Ultrastructural Findings in Metastatic Bronchioloalveolar Carcinoma

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1 Annals of Clinical & Laboratory Science, vol. 33, no. 3, Ultrastructural Findings in Metastatic Bronchioloalveolar Carcinoma Mary Aldrene L. Tan, Saul Teichberg, Beth Roberts, and Steven I. Hajdu Department of Pathology, North Shore University Hospital, Manhasset, New York Abstract. This study was prompted by the recent revision of the definition of bronchioloalveolar carcinoma (BAC) that defines BAC, light microscopically, as a non-invasive carcinoma. Doubt has been raised whether BACs retain certain specific microscopic features after becoming invasive or metastatic. We studied 7 cases of metastatic, non-mucinous BAC by electron microscopy. Of these cases, 5 showed Clara cell granules and 1 revealed lamellar bodies. The remaining case did not show ultrastructural features of BAC. These findings suggest that most BACs retain some of their ultrastructural features after becoming metastatic neoplasms. (received 12 May 2003; accepted 15 May 2003) Keywords: Metastatic bronchioloalveolar carcinoma, invasive bronchioloalveolar carcinoma, electron microscopy, Clara cell granules, lamellar inclusions Introduction Bronchioloalveolar carcinoma (BAC) is cytologically, histologically, ultrastructurally, and by molecular composition, a distinct pulmonary adenocarcinoma [1-7]. BAC was first described by Malassez in 1876 [8]. In 1960, Liebow [9] defined BAC as a pulmonary carcinoma that consists of cuboidal or columnar epithelial cells growing on preexisting alveolar septae [9]. For nearly 4 decades this definition received world-wide acceptance by pathologists and oncologists. In addition, it has been recognized during the past decade, more than ever before, that BAC is an unique neoplasm, with frequent multifocal and multicentric presentations and a disproportionate occurrence in young patients, particularly women and nonsmokers [10-12]. In 1999, a new World Health Organization (WHO) definition, based on light microscopic features, was introduced whereby BAC with invasion cannot be classified as BAC [13]. In 2002, we reported ultrastructural evidence that BACs retain their ultrastructural phenotypes after becoming invasive (solid) carcinomas [7]. Address correspondence to Steven I. Hajdu, M.D., 4 Forest Court, Syosset, New York, 11791, USA: tel ; fax As a further step, the present study was undertaken to investigate whether the ultrastructural features of BAC are retained in extrapulmonary metastases. Materials and Methods Seven consecutive cases of metastatic, non-mucinous BAC were selected for ultrastructural examination. After histopathologic review of all available microscopic slides, the paraffin embedded tissue blocks containing the metastatic carcinoma were identified. In 6 cases, the region of the block containing the carcinoma was excised, rehydrated, and prepared for electron microscopic study. In 1 case, simultaneously with the light microscopic diagnosis of metastatic BAC, the fresh tumor sample was fixed in 0.1 M cacodylate buffered glutaraldehyde, post-fixed in osmium-tetroxide, and prepared for ultrastructural study by standard methods. In all cases, sections (1 µm) of toluidine bluestained tissue were examined by light microscopy to identify the tumor cell populations. Appropriate areas were selected and sectioned for examination with a transmission electron microscope (JEOL JEM 100 CXII ) /03/0300/0289 $ by the Association of Clinical Scientists, Inc.

2 290 Annals of Clinical & Laboratory Science Results Of the 7 patients, 5 were women and 2 were were nonsmokers. Four patients were <50 yr old at time of diagnosis. (Table 1). The primary tumors ranged from 1 to 5 cm and were commonly situated in the upper lobes of the lungs. In 1 case, the primary neoplasm was multifocal; in another case, there was multicentric presentation in 2 lobes of both lungs. Follow-up information and pertinent clinicopathological findings are listed in Table 1. All 7 primary BACs showed 2 distinct growth patterns by light microscopy. The dominant pattern was that of a solid, poorly-differentiated, and invasive adenocarcinoma without recognizable light microscopic histologic features of BAC. However, there were substantial and readily visible areas of welldifferentiated, invasive, and non-invasive BAC with typical alveolar features, as well as atypical bronchioloalveolar hyperplasia. At sites of metastases, the neoplasms were all composed of solid nests of adenocarcinoma. In 5 cases, ultrastructural examination of the neoplastic cells showed intracytoplasmic Clara cell granules (Figs. 1 and 2). In 1 case, the neoplastic cells showed intracytoplasmic lamellar inclusions (Fig. 3). In the remaining case, the ultrastructural features of BAC were not detected. Discussion In this study, consistent with our prior study [7] and as reported by others [4,5,10-12], the patients with BAC were mostly female and were generally younger than most patients with lung cancer. The commonly observed multifocal and multicentric presentation of BACs [11-12] was noted in 2 of our patients. Peripheral location of the tumors, modest size, and predilection for the upper lobes are well known features of BACs. All in all, the Table 1. Summary of clinicopathologic findings in 7 cases of bronchioloalveolar carcinoma of the lung. Case #, age, Smoker/ Site, size (cm), Light micro- Metastases Electron micro- Follow-up gender non-smoker specimen scopic findings scopic findings information (1) 44 yr, M smoker RLL,* 3x3, pleural invasion mediastinal & right lamellar died in 15 mo biopsy scalene lymph nodes inclusions (2) 38 yr, F smoker LUL*, 4x3, pleural invasion brain none no follow-up wedge biopsy vascular invasion (3) 49 yr, F non-smoker RUL, 5x4, pleural invasion peribronchial lymph Clara cell Recurrence in brain; lobectomy vascular invasion nodes & brain granules died in 6 mo (4) 69 yr, M former LUL, 4x4, invasive 1 mediastinal lymph Clara cell NED 9 mo smoker lobectomy vascular invasion node granules (5) 47 yr, F smoker LUL, 2x1, invasive 2 hilar lymph nodes Clara cell NED 6 mo lobectomy granules (6) 66 yr, F non-smoker RLL, multi- invasive 2 mediastinal lymph Clara cell NED 11 mo focal, wedge pleural invasion nodes granules biopsy (7) 73 yr, F smoker LLL, 3x2, invasive pattern 2 hilar lymph nodes Clara cell no follow-up lobectomy granules RUL, 1x0.5 non-invasive wedge biopsy * RLL, right lower lobe; LUL, left upper lobe; NED, no evident disease.

3 EM of metastatic bronchioloalveolar carcinoma 291 Fig. 1 (Case 4). Electron photomicrograph of portions of malignant tumor cells from a bronchioloalveolar carcinoma that had metastasized to a mediastinal lymph node, from material retrieved from paraffin embedded material. The tumor cell cytoplasm contains numerous Clara cell granules (G). Short microvilli are evident at the cell surface. A nucleus is at N. (magnification x 15,550). clinical profile and radiologic presentation of the cases in this study, without pathologic examination, would favor BAC. According to the WHO s recently proposed light microscopic classification of lung tumors [13], none of the tumors in this study would be classified as BAC, because of the presence of microscopic invasion. We showed earlier [7] that neoplastic cells of BAC retain their ultrastructural BAC phenotype after becoming invasive. The results of this study add to that observation by showing that BACs retain some ultrastructural features in neoplastic cells even in metastases. Our findings support the observations made >20 years ago [14,15] that neoplastic cells of metastatic BACs retain specific cytologic features. Generous sampling and diligent ultrastructural examination are prerequisite for finding lamellar membraneous inclusion bodies, indicative of type II pneumocytes differentiation, and Clara cell granules. This is particularly true as the tumor cells spread from the primary site to adjacent and distant tissues by metastasis. The matter is complicated by the fact that lammeliform inclusions are unlikely to be demonstrable in paraffin-retrieved material because the organic solvents extract lipids during processing of tissues. In summary, we have found that metastatic malignant cells of invasive BACs, although not

4 292 Annals of Clinical & Laboratory Science Fig. 2 (Case 7). Electron photomicrograph from a bronchioloalveolar carcinoma cell in a hilar lymph node, from material prepared from paraffin retrieved material as in Fig. 1. The tumor cell cytoplasm contains several Clara cell granules (G). Nucleus is at N; nucleolus is at NU. (magnification x 17,000). recognizable as cells derived from BAC by light microscopy, retain some of the ultrastructural features of well-differentiated non-invasive BAC. Therefore, the idea [13] that loss of the characteristic light microscopic features of BAC when they become invasive (poorly differentiated or dedifferentiated ) indicates that the neoplasm is not BAC should be reconsidered. References 1. Hajdu SI. Adenocarcinoma of the lung, broncho-alveolar type. In Anatomic Pathology Check Sample Chicago, ASCP 1977;II-10: Lozowski W, Hajdu SI. Cytology of bronchioloalveolar carcinoma. Acta Cytologica 1987;31: Goodwin LO, Mason JM, Hajdu SI. Gene expression patterns of paired bronchioloalveolar carcinoma and benign lung tissue. Ann Clin Lab Sci 2001;31: Koga T, Hashimoto S, Sugio K, Yoshino I, Mojta Hedzadeh S, Matsuo Y, Yonemitsu Y, Sugimachi K, Sueishi K. Clinicopathological and molecular evidence indicating the independence of bronchioloalveolar components from other subtypes of human peripheral lung adenocarcinoma. Clin Cancer Res 2001;7: Wong MP, Fung L-F, Wang E, Chow W-S, Chiu S-W, Lam W-K, Ho K-K, Ma E S K, Wan T S K, Chung L-P. Chromosomal aberrations of primary lung adenocarcinomas in nonsmokers. Cancer 2003;97:

5 EM of metastatic bronchioloalveolar carcinoma 293 Fig. 3 (Case 1). Electron photomicrograph of a group of malignant tumor cells, showing Type II pneumocyte-like features, from a bronchioloalveolar carcinoma metastasized to a scalene lymph node that was well preserved by primary fixation in glutaraldehyde. Note the numerous swirled membranous, lamellar inclusions (arrows) in the cytoplasm of the tumor cells and the cell surface lined by short, widely spaced microvilli (MV). A large prominent ribbon-like nucleolus (NU) is found in some profiles of tumor cell nuclei (N). (magnification x 4,500; inset x 15,000) 6. Eimoto T, Teshima K, Shirakusa T, Kikuchi M. Ultrastructure of well-differentiated adenocarcinoma of the lung with special reference to bronchioloalveolar carcinoma. Ultrastruct Pathol 1985;8: Darvishian F, Roberts B, Teichberg S, Hajdu S. Ultrastructural comparison of alveolar and solid areas of bronchioloalveolar carcinoma. Ann Clin Lab Sci 2002; 32: Malassez L. Examen histologique d un cas de cancer encephaloide du poumon (epithelioma), Arch Physiol Norm Pathol 1876;3: Liebow AA. Bronchiolo-alveolar carcinoma. Adv Intern Med 1960;10: Auerbach O, Garfinkel L. The changing pattern of lung carcinoma. Cancer 1991;68: Barsky SH, Cameron R, Osann KE, Tomita D, Homes

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