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Determination of Proliferative Activity of Neoplastic Cells by Ki67 and AgNOR Staining in Ovine Pulmonary Adenocarcinoma Affected Lungs

Betala Baby Manasa Vemuri Rama Devi Kokila Satheesh Kommalapati Lakshmi Kavitha
Vol 8(1), 244-250

Ovine Pulmonary Adenocarcinoma (OPA) is a natural lung cancer of the sheep caused by Jaagsiekte Sheep Retrovirus (JSRV). In the present study, 17 OPA affected lung samples were subjected to histopathological, histochemical and immunohistochemical studies. Grossly, lungs revealed diffuse areas of consolidation or tumor nodules and histologically lung sections showed proliferation of alveolar epithelial cells that were arranged in papillary or acinar pattern. The lung sections were further subjected to immunohistochemical staining for cytokeratin and Ki-67. The proliferative activity of tumor cells was determined by Ki67 and AgNor staining and the mean value of Ki-67 index was 9.53 + 1.88. The mean number of AgNORs for all the cases ranged from 6.5 to 11.9 with a mean + standard deviation values of 9.2 + 1.2. The mean values of Ki-67 index and AgNOR staining were increased in the sections of lung tumor than the normal lung sections indicating the increased proliferative activity of lung tumor epithelial cells.

Keywords : AgNOR Staining Immunohistochemistry Ki67 Lungs Ovine Pulmonary Adenocarcinoma Sheep


Ovine Pulmonary Adenocarcinoma (OPA) is a natural lung cancer of the sheep caused by Jaagsiekte Sheep Retrovirus (JSRV). The tumor originates from alveolar type II pneumocytes and rarely from non-ciliated bronchiolar (Clara) cells. It is characterized by papillary projections of cuboidal to low columnar neoplastic cells in the lumen of the alveoli and bronchioles in lungs (Azizi et al., 2014). An experimental model of lung injury has shown that an increase in dividing alveolar cells associated with lung damage increased the susceptibility of target cells to JSRV infection in adults (Murgia et al., 2011); however, how the tumors are initiated in naturally infected sheep is unknown. The present work was carried out to study the proliferative activity of the neoplastic cells by immunostaining with Ki67 and AgNOR staining in natural cases of ovine pulmonary adenocarcinoma.

The materials for the present study consisted of 17 lung samples that were previously confirmed positive for OPA (Manasa et al., 2015). The paraffin tissue sections were subjected to H&E staining and  immunohistochemical staining with antibodies to multi-cytokeratin (Leica), and Ki67 (Bio Genex)  by using Super sensitive polymer-HRP detection kit (Bio Genex) and modified silver colloid staining for AgNOR’s (Kishnamurthi and Paliwal, 1998). Grossly lungs revealed the presence of diffuse consolidation and nodular areas with hard and firm consistency (Fig. 1).

Fig. 1: OPA-Lung- showing the diffusely scattered consolidated areas on the apical lobe

Sections from the areas of consolidation and tumor nodules of OPA lungs revealed proliferation of alveolar epithelial cells arranged in papillary or acinar pattern. The papillary projections protruded above the epithelial layer into the alveolar spaces (Fig. 2). Proliferations of bronchiolar epithelial cells results in papillary ingrowths of lining epithelium in to the lumen of bronchioles were evident. In the present study, the OPA lung sections were labeled with antibody against multi-cytokeratin that was strongly expressed by the neoplastic cells confirming the epithelial origin of the tumor cells. The positive immunostaining was identified by the presence of brown diffuse cytoplasmic staining and strong expression of cytokeratin was seen in the neoplastic cells of alveolar and bronchiolar epithelial cell proliferations, compared to that of normal epithelial cells of lung.

 Fig. 2: OPA-Lung- Showing papillary projections protruding above the epithelial layer into the alveolar spacing.

The higher expression of cytokeratin protein in epithelial cells of lung tissues indicates the epithelial origin of the neoplastic cells in OPA affected lungs (Fig. 3).

 Fig. 3: OPA-Lungs: Immunostaining for Multi cytokeratin: showing diffuse cytoplasmic staining in the neoplastic epithelial cells

These findings were in consistent with the previous report of Kycko and Reichert (2012) who stated that cytokeratin 19 was expressed in the cytoplasm of epithelial cells of bronchioles in non-neoplastic lung sections, as well as epithelial cells of bronchioles and neoplastic cells in lung sections of OPA affected sheep. In the present study, the metastatic tumor cells in lymph nodes also exhibited strong expression of cytokeratin, confirming the epithelial origin of the tumor (Fig. 4, 5, 6).




Fig. 4: OPA- lymph node: Gross appearance of mediastinal lymph node showing metastasis. Clearly demarcated greyish round tumor mass on the posterior end of the lymph node.  Fig. 5: OPA- lymphnode Note the neoplastic area containing with acinar or tubular structures displacing the normal lymphoid tissue. H&E x100

To determine the proliferative activity of the tumor cells, OPA lung sections were subjected to   immunostaining with antibody against Ki67 protein and AgNOR staining. The proliferative alveolar and bronchiolar epithelial cells showed nuclear immunolabelling for Ki67 (Fig.7).

Fig. 6: OPA- OPA-Lymph node: Immunostaining – Multi cytokeratin – Note diffuse cytoplasmic expression in the neoplastic epithelial cells in the metastatic lymph node Fig.7: OPA-Lung: Immunostaining for KI67: showing positive nuclei in the neoplastic cells of alveolar epithelial projections

The Ki67 index was calculated as the mean of positively labelled nuclei in 10 high-power fields and the mean value of Ki67 index was 9.53 +  1.88. The Ki67 index for the normal lung was 0.45 + 0.14 .The number of Ki67 positive nuclei of tumor cells varied in different areas of the section with more number of Ki67 positive cells in the papillary projections of alveoli and bronchioles. Similarly, De las Heras et al. (2014) reported that all solitary tumors associated with JSRV in sheep had a high Ki67 labeling index with a mean value of 11.4.

On AgNOR staining many small, black silver stained dots for NORs were seen scattered in the nuclei of proliferating cells of alveolar and bronchiolar epithelium in the lung sections of OPA cases (Fig. 8).

 Fig. 8: OPA-Lung: AgNOR staining: showing black silver stained dots for NORs were seen scattered in the nuclei of neoplastic cells of alveolar epithelial projections


Number of AgNORs in 100 randomly selected tumor cells were counted under oil immersion lens. AgNOR counting was also performed for normal sheep lung samples. AgNORs in clusters were counted separately wherever possible and the mean number of AgNOR dots per nucleus was calculated for each sample. The results were analysed by t-test one tailed distribution with 2 sample unequal variance ( Snedecor  and Cochran, 1989).The mean number of AgNORs for all the cases ranged from 6.5 to 11.9 with a mean + standard deviation values of 9.2 + 1.2. The mean number of AgNORs in normal lung sections ranged from 1.36 to 1.46 with a mean + standard deviation values of 1.42 + 0.07.  In the present study, AgNOR counts were significantly higher (P<0.05) in OPA lungs than normal lungs indicating high proliferative activity in tumor lungs. Pawaiya and Ram Kumar (2007) observed many small AgNOR dots that were seen scattered in the nuclei of papilli form alveolar epithelial cells with a mean value of 11.63±0.95 in OPA lung sections.

The commonly used proliferative markers in tumor diagnosis are mitotic figures, AgNOR’s, thymidine-labelling index, Ki67 and PCNA (Bedrossian, 1993; Lohr et al., 1997. Tumor proliferative activity is considered to be a good prognostic factor because it reflects the potential biologic aggressiveness and behaviour of the neoplasm (Ogura et al., 1992). In the present study, Ki67 immunostaining was carried out to study the proliferative activity of the neoplastic cells in OPA lungs. Ki67 is a nuclear antigen associated with cell proliferation and is present throughout the active cell cycle but absent in resting cell (Scholzen and Gerdes, 2000). Expression of Ki67 disappears rapidly after mitosis (Gerdes et al., 1984). The antibody Ki67 which recognizes a nuclear antigen in proliferating cells has been widely used to estimate growth fraction in different cancer lesions (Hall et al., 1988; Walker and Camplejohn, 1988). The level of Ki67 expression is of great prognostic significance in carcinoma of the prostrate, breast and liver, malignant melanoma and lung cancer (Scholzen and Gerdes, 2000). The Ki67 proliferating index has been proposed as an unfavourable prognostic indicator for human primary carcinomas (Hommura et al., 2000) and in several types of malignant neoplasms in veterinary medicine. (Ettinger et al., 2006; Webster et al., 2007) . Choudhury et al. (2011) opined that growth fraction estimated by the Ki67 index reflects more closely the clinical stage of tumor than the degree of differentiation of ovarian tumors. Nucleolar organizer regions (NORs) are loops of DNA present in the nucleoli. They contained genes that code for ribosomal RNA (r RNA) which are transcribed by RNA polymerase I (Egan and Crocker, 1992). Certain  argyrophilic  proteins, called NOR-associated proteins (NORAPs), are associated with this gene. These argyrophilic nuclear organizer region proteins are said to be accumulated in highly proliferating cells of tumors due to its segregation during transcription which could be demonstrated as black dots with silver staining on routine histopathological sections and called as argyrophilic nucleolar organizer regions (AgNORs) (Crocker et al., 1989). The proliferative activity of the tumor cells in the present study was also supported by the observation of high AgNOR counts in addition to Ki67 mean values.

In conclusion, the present study demonstrated the proliferative activity of tumor cells by  immunostaining  with Ki67 and AgNOR staining , that showed  high proliferative activity in the neoplastic cells of OPA affected lung tissues.


The authors are grateful to SERB, Department of Science and Technology (DST), Govt. of India for funding the project and to Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh for providing the facilities.


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