Document kDG57O5N9OVLLYE2J0VReq21J

fespiicflon Basic Science Investigations Respiration 2002;69:261-267 Received: October 18, 2001 Accepted after revision: February 12, 2002 Black Spots of the Parietal Pleura: Morphology and Formal Pathogenesis K.-M. Muller I. Schmitz K. Konstantinidis Institute of Pathology, BG-Clinics Bergmannsheil, University Clinic, Bochum, Germany Key Words Black spots Pleura Coal dust Asbestos Mesothelioma Abstract Background: Dark incorporations (black spots) have been described in various organs. 'Black spots' seen as flat-to-nodular lesions of the parietal pleura are common findings in former miners. They represent areas of coal dust accumulation. An increased incorporation of asbes tos fibres has been described in these areas, causing them to be seen as potential starting points for malignant mesotheliomas. Objectives: The aim of our examina tions was to describe the morphology of black spots in order to understand their formal pathogenesis and dis cuss their role in the development of malignant meso theliomas. Materials: We report the results of the mor phological and energy dispersive X-ray analysis of 12 black spots (4 surgical and 8 autopsy specimens) located in the parietal pleura. Results: Black spots of the pleura develop in close correlation to lymphatic channels and blood vessels. Their formal pathogenesis is character ized by a mild fibrosis and an inflammatory reaction to the incorporated foreign particles. The proliferation of connective tissue may result in the formation of hyaline granulomas. Aluminum, silicone and sometimes fibres are also found in such areas. Mesothelial cells may be irritated. Conclusion: Although there are hints for an increased proliferation of mesothelial cells in some areas with black spots, our findings do not support the classifi cation of black spots as an obligate early lesion in the development of malignant mesotheliomas. Copyright 2002 S. Karger AG, Basel Introduction The term `black spots' refers to dark, circumscribed pigment incorporations or black foci of different origin found in various organs [1]. Such black foci in the parietal pleura resulting from incorporations of mixed dust pig ments were first described over a hundred years ago [2] and are commonly encountered in autopsy specimens from former coal miners. In 1867, Zenker [3] associated `black spots' in the parietal pleura with prolonged occupa tional mixed dust exposure. Other authors regard black spots as the morphological correlate ofpulmonary anthraco-silicosis [4]. Intrapleural mixed dust particle accumu lations in the region of pleural lymphatic channels ensue from inhalation of foreign substances after prolonged occupational dust exposure. AAGGER 2002 S' Karger AG, Basel 0025-7931/02/0693-0261S18.50/0 Fax + 41 61 306 12 34 E-Mail karger@karger. ch Accessible online at: www.karger.comwww.karger.com/journals/res Prof. Dr. med. Klaus-Michael Muller BG-Kliniken Bergmannsheil, Universitatsklinik, Institut fur Pathologie Burkle de la Camp Platz 1 D-44789 Bochum (Germany) Tel. +49 234 3026600, Fax +49 234 3026671, E-Mail patho-bhl@ruhr-uni-bochum.de Boutin et al. [5] discussed the role of black spots as accumulation sites for asbestos fibres and consequently focused on them as potential starting points for malignant mesotheliomas. Compared to normal pleural regions they regarded black spots containing asbestos fibres as proba ble pleural target zones for the development of mesothe liomas and/or pleural plaques. Black spots are also frequently found in the parietal pleura of former coal miners in the German Ruhr region without a concomitant rise in the incidence of non-asbes tos-related mesotheliomas. The aim of our study was to understand the formal pathogenesis of black spots and their possible role in the development of mesotheliomas. There are hints for an increased proliferation of mesothelial cells in areas closely related to dust accumulations in black spots. An exact morphological characterization ofblack spots by means of energy dispersive X-ray microanalysis allows the differentiation of mixed dust accumulations from oth er causes of inflammatory pulmonary lesions. Material and Methods We report the results of the morphological, histological, ultrastructural and energy dispersive X-ray analysis of 12 patients with black spots (4 surgical and 8 autopsy specimens) located in the pari etal pleura. The specimens were fixated in 5% formaldehyde to stabilize organic structures. After standard preparation they were examined under the light microscope, combined with immunohistochemical marking with antibodies against calretinin (polyclonal antibody, Swant, 1:3,000), B-(CD20, monoclonal antibody, Dako, 1:800) and T-lymphocytes (CD45RO, monoclonal antibody, Dako, 1:400), staining machine: `Tech Mate 2000', Dako. Specimens for scanning electron microscopic investigations were cut, mounted onto Thermanoc slides and coated with coal (40 nm, Edwards sputter coater) and examined with a scanning electron microscope (Zeiss/LEO DSM 940) combined with an energy disper sive X-ray analysis system (Oxford, Link system, AN 10/25). EDX- analysis provides a method to identify elements with high sensitivity and precise location in scanning electron microscopy and transmission electron microscopy. With this method, X-rays gener ated by the interaction of the primary beam with the specimen are sorted electronically and give information about the elemental com position of the specimen. For transmission electron microscopy black spots were fixed with 2.5% glutaraldehyde, dehydrated, and embedded in Durcupan. Ultrathin sections were stained with Ultrostain I and Ultrostain II (Leica). A transmission electron microscope (Zeiss/LEO, EM 902) com bined with an EELS analysis system was additionally used to charac terize mixed dust pigment incorporations (TEM: delta E = 0, no ESI mode, 50 kV). Qualitative EELS analysis (energy element loss spectroscopy) can be used to show the presence of elements in an image. When acceler ated electrons are transmitted through the specimen some of them are scattered. In EELS the primary processes of electron excitation is directly studied and give information about elemental composition of the investigated probe. Results Macroscopic and Light-Microscopic Findings Macroscopically, black spots appear as flat, punctate or planar dark pigmentations along the lymphatic channels in the parietal pleura (fig. 1a). Size and shape are subject to variation ranging from round/oval with a diameter of 2 x 2 mm to 3 X 0.2 cm in size. They occur bilaterally either singly or in clusters and are mainly located in the middle or lower lateral and occasionally sternal zones of the thoracic pleura. In rare cases, the entire parietal pleura can be covered by small black spots. Black spots are lim ited to the parietal pleura without invasion of the sur rounding thoracic wall structures. Adhesions to the oppo site visceral pleura are uncommon. The surface of the black spots is generally smooth. During the preparation process they can be easily removed from their undersur face together with an adherent piece of pleura. The vast majority of black spots are only slightly raised ranging in thickness from 0.5 to 1 mm. Nodular black spots ranging in thickness from 2.5 to 4 mm (fig. 1b) are rather rare findings. With careful preparation, the surface of black spots remains covered by an intact mesothelial cell layer. The mesothelial cells covering nodular black spots can focally lose their flat shape, hypertrophy and proliferate. Accumulations of mixed dust pigments contiguous with lymphatic channels are seen embedded in the pleural stroma (fig. 1c). Fig. 1. a Macroscopic view of black spots in parietal pleura. b Nodu lar black spots with central and peripheral scar formations. c Light microscopy of the parietal pleura with mixed dust pigment incorpo ration (P), ectasy of lymphatic channels (L), inflammation (I) and reactive proliferation of the mesothelial cell (inset). d Polarization microscopy. Light refracting particles in the areas of mixed dust accumulation. e B lymphocytes (red) in a black spot marked with CD20 antibodies. f Mesothelial cells (red) on the surface of a black spot marked with calretinin. g TEM. Dark mixed dust accumulation in a black spot consisting of fibre-like formed silicates (*). According to EDX analysis most probably muscovite. x 12,000. Peaks for sili cone and aluminum. Possible mineral is muscovite: Data based on automatically identifying computer analysis and on Rosler [9]. h EDX analysis of a black spot. Peaks for silicone and aluminum. 262 Respiration 2002;69:261-267 Muller/Schmitz/Konstantinidis Black Spots of the Parietal Pleura Respiration 2002;69:261-267 263 predominantly silicone- and aluminum-containing depos its. Fig. 2. Relative concentration of the elements found by means of EDX single spot analysis. Lymphocytic infiltrates can be identified within the circumscribed portion of black spots; however, the in flammatory reaction itself is usually mild. In nodular black spots depending on the incorporation of silicone particles there is evidently stronger fibrosis or even hyalinized granulomatosis. Immunohistochemical Findings Immunohistochemical markers were able to confirm the presence of T- and B-lymphocytes within black spots (fig. 1e). Mesothelial cells on the surface ofblack spots undergo ing hyperplastic reactive changes can be accentuated with, for example, keratin or calretinin antibodies (fig. 1f). Electron Microscopic and Energy Dispersive X-Ray Microanalysis Findings Transmission electron microscopy of black spots re vealed in parts flattened electron-dense incorporations and in parts electron-dense inclusions interspersed be tween collagen fibres and adipocytes (fig. 1g, h). EELS analysis was able to identify comparatively high silicone concentrations and in parts also traces of alumin um and carbon. Scanning electron microscopy was used for the charac terization of mixed dust incorporations. 32 single spot analyses were carried out. Figure 2 shows the elements found and their relative concentrations. Even in adjoining areas, the element dis tribution turned out to be quite heterogenous comprising Pathogenesis ofBlack Spots The start and subsequent development of black spots encompasses several stages, which correlate well with the corresponding histological findings. The initial phase in cludes inhalation of mixed dust particles and their deposi tion in the parietal pleura. The inhaled particles are trans ported in the lymph across the pleural cavity and reach the thoracic pleura via the stomata before being deposited along the lymphatic channels. The areas where the mixed dust particles were laid down appear macroscopically as black spots. In the course of time, the incorporated dust particles trigger a low-grade inflammatory reaction of the parietal pleura. The liberation of mediators leads to fibro blast activation generating in turn a concomitant lowgrade fibrosis in the main pleural layer. On rare occasions, the inflammatory pleural reactions are so pronounced that high-grade fibrotic changes of the main pleural layer ensue. The results are macroscopically nodular black spots. By means of light and scanning electron microsco py, granulomatoid structures with concentrically ar ranged fibres can be documented. Reactive mesothelial cell hyperplasia can be observed on the surface of the black spots. The phasic development of pleural reactions within the regions of black spots is summarized schemati cally in figure 3. Black Spots and Asbestos-Induced Lesions In some of the specimens examined, black spots oc curred together or close to hyaline pleural plaques. The morphological comparison of black spots with hyaline pleural plaques revealed in both cases a marked prolifera tion of collagenous fibres in the main pleural layer. This was, however, the only feature these two types of lesions had in common. The collagen fibre mesh seen in nodular black spots was completely infiltrated by inflammatory cells whereas in hyaline plaques the inflammatory cell infiltrate was limited to the outer borders of the lesions. The oval gaps characteristic of pleural plaques are absent in black spots. Structures resembling black spots can be discerned in close proximity to overt mesotheliomas frequently diag nosed in former coal miners. The dark coal dust incorpo rations of the original visceral pleura represent a thin bor der line between the lung tissue and the greyish-white tis sue of the malignant mesothelioma (fig. 4). In light mi croscopy, sometimes pre-existing dark mixed dust incor- 264 Respiration 2002;69:261-267 Muller/Schmitz/Konstantinidis Pleural stroma with lymphatic channels Intercostal muscle A Lymphatiic* fi stoma Mixed dust Mesothelial cell layer Mixed dust accumu ation Inflammatory reaction Fibrosis Development of a hyalinized granulomatoid structure Reactive mesothelial cell hyperplasia F>g- 3. Phasic development of pleural reac tion within the regions of black spots. porations are found adjacent to the fibrosed main layer of the origin parietal pleura together with an early develop ment phase of an epitheloid mesothelioma (fig. 5). Discussion The parietal pleura covers the endothoracic fascia and borders on the intercostal musculature and the ribs. The surface facing the pleural cavity is covered by mesothelial cells whose diameter lies between 20 and 30 pm. The vas culature is embedded in the connective tissue layer of the pleura. In the costal region, the vascular supply to the parietal pleura is derived from branches of the intercostal arteries and intercostal branches of the internal thoracic artery. The vascular network overlying the ribs is much tighter compared to that overlying the intercostal muscles. The lymphatic stomata in the parietal pleura measure between 2 and 8 pm and are of particular importance as they represent a direct link between the pleural cavity and Black Spots of the Parietal Pleura Respiration 2002;69:261-267 265 muuu^ll^ * Fig. 4. Foci of dark mixed dust incorporations in segments of the original pulmonary pleura between lung tissue and the greyish-white tumor tissue of manifest mesothelioma. Fig. 5. Early development phase of an epithelioid mesothelioma. Pre-existing dark mixed dust incorporations adjacent to the fibrosed main layer of the origin parietal pleura (highly differentiated epithe lioid mesothelioma stained with anticalretinin-antibodies). the lymphatic system for the transport of fluid, fibrin and any corpuscular matter [6]. Black spots are dark patches resulting from carbon-rich mixed dust pigment incorporations in the main connec tive tissue layer of the parietal pleura. They are frequently found in the pleura of former coal miners. Following pig ment deposition they continue to develop along pleural lymphatic channels. According to the results of the energy dispersive X-ray microanalyses, the composition of the stored pigments depends on the occupational exposure to different types of mixed dust particles. Depending on the concentration of crystalline quartz, locally fibrosing reactions are varia bly pronounced progressing not uncommonly to the for mation of typical, hyalinized granulomas. The reaction patterns to the incorporated minerals compare well with the pulmonary findings [7]. These reactive pleural lesions are basically benign. Boutin et al. [5] described asbestos fibre accumulations within black spots. They concluded that black spots were predilection sites for the development of malignant meso theliomas. Indeed, in areas of high concentrations of asbestos fibres in the lung or in the pleura an increased risk for development of a mesothelioma is given [8]. But due to our experiences the morphological finding ofblack spots is not an indicator for an existing mesothelioma or the possibility for the further development of a mesothe lioma. Our results do not support the hypothesis whereby mesotheliomas develop preferentially in the regions of pre-existing black spots. In our collective of former min ers of the Ruhr area we do not find asbestos fibres espe cially amphibole fibres directly located in black spots. But we found silicates, quartz and silicone as well as alumin um-rich minerals such as muscovite. There were no hints that black spots play a possible role in the development of mesotheliomas Irritation of mesothelial cells due to increased pigment incorporation and fibrotic alterations are not uncommon (fig. 4, 5) and have to be regarded as general pathological alterations, whereas mesotheliomas are caused by expo sure to asbestos fibres and not by black spots which are generally free of asbestos fibres. In some cases, coexis tence of these two independent lesions (black spots, meso thelioma) are seen. In cases of high exposure to asbestos fibres and resulting overload and altered lymphatic drain age, asbestos fibres might be found in black spots, too. The occasionally increased proliferation tendencies of mesothelial cells overlying black spots in otherwise tu mor-free tissue can at most be regarded as an indication for the classification of foci as possible sites of malignant tumor development. Up until now it was not possible to establish a firm link between so-called early mesothelio mas and pre-existing black spots. The fact that pigment incorporations like black spots are regularly and repeated ly identifiable within pulmonary pleural tissue without evidence of a co-existing malignant neoplasm on the lung 266 Respiration 2002;69:261-267 Muller/Schmitz/Konstantinidis surface is contradictory to the idea of obligate preneoplas tic lesions as starting points in the development of malig nant mesotheliomas. In connection with the development of malignant me sotheliomas, incorporations of mixed dust particles can at most serve as a marker to map out the course of lymphatic channels. The given physiological conditions allow the pleural transport mechanisms to also trap asbestos fibres in the same places under certain circumstances, such as an occupational asbestos exposure. Mixed dust accumula tions and the potential development of mesotheliomas at a later stage have to be viewed in the first instance as inde pendent occurrences. Principal aspects concerning the role ofblack spots in the development of malignant meso theliomas can only be ascertained by continued observa tions after thoracoscopy has provided the primary diagno sis ofblack spots. This also applies to the histological con firmation of the primary diagnosis of black spots and the documented neoplastic transformation of mesothelial cells adjacent to these foci. References 1 Baffy G, Strate L, Krinsky M: Image of the month. Gastroenterology 2000; 118:252, 453. 2 Worth G, Schiller E: Die Pneumokoniosen. Berlin, Staufen Verlag, 1954, pp 182-183. 3 Zenker F: Uber Staubinhalationskrankheiten der Lungen. Dtsch Arch Klin Med 1867;2:116172. 4 Muller K-M: Pleura; in Remmele W (ed): Pathologie. Berlin, Springer, 1992, vol 3, pp 715 732. 5 Boutin C, Dumortier P, Rey F, Viallat JR, De Vuyst P: Black spots concentrate oncogenic asbestos fibers in the parietal pleura: Thoracos copic and mineralogic study. Am J Respir Crit Care Med 1996;153:444-449. 6 Li J, Zhao Z, Zhou J, Yu S: A study ofthe three dimensional organization of the human dia phragmatic lymphatic lacunae and lymphatic drainage units. Ann Anat 1996; 178:537-544. 7 Konn G, Schejbal V, Oellig W-P: Pneumokoniosen; in Doerr, W, Seifert, G (eds): Spezielle pathologische Anatomie. Pathologie der Lunge. Berlin, Springer, 1983, vol 16/II, pp 647-807. 8 Neumann, N, Gunther S, Muller K-M, Fischer M: Malignant mesothelioma - German meso thelioma register 1987-1999. Int Arch Occup Environ Health 2001;74:383-395. 9 Rosler, H: Lehrbuch der Mineralogie. Leipzig, VEB, Deutscher Verlag fur Grundstoffindustrie, 1979. 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