Tumori, 69: 283-292, 1983 IMMUNOHISTOCHEMISTRY AND ELECTRON MICROSCOPY OF INTRAVASCULAR BRONCHIOLOALVEOLAR TUMOR OF THE LUNG Silvana Pilotti,' Franco Rilke,' Luciano Lombardi 2 and Ugo Pastorino • (' Anatomia Patologica e Citologia, 2 Oncologic Sperimentale A, and s Oncologia Clinica D, Istituto Nazionale per 10 Studio e la Cura dei Tumori, Milano) We report on an intravascular bronchioloalveolar tumor (IVBAT) detected in the lungs of a 45-year-old female. The results of the immunohistochemical and ultrastructural investigations stress the already suggested endothelial origin of the tumor. The clinical and histopathologic differential diagnoses between IVBAT and other rare tumors of the lung are discussed. Similarities of IVBAT with other more or less recently described endothelial tumors of the liver, skin and soft tissues are pointed out. The intravascular bronchioloalveolar tumor (lVBAT) was described for the first time about a decade ago (10) under the term of «so-called pulmonary deciduosis », However, Liebow correctly suggested for that case the diagnosis of IVBAT. Since then, about 30 cases have been investigated at light microscopy and reported (1,2,4, 23), 20 of these by Dail and Liebow (6) in 1975. Eight of the most recent cases were also investigated ultrastructurally (1,2, 4, 11, 22) and 4 also immunohistochemically (2, 23). This report deals with a new case of IVBAT that was investigated by electron microscopy and immunohistochemistry, both of which confirmed its endothelial origin. in diameter, which predominantly involved the posterior aspect of both lungs. The mediastinum, liver, spleen, pancreas, kidneys and retroperitoneum were normal. Skeletal scintiscan was negative. The frozen section of an open-chest biopsy yielded the diagnosis of primary lung tumor comparable with IVBAT. No treatment was given to the patient. Six months later, the patient was still asymptomatic, and chest X-ray showed a slight increase in number and size of the nodules. Three months later, the patient complained of shortness of breath after physical exercise, even though the radiologic picture was essentially unmodified. Case report Material and methods A 45-year-old, asymptomatic woman revealed at a routine chest X-ray several nodules in both lungs. After 20 days of treatment with cortisone without any change in the radiographic pattern, the patient was admitted to the Institute. Thoracic and abdominal CAT scan confirmed the presence of numerous small nodules measuring up to 1 em On Bouin-fixed and paraffin-embedded material, the following stains were performed: hematoxylin and eosin, PAS with and without diastase pretreatment, Alcian blue with and without hyaluronidase pretreatment, Weigert for elastic fibers, and Gomori's silver stain for reticulin fibers. Factor VIII-related antigen (RAG) (Dako Corp., Acknowledgments: The authors thank Ms. B. Johnston for editing and preparing the manuscript. Address tor reprint requests: Dr. S. Pilotti, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milano, Italia. Presented in part at the 1st National Surgical Pathology Specialty Conference held at the Istituto Nazionale per 10 Studio e la Cura dei Tumori, Milano, Italy, on December 11, 1982. Received February 22, 1983. 283 284 Pilotti et at. Santa Barbara, Calif.) was visualized with the PAP method, according to Sternberger et al. (19). The sections were counterstained with AEC (3amino-9-ethylcarbazole) . For electron microscopy, small fragments of neoplastic tissue were fixed in 2.5 % glutaraldehyde in Sorensen's phosphate buffer for 2 h at 4°C, postfixed in 1 % osmic acid in Millonig's phosphate buffer for 2 h at 4 QC, dehydrated in ethanol and embedded in Epon 812. Suitable tumor tissue areas were selected by observation at the light microscope of l-micron-thick sections stained with Azur II-methylene blue. Ultrathin sections were routinely stained with uranyl acetate and lead citrate and examined with a Philips EM 300 electron microscope. Results Gross - The surgical specimen of lung tissue (fig. 1) contained 2 nodules, which measured respectively 0.5 and 1 em in diameter and which were both well-defined, elastic, and pinkish-grey. Microscopic - Both nodules were similar and showed rounded borders (fig. 2), a center that was fibrous-hyaline and almost devoid of cells, and a peripheral rim that was cell rich. In each nodule, the proliferating component was made up of polypoid protrusions that invaded the alveolar Fig. 1 - Gross features of the lung specimen containing two discrete nodules. (fig. 2) and ocasionally the bronchial spaces (fig. 3). These excrescences were made up of cells embedded within an either loose and myxoid or fibrous and hyaline stroma. The peripheral polypoid excrescences did not always entirely fill the alveolar lumens and were made up of 2 types of cells. There were large, round to oval cells that proliferated along the alveolar wall and elongated and/or flattened cells Fig. 2 - The tumor nodule shows well-defined margins, typical polypoid protrusions into the alveolar lining, and a zonal pattern with peripheral cellular areas and central sclerotic areas (Hem.-Eos., X 315). Intravascular bronchioloalveolar tumor 285 3 4 Fig. 3 - Invasion by a polypoid protrusion of a bronchiolar space (Hem.Eos., X 125).• Fig.4 - A micropolypoid intraalveolar neoplastic excrescence made up of cells with eosinophilic vacuolated cytoplasm (Hem.Eos., X 250). Inset: semithin section showing cytoplasms with vacuoli and prominent nucleoli (Azur II - methylene blue, X 625). located at the periphery of the intraalveolar protrusions. The former had clear eosinophilic cytoplasms with well-defined margins that were often vacuolized and round to oval, uniform nuclei that occasionally contained cytoplasmic inclusions. The nucleoli were usually single, eosinophilic, and prominent. Vacuoles were not only present within single cells but also in between 2 or 3 cells with some tendency towards confluence and extracellular position. The vacuoles never stained with Aldan blue or PAS. The second cell type was similar to the hypertrophic alveolar cells that lined the residual free alveolar wall (fig. 4 and inset). Cells of the first type were occasionally encountered within the lumens of blood vessels and/or infiltrated the walls of the arteries. There were no mitotic figures, and karyorrhexis was occasionally present. In the center of the tumor, the polypoid excrescences completely filled the al- Pilotti et al. 286 5 6 Fig. 5 - Just outside the tumor borders there are some strands of eosinophilic tumor cells both within the alveolar walls (arrow) and free in the alveolar lumen (Hem.-Eos., X 200) .• Fig. 6 - FactorVIII-RAG is localized in the cytoplasm of the tumor cells of the micropolypoid excrescences, whereas the surrounding alveolar cells are negative (arrow) (X 250). veolar spaces and were made up of sparse cells of the first type described above, which were single or arranged in short cords. The stroma was abundant and definitely hyalinized in the center of the tumor and chondromyxoid-like in the periphery. In the latter, it was also PAS-positive and Alcian blue-positive, hyaluronidase-sensitive, whereas towards the center, both stains became negative. The silver stain showed a tight reticular meshwork around the cells. The Weigert stain showed the partial preservation of the interalveolar septa. In the grossly normal appearing tissue surrounding the nodules, cells of the first type could be found within the alveolar walls and occasionally in the alveolar lumens (fig. 5). Factor VIII-RAG - The cytoplasm of the first cell type was intensely positive, especially in the peripheral excrescences. The cells of the same type present in the center of the tumor showed somewhat less intensive positive cytoplasm. Positivity was also shown by the endothelial cells of the blood vessels, whereas alveolar cells were completely negative (fig. 6). Electron microscopy - The neoplastic cells had a round or oval outline or presented interdigitating processes connected by frequent junctions of the zonula adherens type (figs. 7 and 8). Their polymorphous nucleus was spherical or lobated with a prominent nucleolus, marginated chromatin, and frequent pseudoinclusions. Large areas of the cytoplasm were filled with 100 to 150 A-thick, criss-crossing intermediate filaments (fig. 8). A few bundles of microfilaments without fusiform densities were present beneath the plasma membrane, from which abundant pinocytotic vesicles budded (fig. 8). Weibel-Palade bodies with Intravascular bronchioloalveolar tumor 287 7 8 9 Fig. 7 - Polymorphous tumoral cells bordering intercellular spaces. A discontinuous layer of basal lamina-like dense material (BL) separates the tumoral cells from the extracellular matrix (EM) (X 7,500). • Fig. 8 Enlargement of the same field as Fig. 7 showing cell projections connected by frequent junctions, abundant intermediate filaments, and pinocytotic vesicles. BL, basal lamina-like dense material (X 10,000).• Fig. 9 - A Weibel-Palade body with tubular structure (X 113,000). 288 10 Pilotti et al. 11 12 Fig. 10 - A tumoral cell with two intracytoplasmic lumens (x 6.000).• Fig. 11 - A tumoral cell pushing inward the epithelial lining of an alveolus (x 5,500).• Fig. 12 - A cluster of tumoral cells bulging into the space of an alveolus lined by type I pneumocytes (X 4,000). Intravascular bronchioloalveolar tumor 289 13 14 Fig. 13 - An alveolus bordered by degenerating type II pneumocytes and containing surfactant and lamellar bodies (X 10,000). Inset: an enlargement of a lamellar body showing the characteristic period structure (X 160,000).• Fig. 14 - Extracellular matrix containing fibrillar material and polymorphous dense bodies (X 11,000). Inset: a dense body showing the same periodic structure as the lamellar bodies of the type II pneumocytes (X 190,000). 290 the characteristic tubular structure were found in a few cells (fig. 9). One or more intracytoplasmic lumens lined by a smooth membrane and containing a scarce floccular dense material were observed in numerous cells (fig. 10). Intercellular spaces with a similar content were bordered by the tumoral cells and their projections. Other cytoplasmic organellae were not prominent. Clusters of tumoral cells pushed inward the epitheliallining of the alveoli (fig. 11) or bulged into the alveolar space through discontinuities of the wall (fig. 12). Alveoli bordered by degenerating epithelial cells and containing cellular debris and a dense material with the structure typical of the lamellar bodies of the type II pneumocytes and of the surfactant secreted by the alveolar cells (7) were also found (fig. 13). The extracellular matrix embedding the tumoral cells and the residual pulmonary structures was particularly abundant in the central areas of the neoplastic nodules and consisted of collagen fibers, filaments without periodicity, clusters of thinly fibrillar material, and abundant polymorphous dense bodies with the same structure of the lamellar bodies of type II pneumocytes (fig. 14). Masses of extracellular matrix protruded into the alveolar spaces. Tumoral cells were separated from the extracellular matrix by a discontinuous layer of basal lamina-like material (figs. 7 and 8). Discussion The clinical and radiologic features of the present case are within the range of those previously reported (1, 2, 4, 6, 11, 22), and are represented by the female sex, the fairly young age, the absence of symptoms at the incidental diagnosis of the disease, the radiologic evidence of small spaceoccupying lesions, and the multiplicity of these within both lungs. At this stage of the diagnostic approach of the disease, for differential diagnostic purposes, a similar clinical course has been reported for the recently identified, pulmonary, metastatic dissemination of leiomyosarcomas predominantly of the uterus (24). Occasionally, other tumors predominantly of the lungs have been reported to be associated with IVBAT (2, 6). With reference to this allegedly common finding, we recently received for consultation * a case of IVBAT that appeared to be * We would like to thank Prof. G. Schlich, Ospedale Pizzardi, Bologna, who kindly submitted the histologic sections. Pilotti et at. radiologically a single nodule, which measured 2 em in diameter and appeared in a 65-year-old male who 2 years later developed a cutaneous malignant melanoma. The tumor remained single, at least until admission to this Institute for treatment of the melanoma. Histologically, the present case did not show features that were different from those already described; the main features were the expanding type of growth, the blood vessel invasion, the difference between central and peripheral areas, the large, somewhat epithelial-like neoplastic cells with vacuolated cytoplasm and large nucleoli, and the chondroid-like, more or lesse abundant stroma. Thus, the overall morphologic features of the IVBAT are very characteristic and can be easily recognized. Before its identification, the tumor was often mistaken as a hamartoma or a chondrosarcoma (4), whose cells always contain glycogen and never show vacuolated cytoplasm. Furthermore, the cells of chondrosarcomas are arranged in loose cords and/or in multinodular aggregates. On the contrary, similarities of IVBAT to sclerosing hemangioma have been reported (18, 20). This benign and histogenetically uncertain tumor (12, 13) and even its solid variant never show a polypoid type of growth. It is made up of large cords of round, uniform, small to medium-sized cells, which have bland nuclei with fine chromatin and inconspicuous nucleoli. These cords are often lined by alveolar cells and seem to represent the progressively thickened alveolar walls. Cytoplasmic vacuoles are rare and occasionally produce a signetring cell-like appearance. Deposits of hemosiderin are common, as are xanthomatous cells (13), which have never been mentioned in IVBAT. Features vaguely reminiscent of sclerosing hemangioma may be seen beyond the peripheral areas of IVBAT, where tumor cells may be found within the alveolar walls. This feature suggested the possible origin of IVBAT from the alveolar wall (1, 18) and the term « sclerosing interstitial vascular sarcoma». Even though the origin of both tumors might be the same, the subsequent growth pattern is entirely different, and pulmonary sclerosing hemangioma is only exceptionally multicentric. The immunohistochemical findings shown by the present case are closely related to those of normal endothelial cells (3, 15, 17), even though it should be remembered that the negative stain for Factor VIII-RAG does not preclude the endothelial nature, particularly when dealing with tumors (3). Intravascular bronchioloalveolar tumor The observation in the present case of abundant pinocytotic vesicles, intermediate filaments, Weibel-Palade bodies, and intra- and extracellular lumens that mimick rudimentary blood vessels is in keeping with a vascular origin of the neoplasia, as suggested by several authors (1, 2, 4, 22). The previously reported absence of Weibel-Palade bodies (2) and the presence of abundant microfilaments (1, 4) may indicate a low differentiation of the neoplastic cells. The hypothesis of the tumor development in the alveolar septa with the eventual invasion of the alveolar lumen is sustained by the display of tumor cells pushing inward and passing through the alveolar wall. As far as the intercellular matrix is concerned, we agree with the assumption that its components may be largely produced by the tumoral endothelial cells (1, 21), as supported by the cytochemical similarities of intercellular matrices of analogous tumors arising in different anatomical sites, such as epithelioid hemangioendothelioma (21) and endovascular papillary angioendotheliorna (5). However, the presence in the intercellular matrices of IVBAT of dense particulate material with the typical structure of the lamellar bodies of the type II pneumocytes suggests an epithelial contribution to its formation. The degenerating alveoli could be the source of the epithelial components of the intercellular matrix. The fairly large number of terms proposed to define this tumor reflects the changing concepts of its histogenesis, which ranged from the alveolar cell (6) to the angioblastic stem cell (4). The first name (IVBAT), which was essentially descriptive and derived from light microscopic evidence (6), was subsequently slightly modified (18) by adding the adjective «sclerosing» (IVSBAT). Other terms were «sclerosing angiogenic tumor» (22) and «sclerosing interstitial vascular sarcoma» (1), which both stressed the endothelial features of the tumor cells and the possible origin from multi potent mesenchymal cells. More recently, the term of « low-grade sclerosing epithelioid angiosarcoma of the lung» (2) has been proposed. With this particular term, the concept was broadened and the message was conveyed that the IVBAT was biologically, morphologically and histogenetically similar to some other tumors described more or less recently in the skin and soft tissues (5, 21) and liver (8, 9, 14, 16,21). In all sites, these tumors featured a long, indolent clinical course and had a low incidence of metastases, which involved the liver, lungs bones, lymph nodes and rarely the spleen. Histo- 291 logically, the proliferating cells were epithelioidlike, with intracytoplasmic vacuoles and rimmed formations, which were similar to the first stages of angiogenesis, and were embedded within a fibro-myxoid or chondroid-like stroma. Intravascular tumor growth was also frequently noticed, and mitotic figures were almost absent. The morphologic similarities among these lesions are indirectly proven also by the differential diagnoses they have in common. The range includes hamartomas, chondrosarcoma and metastatic carcinomas for the lung (4), malignant hamartomas (9), mixed mesenchymal epithelial tumors (4, 16) and cholangiocarcinoma (21) for the liver, and angioendothelioma, angiosarcoma, and metastatic carcinoma (21) for the soft tissues. Even the gross features of the lesion are similar, because the neoplasm in various organs and sites is made up of small, discrete nodules, which are not hemorrhagic but are rather whitish-pink in color, fairly hard in consistency, and occasionally contain gelatinous foci. Calcification is rare in the pulmonary tumors (6) and fairly common in the tumors of the liver, where similarly to the splenic location (14, 16) the tumor may reach considerable size (9, 14, 16). Finally, the common endothelial origin or perhaps the origin from angioblastic stem cells is supported by the results of the ultrastructural and immunohistochemical investigations performed in a number of cases of tumors of the lung (1, 2, 4, 22, 23), the liver (9), and the skin and soft tissues (21). Immunoistocbimica e ultrastruttura del tum ore intravascolare broncbioloalveolare it descritto un caso di «intravascular bronchioloalveolar tumor» (IVBAT) del polmone in una donna di 45 anni, La presentazione clinico-radiologica ed il quadro macro-microscopico di questa rara neoplasia sono sovrapponibili a quelle segnalate in precedenza. Lo studio immunoistocbimico ed ultrastrutturale, dimostrando rispettivamente la presenza di antigene precipitante 0 Factor VIII RAG nelle cellule tumorali, e giunzioni, vescicole pinocitoticbe, lamina basale e corpi di Weibel-Palade nella proliferazione tumorale, ribadiscono entrambi la natura endoteliale della neoplasia. Sono discusse Ie diagnosi differenziali sui piano c1inico con Ie localizzazioni metastaticbe polmonari di tumori della muscolatura Iiscia e su quello istologico con I'emangioma sclerosante. Inoltre sono sottolineate Ie analogie morfologiche, istogeneticbe e di storia naturale tra I'IVBAT e neoplasie descritte recentemente e non, e variamente interpretate in sede epatica, cutanea e di tessuti molli. 292 Pilotti et al. Addendum. After submission of this report, another case of IVBAT in a 24-year-old female was observed and investigated immunocytochemically. The patient was admitted to this Institute with the diagnosis of pulmonary metastases of choriocarcinoma. However, review of the histology of the endometrial curettage material yielded the diagnosis of invasive hydatidiform mole, whereas review of the chest radiographs con- firmed the presence of pulmonary disease compatible with metastases. However, HCG serum levels were within normal limits, and curettage of the uterine cavity was negative. Chemotherapy according to CHAMOCA regimen was given; after a second unsuccessful cycle, which did not modify the chest radiology, an open-chest biopsy was performed, which permitted the final diagnosis. References 13. Katzenstein A.L.A., Gmelich J.T., Carrington C.B.: Sclerosing hemangioma of the lung. A clinicopathologic study of 51 cases. Am. J. Surg. Pathol., 4: 343-356, 1980. 14. Ludwig J., Grier M.W., Hoffman H.N., McGill D.B.: Calcified mixed malignant tumor of the liver. Arch. Pathol., 99: 162-166, 1975. 15. Mukai K., Rosai J., Burgdorf W.H.C.: Localization of factor VIII-related antigen in vascular endothelial cells usng an immunoperoxidase method. Am. J. Surg. Pathol., 4: 273-276, 1980. 16. Persaud V., Bateson E.M., Bankay C.D.: Pleural mesothelioma associated with massive hepatic calcification and unusual metastases. Cancer, 26: 920-928, 1970. 1. 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