IMMUNOHISTOCHEMICAL DETECTION OF CERTAIN TUMOR MARKERS IN SHEEP LUNG TISSUES AFFECTED BY OVINE PULMONARY ADENOCARCINOMA
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Date
2014-11
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA
Abstract
ABSTRACT:
In India sheep farming plays an important role as sustainable source of livelihood for the rural people. Several infectious diseases caused by viruses, bacteria and parasites are prevalent in sheep flocks, resulting in economic losses. In general, tumors are rare in domestic livestock, except small ruminants. In small ruminants two neoplastic diseases - ovine pulmonary adenocarcinoma (OPA) and enzootic nasal adenocarcinoma (ENA) are common. OPA is a natural lung cancer of the sheep caused by Jaagsiekte Sheep Retro Virus (JSRV). Diagnosis of OPA mainly depends on clinical and pathological investigations. Due to the neoplastic nature of lesions in OPA, detection of certain tumor markers by immunohistochemistry helps in the characterization of the neoplastic transformation of the cells in OPA, that will further contribute to understand the pathogenesis of OPA. Successful use of the tumor markers in characterizing the neoplastic properties of the alveolar epithelial cells in OPA will be helpful in its evaluation and diagnosis and to provide further insights into the carcinogenetic mechanisms. Perusal of the available literature revealed a few reports on expression and evaluation of tumor markers in ovine pulmonary adenocarcinoma. Hence, keeping this in view, the present investigation was carried out to study the tissue changes, to determine the cell proliferation activity by AgNOR method and to detect the expression of proliferative and certain other tumor markers by immunohistochemistry in OPA affected lungs of sheep.The materials for the present study consisted of tissue samples of sheep lungs collected from suspected cases of OPA from various slaughter houses located in different places and private organised farms and during postmortem examinations carried out at NTR College of Veterinary Science, Gannavaram. The duration of the study lasted from August 2013 to July 2014. On histopathological and immunohistochemical examinations, 17 (16.2%) lung samples revealed definitive lesions of ovine pulmonary adenocarcinoma in a total of 105 sheep lung samples collected.Grossly, the lung samples from 17 cases of OPA revealed diffuse areas of consolidation or clearly demarcated tumor nodules within the bulk of some or all of the lung lobes. Mostly apical and diaphragmatic lobes showed the lesions and the affected areas of the lungs were solid, hard and light grey in color. On cut surface, the consolidated areas or tumor nodules had a granular appearance with elevated foci along with exudation of clear to milky, opaque, or sero mucoid secretions. The gross lesions noticed in all the cases were similar to classical form of OPA. Mediastinal and tracheobronchial lymph nodes were enlarged and edematous and in one animal gross metastasis was detected. Histologically, all the 17 OPA affected animals showed similar changes with slight variation in the lungs. Sections from the areas of consolidation and tumor nodules of lungs revealed non encapsulated neoplastic areas of varying sizes, composed of cuboidal to columnar epithelium lining the alveolar and bronchiolar walls, and giving papillary or acinar pattern. There were mainly two types of growth patterns of tumor cells viz. papillary and acinar or glandular. Papillary pattern was characterized by the papillary projections protruding above the epithelial layer into the alveolar spaces. The acinar or glandular type of growth pattern was composed of structures resembling acini. The acini were composed of mostly a single layer of cuboidal epithelial cells, separated by interstitial stroma. In addition, bronchioloalveolar growth pattern, characterized by the expansion of neoplastic cells following the alveolar septa, without destruction of alveolar architecture was noticed in some. One or more types of growth patterns were present in the same neoplastic lung tissue.Bronchiolar epithelial cell hyperplasia and papillary ingrowths of the lining epithelium into the lumen was evident in some cases. Alveoli adjacent to the neoplastic areas contained alveolar macrophages with foamy cytoplasm and infiltration of neutrophils was found when there was a secondary bacterial infection. Papillary projections of alveolar epithelial cells were supported by thin connective tissue stalk. The tumor cells were well differentiated with a very few mitotic figures. Individual cells were cuboidal to columnar, with basally located round to oval nuclei with a central nucleolus, and the cytoplasm was vacuolated in some areas. In advanced cases of OPA, proliferation of connective tissue was more prominent. In three cases of OPA, myxomatous nodules of various sizes were present in the tumor stroma and around the adjacent bronchioles. In three cases metastasis of lung tumor was confirmed by microscopical examination of the sections of the mediastinal lymph node. Immunostaining for two viral proteins viz. JSRV-CA and MA was performed on 17 OPA lung samples. Strong positive reaction for JSRV-CA and MA was seen in all the neoplastic cells, alveolar macrophages and desquamated tumor cells and in a few lymphocytes and plasma cells in between the neoplastic acini. Neoplastic epithelial cells in the metastatic lymph nodes also showed positive expression of JSRV- CA and MA. Immunohistochemcal staining for various tumor markers viz. cytokeratin, surfactant proteins, c-Myc, p53 and Ki67 was carried out in OPA lungs. Strong expression of cytokeratin was seen in the neoplastic cells of alveolar and bronchiolar epithelial cell proliferations confirming the epithelial origin of the tumor. Metastatic tumor cells in lymph nodes also exhibited strong expression of cytokeratin. Positive expression of surfactant proteins (SP-A, SP-B, SP-C) were noticed in the tumor cells in the neoplastic areas of lung, that further characterised the neoplastic cells as alveolar type II pneumocytes. Strong nuclear immunolabelling for oncoproteins c-Myc and p53 was also observed in the neoplastic cells of papillary projections of alveolar epithelium in OPA lung sections. The proliferative activity of tumor cells was determined by Ki67 and AgNOR staining. The mean value of Ki67 index and AgNORs was increased in the sections of lung tumor than the normal lung sections.
The present study characterized the tissue changes in ovine pulmonary adenocarcinoma and immunohistochemical studies have confirmed the presence of JSRV infection and characterized the neoplastic cells of OPA. Detection and evaluation of certain tumor markers carried out in the present study by immunostaining can further aid in the prognostic and diagnostic aspects of OPA and for understanding the carcinogenetic mechanisms of OPA.
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