest pain, suspected for pulmonary embolism at the age of 34. Since that incident, nephrogenic arterial hypertension had been noticed in combination with progressively deteriorating renal function. Eight months prior to admission to our department, a cerebral CT-scan. done due to recurrent attacks of headache, revealed subependymal calcifications,corresponding to a cerebral manifestation of tuberous sclerosis. At the time of admission, the chest roentgenogram showed a massive pneumothorax on the right side, the total collapse of the lung only prevented by numerous pleuropulmonary adhesions. The radiolucency of the left lung was increased. The pneumothorax was treated by intercostal suction drainage. Lung function tests with the drainage still in situ yielded an FEVl of 2.31 (78%),a flow of 380 l/min and a resistance of 3.0, corresponding to a slight obstruction. Although the lung failed to re-expand totally, the patient who at that time refused further 347
348 H.H.Popper, F.M.Juettner-Smolle and M.G.Pongratz
therapeutic and diagnostic procedures was discharged with a ‘stable’ apical residual pneumothorax. During follow-up she noticed recurrent attacks of right-sided chest pain, but did not consult a doctor. The blood gas checks revealed hyperventilation (pC0~: 30.5 mm Hg) as a compensation to metabolic acidosis (BE - 3.2, HC03-: 17.7 mm Hg), the other parameters being within normal range. Lung function tests were not performed in order not to aggravate the pneumothorax, which moreover would have interfered with the findings. A CT-scan of the lungs showed multiple emphysematous areas with disseminated blebs on both sides, suggesting incipient lymphangioleiomyomatosis as a pulmonary manifestation of the Pringle-Bourneville syndrome. Thoracotomy was performed both in order to obtain a histological diagnosis and to achieve re-expansion of the lung: the adhesions were detached and the lung was examined. Multiple emphysematous blebs up to 1cm in diameter were found, disseminated in all three lobes with no apparent predilection of localization. They were situated both deeply within the parenchyma and subpleurally. No obvious air-leakage was detectable. A specimen from the middle lobe was taken for histological investigation. A parietal pleurectomy was done to prevent relapse of the pneumothorax. After an uneventful recovery the patient was discharged with both lungs fully expanded. Up to this time, 9 months after surgery, no relapse has occurred.
Materials and methods Lung tissue was fixed in 10% neutral formaldehyde solution. Formaldehyde was injected with a syringe under a constant pressure of 1 5 cm H20, and then the tissue was immersed in formaldehyde solution. After fixation, the tissue was cut in small pieces which were embedded in paraffin by conventional techniques. The sections were stained with hematoxylin and eosin (H & E), elastica van Gieson stain, trichrome stain according to Masson, and the periodic acid-Schiff reaction (PAS). Sections from the open lung biopsy were processed for routine immunohistochemistry according to the method described by Cordell and co-workersg. Briefly, paraffin sections were dewaxed and rehydrated in phosphate buffered saline (PBS, pH 7.4). Endogenous alkaline phosphatase was blocked by levamisole. The sections were incubated with mouse antibodies against cytokeratins-mouse anti-human cytokeratin RPNl 00, diluted 1: 100 (Amersham) and mouse anti-human cytokeratin EAB 902, diluted 1:40 (Enzo), vimentin (clone V9, diluted 1: 10: Dakopatts), desmin (diluted 1:50; Mono-
san), human factor-VIII-related antigen (factor-VIIIRAg, diluted 1:50; Dakopatts),lysozyme (diluted 1:300: Dakopatts), neuron-specific enolase (NSE, mouse antihuman, diluted 1:30: Innogenetics), s-100 protein (rat anti-bovine A 588. diluted 1: 500;Dakopatts), carcinoembryonic antigen (CEA, mouse anti-CEA 180 kD, BAM 130c, diluted 1:50; Behring), chromogranin (CGA, mouse anti-human chromogranin A, Clone LK2Hl 0, diluted 1:50: Boehringer Mannheim), and laminin (rat anti-laminin HCMS, diluted 1: 50; Medac), respectively. The sections were then washed in PBS, incubated with secondary antibodies against mouse or rat IgG (diluted 1: 100; Vector) and washed again. The rat antibodies were coupled by a third antibody against mouse IgG. Then the slides were incubated with an alkaline phosphatase-anti-alkaline phosphatase complex (APAAP, diluted 1: 100, developed in mice: Dakopatts). The reaction product was visualized by naphthol-ASMX-phosphate as substrate and fast red as chromogen. The nuclei were counterstained with haematoxylin. In addition, reactions with peanut and Ulex europaeus I agglutinins (PNA, AL 2301 diluted 1:300; UEA-I, H2201, diluted 1: 100,both EY-Lab) were performed. The sections were dewaxed and rehydrated as described above, endogenous peroxidase was blocked by H202 diluted in methanol, and incubated with the peroxidasecoupled agglutinins. No protease pretreatment was necessary. After rinsing with PBS, the reactions were developed with diaminobenzidine. For negative controls the primary antibodies or agglutinins were replaced by serum diluted 1:10 in PBS. Surrounding normal lung parenchyma served as positive controls (cytokeratins. factor-VIII-RAg, laminin and both agglutinins). Further positive controls were: a lung carcinoid for NSE and CGA; an adenocarcinoma for CEA; a naevus cell naevus for S-100 protein: a central bronchus with nerves and blood vessels for vimentin and desmin; and lung tissue with bronchopneumonia for lysozyme. For electronmicroscopic evaluation, formalin-fixed, paraffin-embedded material was dewaxed, post-fixed in 1%osmium tetroxide solution and embedded in Epon 812. Ultrathin sections were cut with an ultramicrotome OmU2 (Reichert), contrasted with uranylacetate and lead citrate and examined with a Philips 400 electronmicroscope.
Results Macroscopically, the excised lung tissue resembled emphysema, with dilated alveolar spaces already visible on gross inspection. No nodules were seen. In H & E sections. however, small and large nodules were evident, the largest measuring 0.8 mm in diameter (Figure 1).
Miconodular pneumocyte hyperplasia 349
Figure 1. Open lung biopsy: there are four lesions-three early micronodular hyperplasia of pneumocytes lesions are indicated by arrows and the largest one by arrowheads. Also clearly visible are cystic dilated alveoli and alveolar ducts (A), and lymphatic vessels (L). H& E. x 10.
Figure 2. Early micronodular hyperplasia of pneumocytes lesions: a cell proliferation is evident in the alveolar septa, but b cells are also found within the alveolar walls. H & E. a x 170. b x 45.
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H.H.Popper, F.M.Juettner-Smolle and M.G.Pongratz
The nodules were composed of epithelial cells with cuboidal shape and vesicular round nuclei. The cells possessed no dome-shaped apical cytoplasm, as is found in Clara cells. Cellular or nuclear atypia was absent. The cells were found on the surface of the alveoli, but were developed also within the alveolar septa: in early lesions there was a marked proliferation of epithelial cells on the surface of the alveoli and only a little penetration of cells into the septa (Figure 2), whereas in fully developed lesions more cells were found within the alveolar walls. On light microscopic examination the proliferating cells resembled transformed cuboidal pneumocytes. The cell borders in most instances were indistinct in H & E sections (Figure 3), and the lesion showed a compact adenoma-like cell growth. The cytoplasm was heavily stained by the PAS reaction, and diastase pretreatment abolished this staining pattern, suggesting glycogen storage. Within the nodules a network of capillaries was found, surrounding small cords and nests of epithelial cells. At the borders of the larger proliferations, degenerated elastic fibres and condensed collagen were found by both light and electronmicroscopy(Figure4). Peripheral
Figure 4. Degeneration of elastic fibres and collagen condensation in the vicinity of the largest micronodular hyperplasia of pneumocytes nodule. Elastic van Gieson. x 160.
Figure 3. Largest lesion of micronodular hyperplasia of pneumocytes: the nodule is composed of cells with round nuclei with fine dlspersed chromatin, the cytoplasm is finely granular, sometimes vacuolar or clear. A rich capillary network is found. H & E. x288.
alveoli and ducts and lymphatic vessels were dilated by the epithelial cell proliferation probably due to occlusion or stenosis of proximal airways and vessels (Figure 1). In the immunohistochemical investigations, all proliferating cells were intensely stained by cytokeratin antibodies (Figure 5 ; Table 1). Antibodies directed against vimentin, NSE, S-100 protein, desmin, CEA, factor-VIII-RAg, lysozyme and CGA gave negative results. With vimentin antibodies the endothelial cells of blood vessels, including capillaries and interstitial cells, were stained, whereas the proliferating epithelial cells remained unstained. Laminin antibodies revealed normal basal lamina but not, as expected, a doubling or splitting of basal laminas along the ingrowth of the pneumocytes. Reactions with peanut and Ulex europueus I agglutinin gave a strong staining of proliferating cells and surrounding type 11pneumocytes. This corresponds well with the normal staining pattern of type I1 pneumocytes for these agglutinins.
Miconodular pneumocyte hyperplasia
3 51
Figure 5. Immunohistochemistry with cytokeratin antibodies. a In this early lesion most cells proliferate on the alveolar surface, but some cells form clusters, nests and cords within the septa. Mesenchymal cells are unstained. b In this more advanced lesion many more cells are growing inside the alveolar walls than outside. a x 170. b x 340.
the nodular proliferation, ruling out a neuroendocrine cell proliferation. The cytoplasm of the proliferating cells was clear or finely granular with abundant glycogen deposits. The cells often formed pseudolumina (Figure 6) and developed short microvilli. Desmosomes were detected between cells, and hemidesmosomes within the cytoplasm.
In electronmicroscopic investigations most cells looked like transformed cuboidal pneumocytes. Only a few normal type II pneumocytes with lamellar bodies were interspersed. The proliferating cells lacked lipophilic electron-dense bodies and a dome-shaped cytoplasm-characteristic for Clara cells. No neurosecretory granules or abundant mitochondria were found within Table 1. Immunohistochemistry of micronodular hyperplasia of type I1 pneumocytes: reactions were graded as strongly positive ( + +), positive ( ), focally positive (f ) and negative (- )
+
+
Cytokeratin Desmin Vimentin Micronodular hyperplasia of pneumocytes Normal pneumocytes Alveolar macrophages Fndothelial cells Interstitial cells Smooth muscle cells
++ + -
-
-
-
-
+
Factor-VIIIRAg Lysozyme PNA UEA-1
-
+ + + +
PNA =peanut agglutinin: UEA-1 = Ulex europueus I agglutinin.
-
-
+
-
-
+
f+ f+ -
+ +
-
-
-
-
-
-
-
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H.H.Popper, F.M.luettner-Smolle and MGPongratz
Flgure 6. Micronodular hyperplasia of pneumocytes. part of the largest nodule: a pseudolumen is formed by the cuboidal transformed pneumonocytes in the right lower area, the intercellular spaces are dilated and the cells project many microvilli into the pseudolumina. A few desmosomes and hemidesmosomes are visible. The cells possess a rough endoplasmic reticulum and rich glycogen deposits (clearly visible despite suboptimal preservation). Lamellar bodies are not found in this area. x 10440.
Miconodular pneumocyte hyperplasia
Discussion The tumorous lesion presented is unique, but a few other entities must be ruled out before establishing the diagnosis of micronodular pneumocyte hyperplasia. Among these entities are desquamative interstitial pneumonia, chemodectoma-like bodies of the lung, adenomas/pneumocytomas and lymphangioleiomyomatosis. Desquamative interstitial pneumonia, an entity which Liebow first published in the English literature, is a diffuse proliferation of pneumocytes'". The pneumocyte proliferation is limited by the alveolar wall and its basement membrane. Penetration of the alveolar walls is quite uncommon. In a few cases of diffuse alveolar damage, Katzenstein and co-workers demonstrated a pseudo-invasion of alveolar walls by normal and transformed cuboidal pneumocytes". This pseudo-invasion was shown to be due to a doubling of the partially destroyed basal lamina, where mostly transformed cuboidal pneumocytes grew along the new basal lamina and were sometimes located below the remnants of the original one. But, in contrast to our case, desquamative interstitial pneumonia is a diffuse process with concomitant inflammatory, mostly granulocytic infiltrates in the alveolar septa and not a nodular proliferation of epithelial cells. Chemodectoma-like bodies of the lungs are rare, and most are found incidentallyI2. The cells contain small, oval, sometimes cleaved nuclei with finely granular chromatin and a clear or granular pinkish cytoplasm. Cell borders are poorly definedI3.The cell proliferations are typically arranged around veins and, in contrast to micronodular hyperplasia of pneumocytes, lack a capillary network. In addition, thrombo-emboli, heart failure, emphysema, chronic bronchitis or malignancies, usually associated with chemodectoma-like bodied4, were not found in our case. On electronmicroscopic examination, chemodectoma-like bodies show welldeveloped desmosomesl s, a prominent Golgi apparatus and numerous filaments, 6-10 nm in length'4.16.Desmosomes were present in our case, but the Golgi apparatus was not prominent, and small lumina with microvilli formation were strong evidence against the diagnosis of chemodectoma-like body. The strongest evidence, however, against the diagnosis of chemodectoma-like body was the strong cytokeratin reaction. Chemodectoma-lie bodies lack cytokeratin, S-1 00 protein and CGA reactivities, but contain ~ i r n e n t i i ' ~ . Recently, different types of peripheral lung adenomas have been reported: one of them representing the new interpretation of the sclerosing hemangioma' '-I9, the others representing new en ti tie^^"-^^. There is no similarity between micronodular hyperplasia of pneumo-
3 53
cytes and alveolar a d e n ~ m a ~Micronodular ~.~~. hyperplasia of pneumocytes is a multifocal proliferation, whereas alveolar adenomas are solitary. All alveolar adenomas described so far have imitated alveoli, whereas micronodular hyperplasia of pneumocytes forms only rudimentary lumina and no alveolar structures. Elastic fibres are not formed in either and are degenerated at the borders of micronodular hyperplasia of pneumocytes. Immunohistochemically both entities express similar markers. Some markers were not investigated in alveolar adenomas and surfactant apoprotein antibodies were not available for evaluation of micronodular hyperplasia of pneumocytes. Invasive capability clearly demonstrable for cells in micronodular hyperplasia of pneumocytes was not described in alveolar adenomas. However, the possibility that micronodular hyperplasia of pneumocytes might be the early stage of alveolar adenoma cannot be ruled out. Our knowledge of this tumour is limited and the clinical follow-up of micronodular hyperplasia of pneumocytes is too short. Transformed cuboidal pneumocytes are capable of differentiation into cells of the terminal bronchiolar and alveolar region, amongst them pneumocytes of types I1 and I, and Clara cells25. A recent report by Weng et a1.2h on an atypical pneumocyte type I1 hyperplasia is also unrelated to micronodular hyperplasia of pneumocytes. Alveolar structures and nuclear atypia were described, features not found in micronodular hyperplasia of pneumocytes. Another tumour composed of neoplastic Clara cells was induced experimentally in mice by ethylnitrosourea22. This tumour showed a papillary and tubular structure, and the cells possessed a dome-shaped apical cytoplasm and electron-dense granules as well as numerous mitochondria. Thus, again the structure and cellular components were different from micronodular hyperplasia of pneumocytes. A papillary type of pneumocytic adenoma was reported by Noguchi and colleagues21.It consisted of a mixture of Clara cells, secretory cells and type I1 pneumocytes. In the light of reports which re-evaluated sclerosing haemangioma of the lung17-19,both entities may represent a spectrum of the newly proposed pneumocytoma, with and without sclerosis and haemorrhage. The papillary adenoma as well as the sclerosing pneumocytoma showed a papillary proliferation, were composed of a mixture of cells derived from the bronchoalveolar junction and are, therefore, quite different from micronodular hyperplasia of pneumocytes. Lymphangioleiomyomatosis, a very common pulmonary lesion in tuberous sclerosis27,can easily be distinguished from micronodular hyperplasia of pneu-
3 54
H.H.Popper, F.M.]uettner-Smolle and M.C.Pongratz
mocytes by the strong positive reaction of proliferating smooth muscle cells with desmin and muscle-specific LYactin, and by the negative reaction with cytokeratin antibodies. In our opinion micronodular hyperplasia of pneumocytes represents a new entity: although a similar or probable identical lesion has been mentioned by Corrin et ~ 1 . who ~ ~ called . it an adenomatoid atypical proliferation, this lesion was not characterized with appropriate methods. Micronodular hyperplasia of pneumocytes in our opinion does not possess malignant potential. Cellular and nuclear atypia were not present, progression has not been seen so far and invasion into blood or lymphatic vessels was not found. Invasive growth within alveolar septa is an unexplained feature of micronodular hyperplasia of pneumocytes, not related to a doubling of basal laminas or an infolding of alveolar walls. The lesion is probably one of the numerous tumours and tumour-like lesions associated with tuberous sclerosis. As far as genetics can explain this disease, there exists a genetically transmitted chromosome instability and some complex chromosomal aberrations in tuberous sclerosis2s.This chromosome instability might be the cause of secondary somatic mutations in postnatal life, leading to various skin, kidney and lung tumours and tumour-like lesions. Besides hamartomas, fibromas and lymphangioleiomyomatosis, micronodular hyperplasia of pneumocytes is a further tumour-like lesion associated with tuberous sclerosis.
Acknowledgements This work was in part supported by the Austrian Research Fund (project P 6065) to H.H.P.
References 1 . Hunt A, Lindenbaum RH. Tuberous sclerosis: a new estimate of prevalence within the Oxford region. 1. Med. Genet. 1984: 21: 272-277. 2. Baraitser M. Patton MA. Reduced penetrance in tuberous sclerosis. 1. Med. Genetics 1985: 22; 29-31. 3. Fryer AE. Connor JM. Povey S et al. Evidence that the gene for tuberous sclerosis is on chromosome 9. Lancet 1987: i:659-661. 4. McKusick VA. Mendelian Inheritance in Man. 6th ed. Baltimore: Johns Hopkins University Press. 1987: 895-896. 5. Rumancik WM. Bosniak MA, Rosen RJ. Hulnick D. Atypical renal and pararenal hamartomas associated with lymphangiomyomatosis. AIR 1984: 142: 971-972. 6 . Lieberman BA, Chamberlain DW. Goldstein RS. Tuberous sclerosis with pulmonary involvement. Can. Med. Assoc. 1. 1984: 1230; 287-289. 7. Luna CM. Gene R, Jolly EC. Nahmod N. Detkanch HA, Patino G. Elsner B. Pulmonary lymphangiomyomatosis associated with tuberous sclerosis. Treatment with tamoxifen and tetracyclinepleurodesia. Chest 1985: 88; 473-475.
8. Malik SD. Pardee N. Mattin CJ. Involvement of the lungs in tuberous sclerosis. Chest 1970: 58; 538. 9. Cordell JL. Falini B. Erber WN. Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). 1. Histochem. Cytochem. 1984: 32; 219-229. 10. Liebow AA. Steer A. Billingsley JG. Desquamative interstitial pneumonia. Am. 1. Med. 1965: 39, 369-404. 1 1 . Katzenstein A-L. Myers JL, Mazur MT. Acute interstitial pneumonia. A clinicopathologic. ultrastructural. and cell kinetic study. Am. 1. Surg. Pathol. 1986: 10; 256-266. 12. Korn D. Bensch K. Liebow AA. Castleman B. Multiple minute pulmonary tumors resembling chemodectomas. Am. 1. Pathol. 1960: 37; 641-672. 1 3 . Dail DH. [Jncommon tumors. In: Dail DH. Hammar SP eds. Pulmonary Pathology. Berlin: Springer, 1988: 903-91 0. 14. Churg AM. Warnock ML. So-called ‘minute pulmonary chemodectoma’: a tumor not related to paragangliomas. Cancer 1976: 37; 1759-1 769. 15. Costero I. Barroso-Moguel R, Martinez-PalornoA. Pleural origin of some of the supposed chemodectomoid structures of the lung. Beitr. Pathol. 1972: 146: 351-365. 16. Kuhn CIII, Askin FB. The fine structure of so-called minute pulmonary chemodectomas. Hum. Pathol. 1975: 6; 681-691. 17. Nagata N, Daikaru M. Ishida T. Sueishi K. Tanaka K. Sclerosing hemangioma of the lung. Immunohistochemical characterization of its origin as related to sufactant apoprotein. Cancer 198 5: 5 5: 1 1 6-1 23. 18. Satoh Y. Tsuchiya E. Weng SY et al. Pulmonary sclerosing hemangioma of the lung-a type-I1 pneumocytoma by immunohistochemical and immunoelectron microscopic studies. Cancer 1989: 6 4 1310-1317. 19. Semeraro D. Gibbs AR. Pulmonary adenoma-a variant of sclerosing haemangiorna of lung. I. Clin. Pathol. 1989: 42: 12221223. 20. Franco V. Aragona F. Genova G. Clara cell and type 11 pneumocyte adenoma. An immunocytochemical and ultrastructural study of three cases. Pathologica 1987: 78; 593-602. 21. Noguchi M. Kodama T. Shimosato V et al. Papillary adenoma of type 2 pneumocytes. Am. 1. Surg. Pathol. 1986: 1 0 134-1 39. 22. Palmer KC. Clara cell adenomas of the mouse lung. Interaction with alveolar type 2 cells. Am. 1. Pathol. 1985: 120; 455-463. 23. Siebenmann RE, Odermatt B. Hegglin J, Binswanger RO. Alveolar cell adenoma. a recently identiied benign lung tumour. Pathologe 1990: 11; 48-54. 24. Yousem SA. Hochholzer L. Alveolar adenoma. Hum. Pathol. 1986: 17; 1066-1071. 25. Johnson NF. Repopulation of denuded tracheal grafts with alveolar type I1 cells. In: Mauderley JL, Mehinney JA. Bechtold WE, Sun JD, Coons TA. eds. Inhalation Toxicology Research Institute Annual Report. Springfield. VA: Natl Technical Information Service, US Department of Commerce, 1987-1988: 416-419. 26. Weng S. Tsuchiya E. Satoh Y, Kitagawa T. Nakagawa K. Sugano H. Multiple atypical adenomatous hyperplasia of type-I1 pneumocytes and bronchiolo-alveolar carcinoma. Histopathology 1990: 16: 101-103. 27. Corrin B. Liebow AA. Friedman PJ. Pulmonary lymphangiomyomatosis. A review. Am. I. Pathol. 1975: 79; 347-382. 28. Scudiero DA. Moshell AN, Scarpinato RG et al. Lymphoblastoid lines and skin fibroblasts from patients with tuberous sclerosis are abnormally sensitive to ionizing radiation and to a radiomimetic chemical. 1. Invest. Dermatol. 198 1 : 78; 234-238.
AWNIS
Micronodular hyperplasia of type I1 pneumocytes-a lung lesion associated with tuberous sclerosis
030901679100063E
new
H.H.POPPER, F.M. JUETTNER-SMOLLE* & M.G.PONGRATZt Institute of Pathology, *Department of Thoracic and Hyperbaric Surgery, University of Graz and ?Department of Pulmology, General Hospital of Graz, Austria Date of submission 6 November 1990 Accepted for publication 20 November 1990
POPPER H . H . , JUETTNER-SMOLLE F.M. & PONGRATZ M . G .
(1991) Histopathology 18, 347-354
Micronodular hyperplasia of type II pneumocytes-a tuberous sclerosis
new lung lesion associated with
Open lung biopsy in a 38-year-old female with Pringle-Bourneville syndrome and recurrent pneumothorax revealed a micronodular pneumocyte I1 hyperplasia, a new entity probably associated with the tuberous sclerosis syndrome. The lesion caused an obstruction of the alveolar lymphatic vessels and alveolar ducts, resulting in an emphysema-like picture. This cystic dilation of alveoli and draining lymphatics followed by rupture caused the recurrent pneumothorax. The epithelial pneumocytic nature of the lesion was confirmed by immunohistochemistry and electronmicroscopy. Keywords: tuberous sclerosis, Pringle-Bourneville syndrome, micronodular pneumocytic hyperplasia, pneumothorax
Introduction Tuberous sclerosis is a very rare hereditary autosomal dominant disease with an incidence of approx. 1: 15 0001,a high but incomplete penetrance and great variability in expression, even within familie~l-~. It is characterized by both dysplastic development and heterotopia of the ectodermal cells of the central nervous system. Clinically, it is often accompanied by other developmental abnormalities, l i e sebaceous adenoma and fibromas of the skin, fibrovascular subungual nodules, renal tumours5 (angiomyolipofibromas),mental retardation and epilepsy6.Pulmonary involvement, such as lymphangioleiomyomatosis,fibrosis and hamartomas. is rarely seen6-8. There exist some reports concerning lymphangioleiomyomatosis,but no description of a micronodular hyperplasia of pneumocytes.
Case report A 38-year-old female was admitted to the department of pneumology for severe chest pain due to recurrent rightAddress for correspondence:Dr H.H.Popper, Institute of Pathology, University of Graz. School of Medicine, Auenbruggerplatz 25, A-8036 Graz. Austria.
sided pneumothorax. Pringle-Bourneville syndrome with prominent cutaneous manifestations and bilateral cystonephrosis had been diagnosed in her first year of life. The history revealed a needle biopsy of the left kidney at the age of 20 and an episode of severe left-sided chest pain, suspected for pulmonary embolism at the age of 34. Since that incident, nephrogenic arterial hypertension had been noticed in combination with progressively deteriorating renal function. Eight months prior to admission to our department, a cerebral CT-scan. done due to recurrent attacks of headache, revealed subependymal calcifications,corresponding to a cerebral manifestation of tuberous sclerosis. At the time of admission, the chest roentgenogram showed a massive pneumothorax on the right side, the total collapse of the lung only prevented by numerous pleuropulmonary adhesions. The radiolucency of the left lung was increased. The pneumothorax was treated by intercostal suction drainage. Lung function tests with the drainage still in situ yielded an FEVl of 2.31 (78%),a flow of 380 l/min and a resistance of 3.0, corresponding to a slight obstruction. Although the lung failed to re-expand totally, the patient who at that time refused further 347
348 H.H.Popper, F.M.Juettner-Smolle and M.G.Pongratz
therapeutic and diagnostic procedures was discharged with a ‘stable’ apical residual pneumothorax. During follow-up she noticed recurrent attacks of right-sided chest pain, but did not consult a doctor. The blood gas checks revealed hyperventilation (pC0~: 30.5 mm Hg) as a compensation to metabolic acidosis (BE - 3.2, HC03-: 17.7 mm Hg), the other parameters being within normal range. Lung function tests were not performed in order not to aggravate the pneumothorax, which moreover would have interfered with the findings. A CT-scan of the lungs showed multiple emphysematous areas with disseminated blebs on both sides, suggesting incipient lymphangioleiomyomatosis as a pulmonary manifestation of the Pringle-Bourneville syndrome. Thoracotomy was performed both in order to obtain a histological diagnosis and to achieve re-expansion of the lung: the adhesions were detached and the lung was examined. Multiple emphysematous blebs up to 1cm in diameter were found, disseminated in all three lobes with no apparent predilection of localization. They were situated both deeply within the parenchyma and subpleurally. No obvious air-leakage was detectable. A specimen from the middle lobe was taken for histological investigation. A parietal pleurectomy was done to prevent relapse of the pneumothorax. After an uneventful recovery the patient was discharged with both lungs fully expanded. Up to this time, 9 months after surgery, no relapse has occurred.
Materials and methods Lung tissue was fixed in 10% neutral formaldehyde solution. Formaldehyde was injected with a syringe under a constant pressure of 1 5 cm H20, and then the tissue was immersed in formaldehyde solution. After fixation, the tissue was cut in small pieces which were embedded in paraffin by conventional techniques. The sections were stained with hematoxylin and eosin (H & E), elastica van Gieson stain, trichrome stain according to Masson, and the periodic acid-Schiff reaction (PAS). Sections from the open lung biopsy were processed for routine immunohistochemistry according to the method described by Cordell and co-workersg. Briefly, paraffin sections were dewaxed and rehydrated in phosphate buffered saline (PBS, pH 7.4). Endogenous alkaline phosphatase was blocked by levamisole. The sections were incubated with mouse antibodies against cytokeratins-mouse anti-human cytokeratin RPNl 00, diluted 1: 100 (Amersham) and mouse anti-human cytokeratin EAB 902, diluted 1:40 (Enzo), vimentin (clone V9, diluted 1: 10: Dakopatts), desmin (diluted 1:50; Mono-
san), human factor-VIII-related antigen (factor-VIIIRAg, diluted 1:50; Dakopatts),lysozyme (diluted 1:300: Dakopatts), neuron-specific enolase (NSE, mouse antihuman, diluted 1:30: Innogenetics), s-100 protein (rat anti-bovine A 588. diluted 1: 500;Dakopatts), carcinoembryonic antigen (CEA, mouse anti-CEA 180 kD, BAM 130c, diluted 1:50; Behring), chromogranin (CGA, mouse anti-human chromogranin A, Clone LK2Hl 0, diluted 1:50: Boehringer Mannheim), and laminin (rat anti-laminin HCMS, diluted 1: 50; Medac), respectively. The sections were then washed in PBS, incubated with secondary antibodies against mouse or rat IgG (diluted 1: 100; Vector) and washed again. The rat antibodies were coupled by a third antibody against mouse IgG. Then the slides were incubated with an alkaline phosphatase-anti-alkaline phosphatase complex (APAAP, diluted 1: 100, developed in mice: Dakopatts). The reaction product was visualized by naphthol-ASMX-phosphate as substrate and fast red as chromogen. The nuclei were counterstained with haematoxylin. In addition, reactions with peanut and Ulex europaeus I agglutinins (PNA, AL 2301 diluted 1:300; UEA-I, H2201, diluted 1: 100,both EY-Lab) were performed. The sections were dewaxed and rehydrated as described above, endogenous peroxidase was blocked by H202 diluted in methanol, and incubated with the peroxidasecoupled agglutinins. No protease pretreatment was necessary. After rinsing with PBS, the reactions were developed with diaminobenzidine. For negative controls the primary antibodies or agglutinins were replaced by serum diluted 1:10 in PBS. Surrounding normal lung parenchyma served as positive controls (cytokeratins. factor-VIII-RAg, laminin and both agglutinins). Further positive controls were: a lung carcinoid for NSE and CGA; an adenocarcinoma for CEA; a naevus cell naevus for S-100 protein: a central bronchus with nerves and blood vessels for vimentin and desmin; and lung tissue with bronchopneumonia for lysozyme. For electronmicroscopic evaluation, formalin-fixed, paraffin-embedded material was dewaxed, post-fixed in 1%osmium tetroxide solution and embedded in Epon 812. Ultrathin sections were cut with an ultramicrotome OmU2 (Reichert), contrasted with uranylacetate and lead citrate and examined with a Philips 400 electronmicroscope.
Results Macroscopically, the excised lung tissue resembled emphysema, with dilated alveolar spaces already visible on gross inspection. No nodules were seen. In H & E sections. however, small and large nodules were evident, the largest measuring 0.8 mm in diameter (Figure 1).
Miconodular pneumocyte hyperplasia 349
Figure 1. Open lung biopsy: there are four lesions-three early micronodular hyperplasia of pneumocytes lesions are indicated by arrows and the largest one by arrowheads. Also clearly visible are cystic dilated alveoli and alveolar ducts (A), and lymphatic vessels (L). H& E. x 10.
Figure 2. Early micronodular hyperplasia of pneumocytes lesions: a cell proliferation is evident in the alveolar septa, but b cells are also found within the alveolar walls. H & E. a x 170. b x 45.
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H.H.Popper, F.M.Juettner-Smolle and M.G.Pongratz
The nodules were composed of epithelial cells with cuboidal shape and vesicular round nuclei. The cells possessed no dome-shaped apical cytoplasm, as is found in Clara cells. Cellular or nuclear atypia was absent. The cells were found on the surface of the alveoli, but were developed also within the alveolar septa: in early lesions there was a marked proliferation of epithelial cells on the surface of the alveoli and only a little penetration of cells into the septa (Figure 2), whereas in fully developed lesions more cells were found within the alveolar walls. On light microscopic examination the proliferating cells resembled transformed cuboidal pneumocytes. The cell borders in most instances were indistinct in H & E sections (Figure 3), and the lesion showed a compact adenoma-like cell growth. The cytoplasm was heavily stained by the PAS reaction, and diastase pretreatment abolished this staining pattern, suggesting glycogen storage. Within the nodules a network of capillaries was found, surrounding small cords and nests of epithelial cells. At the borders of the larger proliferations, degenerated elastic fibres and condensed collagen were found by both light and electronmicroscopy(Figure4). Peripheral
Figure 4. Degeneration of elastic fibres and collagen condensation in the vicinity of the largest micronodular hyperplasia of pneumocytes nodule. Elastic van Gieson. x 160.
Figure 3. Largest lesion of micronodular hyperplasia of pneumocytes: the nodule is composed of cells with round nuclei with fine dlspersed chromatin, the cytoplasm is finely granular, sometimes vacuolar or clear. A rich capillary network is found. H & E. x288.
alveoli and ducts and lymphatic vessels were dilated by the epithelial cell proliferation probably due to occlusion or stenosis of proximal airways and vessels (Figure 1). In the immunohistochemical investigations, all proliferating cells were intensely stained by cytokeratin antibodies (Figure 5 ; Table 1). Antibodies directed against vimentin, NSE, S-100 protein, desmin, CEA, factor-VIII-RAg, lysozyme and CGA gave negative results. With vimentin antibodies the endothelial cells of blood vessels, including capillaries and interstitial cells, were stained, whereas the proliferating epithelial cells remained unstained. Laminin antibodies revealed normal basal lamina but not, as expected, a doubling or splitting of basal laminas along the ingrowth of the pneumocytes. Reactions with peanut and Ulex europueus I agglutinin gave a strong staining of proliferating cells and surrounding type 11pneumocytes. This corresponds well with the normal staining pattern of type I1 pneumocytes for these agglutinins.
Miconodular pneumocyte hyperplasia
3 51
Figure 5. Immunohistochemistry with cytokeratin antibodies. a In this early lesion most cells proliferate on the alveolar surface, but some cells form clusters, nests and cords within the septa. Mesenchymal cells are unstained. b In this more advanced lesion many more cells are growing inside the alveolar walls than outside. a x 170. b x 340.
the nodular proliferation, ruling out a neuroendocrine cell proliferation. The cytoplasm of the proliferating cells was clear or finely granular with abundant glycogen deposits. The cells often formed pseudolumina (Figure 6) and developed short microvilli. Desmosomes were detected between cells, and hemidesmosomes within the cytoplasm.
In electronmicroscopic investigations most cells looked like transformed cuboidal pneumocytes. Only a few normal type II pneumocytes with lamellar bodies were interspersed. The proliferating cells lacked lipophilic electron-dense bodies and a dome-shaped cytoplasm-characteristic for Clara cells. No neurosecretory granules or abundant mitochondria were found within Table 1. Immunohistochemistry of micronodular hyperplasia of type I1 pneumocytes: reactions were graded as strongly positive ( + +), positive ( ), focally positive (f ) and negative (- )
+
+
Cytokeratin Desmin Vimentin Micronodular hyperplasia of pneumocytes Normal pneumocytes Alveolar macrophages Fndothelial cells Interstitial cells Smooth muscle cells
++ + -
-
-
-
-
+
Factor-VIIIRAg Lysozyme PNA UEA-1
-
+ + + +
PNA =peanut agglutinin: UEA-1 = Ulex europueus I agglutinin.
-
-
+
-
-
+
f+ f+ -
+ +
-
-
-
-
-
-
-
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H.H.Popper, F.M.luettner-Smolle and MGPongratz
Flgure 6. Micronodular hyperplasia of pneumocytes. part of the largest nodule: a pseudolumen is formed by the cuboidal transformed pneumonocytes in the right lower area, the intercellular spaces are dilated and the cells project many microvilli into the pseudolumina. A few desmosomes and hemidesmosomes are visible. The cells possess a rough endoplasmic reticulum and rich glycogen deposits (clearly visible despite suboptimal preservation). Lamellar bodies are not found in this area. x 10440.
Miconodular pneumocyte hyperplasia
Discussion The tumorous lesion presented is unique, but a few other entities must be ruled out before establishing the diagnosis of micronodular pneumocyte hyperplasia. Among these entities are desquamative interstitial pneumonia, chemodectoma-like bodies of the lung, adenomas/pneumocytomas and lymphangioleiomyomatosis. Desquamative interstitial pneumonia, an entity which Liebow first published in the English literature, is a diffuse proliferation of pneumocytes'". The pneumocyte proliferation is limited by the alveolar wall and its basement membrane. Penetration of the alveolar walls is quite uncommon. In a few cases of diffuse alveolar damage, Katzenstein and co-workers demonstrated a pseudo-invasion of alveolar walls by normal and transformed cuboidal pneumocytes". This pseudo-invasion was shown to be due to a doubling of the partially destroyed basal lamina, where mostly transformed cuboidal pneumocytes grew along the new basal lamina and were sometimes located below the remnants of the original one. But, in contrast to our case, desquamative interstitial pneumonia is a diffuse process with concomitant inflammatory, mostly granulocytic infiltrates in the alveolar septa and not a nodular proliferation of epithelial cells. Chemodectoma-like bodies of the lungs are rare, and most are found incidentallyI2. The cells contain small, oval, sometimes cleaved nuclei with finely granular chromatin and a clear or granular pinkish cytoplasm. Cell borders are poorly definedI3.The cell proliferations are typically arranged around veins and, in contrast to micronodular hyperplasia of pneumocytes, lack a capillary network. In addition, thrombo-emboli, heart failure, emphysema, chronic bronchitis or malignancies, usually associated with chemodectoma-like bodied4, were not found in our case. On electronmicroscopic examination, chemodectoma-like bodies show welldeveloped desmosomesl s, a prominent Golgi apparatus and numerous filaments, 6-10 nm in length'4.16.Desmosomes were present in our case, but the Golgi apparatus was not prominent, and small lumina with microvilli formation were strong evidence against the diagnosis of chemodectoma-like body. The strongest evidence, however, against the diagnosis of chemodectoma-like body was the strong cytokeratin reaction. Chemodectoma-lie bodies lack cytokeratin, S-1 00 protein and CGA reactivities, but contain ~ i r n e n t i i ' ~ . Recently, different types of peripheral lung adenomas have been reported: one of them representing the new interpretation of the sclerosing hemangioma' '-I9, the others representing new en ti tie^^"-^^. There is no similarity between micronodular hyperplasia of pneumo-
3 53
cytes and alveolar a d e n ~ m a ~Micronodular ~.~~. hyperplasia of pneumocytes is a multifocal proliferation, whereas alveolar adenomas are solitary. All alveolar adenomas described so far have imitated alveoli, whereas micronodular hyperplasia of pneumocytes forms only rudimentary lumina and no alveolar structures. Elastic fibres are not formed in either and are degenerated at the borders of micronodular hyperplasia of pneumocytes. Immunohistochemically both entities express similar markers. Some markers were not investigated in alveolar adenomas and surfactant apoprotein antibodies were not available for evaluation of micronodular hyperplasia of pneumocytes. Invasive capability clearly demonstrable for cells in micronodular hyperplasia of pneumocytes was not described in alveolar adenomas. However, the possibility that micronodular hyperplasia of pneumocytes might be the early stage of alveolar adenoma cannot be ruled out. Our knowledge of this tumour is limited and the clinical follow-up of micronodular hyperplasia of pneumocytes is too short. Transformed cuboidal pneumocytes are capable of differentiation into cells of the terminal bronchiolar and alveolar region, amongst them pneumocytes of types I1 and I, and Clara cells25. A recent report by Weng et a1.2h on an atypical pneumocyte type I1 hyperplasia is also unrelated to micronodular hyperplasia of pneumocytes. Alveolar structures and nuclear atypia were described, features not found in micronodular hyperplasia of pneumocytes. Another tumour composed of neoplastic Clara cells was induced experimentally in mice by ethylnitrosourea22. This tumour showed a papillary and tubular structure, and the cells possessed a dome-shaped apical cytoplasm and electron-dense granules as well as numerous mitochondria. Thus, again the structure and cellular components were different from micronodular hyperplasia of pneumocytes. A papillary type of pneumocytic adenoma was reported by Noguchi and colleagues21.It consisted of a mixture of Clara cells, secretory cells and type I1 pneumocytes. In the light of reports which re-evaluated sclerosing haemangioma of the lung17-19,both entities may represent a spectrum of the newly proposed pneumocytoma, with and without sclerosis and haemorrhage. The papillary adenoma as well as the sclerosing pneumocytoma showed a papillary proliferation, were composed of a mixture of cells derived from the bronchoalveolar junction and are, therefore, quite different from micronodular hyperplasia of pneumocytes. Lymphangioleiomyomatosis, a very common pulmonary lesion in tuberous sclerosis27,can easily be distinguished from micronodular hyperplasia of pneu-
3 54
H.H.Popper, F.M.]uettner-Smolle and M.C.Pongratz
mocytes by the strong positive reaction of proliferating smooth muscle cells with desmin and muscle-specific LYactin, and by the negative reaction with cytokeratin antibodies. In our opinion micronodular hyperplasia of pneumocytes represents a new entity: although a similar or probable identical lesion has been mentioned by Corrin et ~ 1 . who ~ ~ called . it an adenomatoid atypical proliferation, this lesion was not characterized with appropriate methods. Micronodular hyperplasia of pneumocytes in our opinion does not possess malignant potential. Cellular and nuclear atypia were not present, progression has not been seen so far and invasion into blood or lymphatic vessels was not found. Invasive growth within alveolar septa is an unexplained feature of micronodular hyperplasia of pneumocytes, not related to a doubling of basal laminas or an infolding of alveolar walls. The lesion is probably one of the numerous tumours and tumour-like lesions associated with tuberous sclerosis. As far as genetics can explain this disease, there exists a genetically transmitted chromosome instability and some complex chromosomal aberrations in tuberous sclerosis2s.This chromosome instability might be the cause of secondary somatic mutations in postnatal life, leading to various skin, kidney and lung tumours and tumour-like lesions. Besides hamartomas, fibromas and lymphangioleiomyomatosis, micronodular hyperplasia of pneumocytes is a further tumour-like lesion associated with tuberous sclerosis.
Acknowledgements This work was in part supported by the Austrian Research Fund (project P 6065) to H.H.P.
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