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Year : 2008  |  Volume : 51  |  Issue : 3  |  Page : 329-336
Spectrum of pulmonary adenocarcinoma with ultrastructural correlation: An autopsy study from northern India

1 Department of Histopathology, Post Graduate Institute of Medical Education and Research, (PGIMER), Chandigarh, India
2 Department of Pulmonary Medicine, Post Graduate Institute of Medical Education and Research, (PGIMER), Chandigarh, India

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Background: In the present study, we have evaluated the use of electron microscopy in subtyping pulmonary adenocarcinomas, comparing the ultrastructural findings with the diagnosis rendered by light microscopy. Materials and Methods: The gross and histologic features of 16 autopsy cases of pulmonary adenocarcinoma were analyzed and compared with electron microscopic features. The cytologic phenotypes of these cases of well-differentiated pulmonary adenocarcinoma were determined by electron microscopic examination. More than 200 cells in each case were examined, and the tumors were classified according to the predominant feature noted. Results: Eight cases were of Clara cell origin and one case each of type II pneumocyte and bronchial surface cell type. The remaining 6 cases lacked definite discernible features of differentiation towards any specific cell type, other than presence of small nuclear clefts in occasional nuclei. Tumors with Clara cell differentiation were low cuboidal with apical snouts. Type II pneumocyte tumor failed to reveal any characteristic definable as light microscopic feature. Conclusion: Ultrastructural examination is the only definite means of identification of various cell types in the respiratory epithelium and hence forms an invaluable tool in classification of pulmonary adenocarcinoma.

Keywords: Bronchioloalveolar carcinoma, Clara cell, electron microscopy, lung adenocarcinoma, type II pneumocyte, ultrastructure

How to cite this article:
Gupta K, Joshi K, Jindal SK, Rayat CS. Spectrum of pulmonary adenocarcinoma with ultrastructural correlation: An autopsy study from northern India. Indian J Pathol Microbiol 2008;51:329-36

How to cite this URL:
Gupta K, Joshi K, Jindal SK, Rayat CS. Spectrum of pulmonary adenocarcinoma with ultrastructural correlation: An autopsy study from northern India. Indian J Pathol Microbiol [serial online] 2008 [cited 2019 Apr 24];51:329-36. Available from:

   Introduction Top

Lung cancer is currently the most frequently diagnosed major cancer and the most common cause of cancer mortality in males, worldwide. [1] This is largely due to the effects of cigarette smoking. Adenocarcinoma is the predominant histological subtype of lung cancer in many countries. [2] It is the most prevalent form of lung cancer in younger males (<50 years of age) and in women of all ages. [3] The classification is based on the histological characteristics of tumors seen in surgical or needle biopsy, and is primarily based on light microscopy. Immunohistochemistry and electron microscopic findings provide important useful features that are helpful in classification of cases with overlapping microscopic findings.

Difficulties in adenocarcinoma subclassification arise due to the fact that this group is highly heterogeneous histologically, with only a minority of cases showing a pure histological pattern. Studies have documented that these neoplasms differ in cell type, degree of differentiation, growth pattern, and immunohistochemical staining characteristics, with regional differences in any given neoplasm. [4]

Although adenocarcinoma is subdivided histologically into acinar (tubular), papillary, bronchioloalveolar (BAC), solid type with mucin, and mixed subtypes by WHO classification, [1],[5] it differs cytologically. The adenocarcinoma cells are supposed to differentiate towards a variety of epithelial cells seen in the bronchus and bronchioloalveoli. It is subclassified into six cytologic subtypes: [6]

  1. Bronchial cell surface type with little or no mucus
  2. Goblet cell type
  3. Bronchial gland cell type
  4. Clara cell type
  5. Type II alveolar epithelial cell type
  6. Mixed cell type

Besides the presence of characteristic cytological features, each of the above subtypes exhibits unique ultrastructural features.

This study presents the gross microscopic findings and detailed ultrastructural features of 16 autopsy cases of lung adenocarcinoma and compares the diagnosis based on light microscopic features with that based on ultrastructural findings. Also, the ultrastructural findings in a given case were compared with light microscopy both in the well-differentiated and poorly differentiated areas to document the presence of regional differences.

   Materials and Methods Top

Retrospective study from archival material of our department was carried out on 16 autopsy cases of lung adenocarcinoma over a span of 15 years. The tissue was fixed in 10% buffered formalin, processed routinely, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H and E) and Alcian blue with periodic acid-Schiff (AB-PAS). For ultrastructural examination, representative section was taken from paraffin block, put in xylene for 30 minutes, with frequent changes to remove paraffin at room temperature (RT). This was followed by putting the sections for 20 minutes each in graded alcohol - absolute, 90%, 70%, and 50% - to bring the section to water and then finally putting them in Sorensen's buffer for 20 minutes. Later the sections were post-fixed in 1% osmium tetroxide and embedded in epoxy resin (Tab 812). Ultra-thin sections selected from the representative areas were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with Zeiss 906 electron micrmoscope.

Observations and results

Clinical details and gross findings

The age ranged from 30 to 80 years, with a male-to-female ratio of 2:1 [Table 1]. On gross examination the lungs were heavy, with weight ranging from 975 to 1600 g [Table 1]. The tumor was present bilaterally as multiple small grayish white nodules [Figure 1] randomly distributed throughout the lung parenchyma in 6 cases. Eight cases revealed presence of large necrotic tumor located in the right lung, with 4 cases showing the growth in the right main bronchus. One case revealed a necrotic mass measuring 8 6 cm located in the upper lobe of left lung. The tumor was present as a subpleural nodule in the right lung, measuring 2.5 cm in size, with an overlying scar and widespread tumor emboli on microscopy in one of the cases.

Light microscopic findings [Table 2]

Histology of the tumors showed features of adenocarcinoma (14 cases) and adenosquamous carcinoma (2 cases). Bronchioloalveolar pattern was noted in 5 cases, with neoplastic cells creeping along the preexisting alveolar septae [Figure 2A]. Predominance of papillary architecture was noted in one case [Figure 2B]. Rest of the cases revealed variable admixture of low columnar-to-cylindrical cells arranged in acini, tubules, and varying-sized glands [Figure 2C]. Mixed pattern with equal admixture of the above types was noted in 3 cases. In one case, the tumor revealed large areas with poorly differentiated morphology, besides the presence of bronchioloalveolar pattern. The poorly differentiated areas comprised of cells present discretely with bizarre morphology, including tumor giant cells also. Adjoining areas in this case also revealed presence of atypical adenomatous hyperplasia (AAH) [Figure 2D]. Mucin production was noted in 10 cases, further confirmed by AB-PAS stain [Figure 3A]. The nuclei were moderately to severely hyperchromatic, vesicular, and pleomorphic. Multinucleated tumor giant cells were occasionally observed. Mitotic figures were seen with focal areas showing brisk mitotic activity. The cell type based on light microscopy was low columnar with apical snouts in 6 cases [Figure 3B], and tall columnar with no definite apical snouts in 7 cases [Figure 3C]. In 2 cases, the morphology was like the bronchial surface cell type with little or no mucus production. The cells were tall columnar, with morphology resembling the ciliated columnar cells although no cilia were visible. One case showed morphology similar to the bronchial gland, with cuboidal-to-polygonal cells arranged in acini, tubules, and solid nests [Figure 3D].

The 'adeno' component in 2 cases of adenosquamous carcinoma was composed of low columnar cells with morphology resembling Clara cells on light microscopy in one case, and the other case showed tall columnar cells.

Electron microscopic findings [Table 2]

Ultrastructurally, the nuclei were large, with irregular folded membrane and prominent nucleoli. The features of Clara cell differentiation seen in 8 cases included cuboidal-to-columnar cells, often with prominent apical snouting and presence of intranuclear tubular inclusions, many showing branching and others traversing the entire diameter of the nucleus [Figure 4A]. In addition, all these 8 cases showed round-to-oval or irregular electron-dense granules ranging in size from 400 to 600 nm within the cytoplasm [Figure 4B]. Besides these, the cytoplasm also contained usual cell organelles-like ribosomes and endoplasmic reticulum. Tonofilaments were absent.

One case revealed features of type II alveolar epithelial cell with numerous lamellar inclusion bodies and intermediate forms between multivesicular and lamellar bodies present within the cytoplasm [Figure 4C]. These measured 400 to 600 nm in diameter, with evenly spaced, curved, membrane-like lamellar structures with 10 to 16 nm periodicity. These bodies were distinctly different from altered membranes present in swollen mitochondria. These ultrastructural features are similar to those seen in type II pneumocyte of normal lung tissue.

One of the cases with tall columnar cell morphology and BAC pattern showed abundance of mitochondria and endoplasmic reticulum, along with few mucin vacuoles within the cytoplasm, which are consistent with features of bronchial surface cell type of cell [Figure 4D]. However, no well-defined basal bodies indicative of originating from ciliated bronchial epithelial cells were identified. The rest of the 6 cases with variable light microscopic pattern were not classifiable into a definite cell type as the electron microscopic features of a particular cell type were very occasionally observed.

The 'adeno' component in 1 of the 2 cases of adenosquamous carcinoma showed features of Clara cell differentiation with intranuclear tubular inclusions and electron-dense granules. In the other case, the neoplastic cells of 'adeno' component revealed very occasional small intranuclear clefts. The other 2 cases with cytologic appearance of bronchial surface cell type on light microscopy, showed on electron microscopy only very occasional tubular intranuclear inclusions and electron-dense granules in the cytoplasm - the features being relatively nondescript to fall into a definite category of Clara cell type.

The adenocarcinoma with a predominant papillary pattern of growth also showed well-defined and deep intranuclear tubular inclusions confirming to the features of Clara cell differentiation, ultrastructurally.

Ultrastructural examination of 5 cases with bronchioloalveolar pattern showed features confirming to Clara cells in 2 cases, and features of type II alveolar epithelial cell with presence of lamellar bodies and that of bronchial surface cell type in 1 case each respectively. These were the only 2 cases belonging to each of this category in the present study. The ultrastructural features in the fifth case with BAC pattern showed very occasional nuclear clefts only.

A single case which demonstrated presence of both poorly differentiated and well-differentiated areas showed features similar to the Clara cell differentiation in sections taken from both the bronchioloalveolar and poorly differentiated areas and also from the metastatic site.

The mucin production by the neoplastic cells seen on light microscopy and confirmed by PAS-AB stain in 10 cases could not be substantiated on electron microscopy as mucin vacuoles in the cytoplasm were not well evident.

No ciliated cells or basal bodies were noted ultrastructurally. The stroma of the tumors was rich in elastin and collagen.

   Discussion Top

Primary lung adenocarcinomas are highly heterogeneous histologically, with only a minority of cases showing a pure histological pattern. Mixed pattern adenocarcinomas are more common than tumors showing a single pattern (e.g., acinar, papillary, BAC, and solid adenocarcinoma with mucin production). [1] The adenocarcinomas are also heterogeneous with respect to their progenitor cells. [7] The subtype bronchioloalveolar carcinoma is considered to originate from different progenitor cells. With reference to cytological features of tumor cells, Shimosato [8] et al. subclassified primary lung adenocarcinomas into 5 subgroups, namely, bronchial cell surface type, goblet cell, bronchial gland cell, Clara cell, and type II pneumocyte cell. All of the five cell types are seen in peripheral adenocarcinomas. Clara cell type is the most frequently seen, followed by bronchial surface cell (non-goblet) type. Type II pneumocyte cell type is extremely rare. [8] Hirsch et al. [9] in their study of 200 cases of malignant lung tumors have documented tumors with morphological features of more than one cell type in 24 patients, thereby concluding that morphological heterogeneity is a considerable problem in classification of malignant lung tumors. In the present study, ultrastructurally, tumors of Clara cell origin were the commonest, followed by both type II pneumocyte and bronchial surface cell type. We found cytologic differentiation towards Clara cell in 6 cases based on light microscopic findings; however, on ultrastructural examination, 8 cases belonged to this category. On light microscopy, 7 cases had tall columnar type morphology without the presence of apical snouts. Two cases cytologically belonged to bronchial surface cell type with mucin production, based on light microscopy. No cilia were identified in these. However, in these 2 cases, we were unable to find any definite ultrastructural features to justify placing them in any definite category.

The neoplastic cells of bronchioloalveolar carcinoma have a remarkable potential for divergent differentiation. This tumor may express alveolar and bronchiolar differentiation within a given cell. [10] The 1999 and 2004 WHO classifications are more restrictive in the definition of BAC; this category includes only noninvasive tumors with lepidic spread. This present definition excludes cases with stromal, vascular, or pleural invasion. [1],[5],[11] Tumors showing these features are classified as adenocarcinoma mixed type with predominant BAC pattern. Most tumors described as BAC in the past would now belong to this category. In a study done in 1986, Clayton et al. [12] have concluded that electron microscopy is useful in confirming and/or establishing the Clara cell or type II pneumocyte as origin in tumors with BAC pattern. Five cases which had BAC pattern on light microscopy showed features of Clara cell in two cases, type II pneumocyte and bronchial surface cell type in one case each.

Study by Darvishian et al. [13] on 7 cases of nonmucinous BAC revealed that the neoplastic cells in BAC retain their ultrastructural phenotypes after becoming an invasive carcinoma with loss of alveolar differentiation. Another study by Tan et al. [14] suggested that most BACs retain their ultrastructural features after becoming metastatic neoplasm. In this study, in 1 case of BAC admixed with a poorly differentiated component, the ultrastructural phenotype was same in the tissue taken from both the BAC and the poorly differentiated component and also from the metastatic site. More recent studies [15] have suggested different ultrastructural phenotypes in a BAC with both poorly differentiated and invasive component as compared to areas with classic BAC pattern.

Auerbach et al. [16] in a study done in 1982 have evaluated the use of electron microscopy by comparing the light microscopic findings with electron microscopic features in well- differentiated, moderately differentiated, and poorly differentiated lung carcinoma, including squamous cell, small cell, carcinoid, and large cell type. They have emphasized the advantages of resolution that ultrastructural analysis offers in classification of pulmonary adenocarcinomas. The sample size for electron microscopy remains a limiting factor in tumors unclassifiable after ultrastructural examination. In the present study, ultrastructurally, 8 cases showed features of Clara cell, 1 case revealed type II pneumocyte features, and 1 case had abundant cellular organelles-like endoplasmic reticulum and mitochondria, thus belonging to the bronchial surface cell type. The rest of the 6 cases showed variable ultrastructural features, making it difficult to place them into a definite cellular type.

Mucin production identified in 10 cases both on light microscopy and by PAS-AB staining, could not be substantiated ultrastructurally. This is likely to be attributed to the fact that tissue for electron microscopy was taken from formalin-fixed material and hence preservation was compromised to some extent. The cytologic cell subtypes associated with conspicuous mucin production are bronchial surface cell type, bronchial gland type, and goblet cell type. [6] The Clara cell and type II pneumocyte types are put into the category of nonmucinous tumors. [12] In our study, 4 cases with Clara cell differentiation on light microscopy revealed presence of mucinous vacuoles within them on PAS-AB stain, thus supporting the concept of tumor heterogeneity within pulmonary adenocarcinoma. Moreover, it is documented that many adenocarcinomas less than 1.5 cm in diameter are composed of tumor cells of a single-cell type, whereas larger tumors often consist of tumor cells of two or more-cell types because of metaplastic changes in tumor cells from one cell type to another, such as Clara cell type to mucus-producing cells, [7],[17] which are complicated by anaplastic changes in tumor cells.

In addition to electron microscopy, immunohistochemistry also has an important role to play in the classification of pulmonary adenocarcinoma. Thyroid transcription factor-1 (TTF-1) has a potential role, with recent studies documenting its expression in 72% of adenocarcinomas and 89% of small cell carcinomas, and a very low frequency in squamous carcinomas and large cell carcinomas. [18] Furthermore, Amin MB et al., [19] in a study, have shown that TTF-1, cytokeratin 7 (CK 7) positivity and CK20 negativity helps in picking up cases of adenocarcinoma with micropapillary pattern. In a study by Yatabe Y et al., TTF-1-positive adenocarcinomas have been shown to have a different molecular pathway of pathogenesis as compared to TTF-1-negative adenocarcinomas. [20]

In conclusion, the study emphasizes the importance of electron microscopy in classifying lung adenocarcinomas. The direction of differentiation is assumed by light microscopic observation and is confirmed by ultrastructural findings.

   References Top

1.Brambilla E, Travis WD, Colby TV, Corrin B, Shimosato Y. The new World Health Organization classification of lung tumors. Eur Respir J 2001;18:1059-68.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Travis WD, Travis LB, Devesa SS. Lung cancer. Cancer 1995;75:191-202.  Back to cited text no. 2  [PUBMED]  
3.Ramalingam S, Pawlish K, Gadgeel S, Demers R, Kalemkerian GF. Lung cancer in young patients: Analysis of a surveillance, epidemiology, and end results database. J Clin Oncol 1998;16:651-7.  Back to cited text no. 3    
4.Sarantopoulos GP, Gui D, Shintaku P, Hong L, Wang YY, Yap CS, Fishbein MC. Immunohistochemical analysis of Lung carcinomas with pure or partial bronchioloalveolar differentiation. Arch Pathol Lab Med 2004;128:406-14.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Travis WD, Muller-Hermelink HK, Harris CC, editors. The World health Organization Classification of tumors. Pathology and genetics of tumors of the lung, pleura, thymus and heart. Lyon: IARC Press; 2004.  Back to cited text no. 5    
6.Shimosato Y. Pulmonary neoplasms In : Sternberg SS, editor. Diagnostic surgical pathology. 3 rd ed, Vol I. Philadelphia, PA: Lippincott Williams and Wilkins; 1999. p. 1069-115.  Back to cited text no. 6    
7.The World health Organization-Histological typing of lung tumors, 2 nd ed. Geneva: World Health Organization; 1981.  Back to cited text no. 7    
8.Shimosato Y, Kodama T, Kameya T. Morphogenesis of peripheral type adenocarcinoma of the lung. In : Shimosato Y, Melamed MR, Nettesheim P, editors. Morphogenesis of lung cancer. Vol I. Boca Raton, FL: CRC Press; 1982. p. 65-89.  Back to cited text no. 8    
9.Hirsch FR, Ottesen G, Jan PA, Olsen J. Tumor heterogeneity in lung cancer based on light microscopic features. Virchow Arch 1983;402:147-53.  Back to cited text no. 9    
10.Espinoza CG, Balis JU, Saba SR, Paciga JE, Shelley SA. Ultrastructural and immunohistochemical studies of Bronchioloalveolar carcinoma. Cancer 1984;54:2182-9.  Back to cited text no. 10  [PUBMED]  
11.Franklin WA. Diagnosis of lung cancer: Pathology of invasive and non-invasive neoplasia. Chest 2000;117:808-98.  Back to cited text no. 11    
12.Clayton F. Bronchioloalveolar carcinomas: Cell types, patterns of growth, and prognostic correlates. Cancer 1986;57:1555-64.  Back to cited text no. 12  [PUBMED]  
13.Darvishian F, Roberts B, Teichberg S, Steven HI. Ultrastructural comparison of "alveolar" and "solid" areas in bronchioloalveolar carcinoma. Ann Clin Lab Sci 2002;32:225-30.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Tan MAL, Teichberg S, Roberts B, Hajdu SI. Ultrastructureal findings in metastatic bronchioloalveolar carcinoma. Ann Clin Lab Sci 2003;33:289-94.  Back to cited text no. 14    
15.Hadju SI. The story of Bronchioloalveolar carcinoma. Ann Clin Lab Sci 2005;35:336-38.  Back to cited text no. 15    
16.Aurerbach O, Frasca JM, Parks VR, Carter HW. A comparison of World health organization (WHO) classification of lung tumors by light and electron microscopy. Cancer 1982;50:2079-88.  Back to cited text no. 16    
17.Shimosato Y. Lung cancer: Its histogenesis, differentiation and prognostic factors. Trans Soc Pathol Jpn 1983;72:29-57.  Back to cited text no. 17    
18.Kaufmann O, Dietel M. Thyroid transcription factor-1 is the superior immunohistochemical marker for pulmonary adenocarcinomas and large cell carcinomas compared to surfactant proteins A and B. Histopathology 2000;36:8-16.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]
19.Amin MB, Tamboli P, Merchant SH, Ordonez NG, Ro J, Ayala AG, et al . Micropapillary component in lung adenocarcinoma: A distinctive histologic feature with possible prognostic significance. Am J Surg Pathol 2002;26:358-64.  Back to cited text no. 19    
20.Yatabe Y, Mitsudomi T, Takahashi T. TTF-1 expression in pulmonary adenocarcinomas. Am J Surg Pathol 2002;26:767-73.  Back to cited text no. 20  [PUBMED]  [FULLTEXT]

Correspondence Address:
Kirti Gupta
Department of Pathology, PGIMER, Chandigarh - 160 012
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.42505

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  [Figure 1], [Figure 2A], [Figure 2B], [Figure 2C], [Figure 2D], [Figure 3A], [Figure 3B], [Figure 3C], [Figure 3D], [Figure 4A], [Figure 4B], [Figure 4C], [Figure 4D]

  [Table 1], [Table 2]


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