A total of twelve tissue samples were confirmed as SCC (horn and eye) on the basis of histopathology and eleven of them were confirmatory for SCC in Immunohistochemical examination (Table 4).
Kalim et al., (2021) reported 30 cases of horn cancer out of which 21 cases (67%) were of non-descript breed. The findings of the present study were in accordance with
Kalim et al., (2021). High occurrence of horn cancer in non-descript animals could be due to more population of non- descript animals in Chhattisgarh state.
Kalim et al., (2021) reported all 30 clinical cases of horn cancer only in bullocks however this observation was contradictory to our results shown in Table 3.
Haematological examination
The observation of mean ± SE values of Haemoglobin (Hb), packed cell volume (PCV), Total erythrocytes count (TEC) and Total leukocyte count (TLC) were estimated (Table 5). The results revealed no significant hematological alterations were observed in bovines affected with squamous cell carcinoma, although in cases of horn as well as ocular cancer, neutrophilia (>60%,) was observed, abundant lymphocytes (45%) were seen in neoplasm of horn and abnormal values of monocytes was observed which may be due to bacteremia, hemolysis and hemorrhage observed. Higher mean value for neutrophils and monocytes were observed in tumour affected bovines in comparison with healthy bovines.
Findings such as neutrophilia was in accordance with
Gomes et al., (2012). Eosinophilia was reported by
Somvanshi (1991) in advance cases of horn cancer but in our study eosinophilia was not observed in any case of neoplasm of horn and eye.
Gross changes
A total of 12 unilateral neoplastic growths suspected to have SCC at horn were examined grossly, which revealed large, irregular masses at the base of horn. Cauliflower like growth at the base of horn were observed in most of the cases. Two cases of large cauliflower like mass of about 20 cm diameter were seen (Fig 1, 2). Ocular neoplasms were small, pinkish neoplastic growths, varied from soft to hard in consistency. Large firm, cauliflower like mass, extending deep into the frontal and nasal sinus with verrucous surface was seen in left eye (Fig 3), tumourous mass at medial canthus of right eye extending towards limbus region. One case of neoplastic growth at vulva large cylindrical mass and hard in consistency about 10 cm in length attached proximally to the vulvar lips (Fig 4) and extended to perineal region.
Gross findings were in accordance with the observations of
Sharma et al., (2020); Fornazari et al., (2017) and
Pugliese et al., (2014). Gross pathological findings of neoplasm of horn was found to be unilateral in accordance with
Kalim et al., (2021). Pink Cauliflower like soft, friable growth with bleeding at the base of the horn were also reported by
Giri et al., (2011); Kumar et al., (2013) and
Sharma et al., (2020). Verrucous surface of tumourous growth in eye was reported by
Fornazari et al., (2017). Tumourous growth observed in vulva (BovVC1) was observed as hard, black cylindrical, ulcerated nodular growth in vulva was also observed by
Sreelekshmy et al., (2014).
Hisopathological changes
Histopathological findings in SCC of horn were severe keratinization of horn epithelium with concentric keratin pearls also reffered as “cell nests” (Fig 5a). Characteristic epithelial pearls were observed in well differentiated squamous cell carcinoma (Fig 6a and b). More keratin deposition towards the center was prominent (5a and b). Moderately differentiated SCC of horn was characterised with presence of epithelial pearls not as numerous and well defined as compared to well differentiated SCC (Fig 7). Histopathological findings in OSCC were distinctive keratin pearls with more keratin deposition towards the center in BovEC 4 and characteristic epithelial pearls with concentric layers of keratinization in well differentiated OSCC.
Mineralization was also observed around the periphery of keratin pearls (Fig 8). Multiple islands of invasive tumour cells with mononuclear cell infiltration and keratinization at the center of neoplastic islets in moderately differentiated OSCC (Fig 9). Numerous mitotic figures were also noticed with anaplasia and neovascularization (Fig 10). Large tumour islands with mononuclear cell infiltration were seen in moderately differentiated OSCC. Poorly differentiated OSCC is characterized with absence of cell nests. Microscopic examination of vulvar neoplasm revealed lipoma with multiple fat vacuoles and adipocytes tissue separated with thin, inconspicuous fibrous septa (Fig 11 and Fig 12).
Results of microscopic examination were consistent with the findings of
Velavan et al., (2024); Kalim et al., (2021); Sharma et al., (2020); Reddy et al., (2017); Kumar et al., (2013) and
Joshi et al., (2009). The formation of keratin pearl is due to the lack of cohesion among epithelial cells as a result of malignant changes leading to the concentric arrangement of squamous cells and these cells fate is to form keratin and lay down keratin in concentric manner resembling keratin pearls. Irregular islands of squamous epithelium with cords / islands and clusters of tumourous squamous cells were also reported by
Manasa et al., (2023); Kalim et al., (2021) and
Devi et al., (2010). Islands of tumourous epithelial cells, proliferation of fibroblasts, connective tissue and infiltration of lymphocytes and plasma cells were also reported in OSCC
(Lakshmi.et_al2020;
Sharma et al., 2020; Fornazari et al., 2017 and
Islam et al., 2017). Invasion of dermal stroma and highlighted keratinization with infiltration of neoplastic squamous cells observed in OSCC were also observed by
Fornazari et al., (2017). Numerous mitotic figures, anaplasia and neovasculari- zation were in accordance with
Velavan et al., (2024); Sharma et al., (2020); Fornazari et al., (2017) and
Giri et al. (2011).
Immunohistochemistry
Immunoexpression of Pancytokeratin (Pan-CK)
Variations were observed in the immunohistochemical reactivity of Pan-CK between SCC of horn and eye. Immunohistochemical scoring method was mentioned in Table 3. Mostly cases of SCC of horn revealed positive immunoexpression in >75% neoplastic cells and was given score of 4+ (BovHC3; Fig 13), 3+ (BovHC4, BovHC6). High cytoplasmic reactivity of Pan- CK in more than 50% of tumourous cells in well differentiated SCC of horn. Pan-CK immunoexpression was more prominent in epithelial pearls in case of well differentiated SCC of horn (Fig 13) and more intensely stained periphery/ border inside large keratin/epithelial pearl (Fig 13), well differentiated SCC of horn depicted strong cytoplasmic staining in almost all neoplastic cells and was given the score of 4+ (Fig 13, 14). Poorly differentiated SCC of horn with absence of keratin pearls/cell nests showed strong staining in about 75% of neoplastic cells , given the score of 3+ ; Pan-CK immunoexpression in epidermal layer with 2+ (BovHC1). Reddish brown staining of Pan-CK in layered keratinization and tumour islands of BovHC9 and strong Pan-CK immunoexpression inside epithelial pearl scored as 3+ (Table 6) were also found with clear demarcation observed. BovEC4 revealed strong immunoreactivity of Pan-CK in the cell nests (keratin pearls) as well as in tumour islands (Fig 17 and 18). Lipoma of vulva exhibited almost no cytoplasmic reactivity for Pan-CK (IHS 0; Fig 21).
Immunoexpression of EGFR
Strong immunoexpression of EGFR with the score of 4+ in well differentiated SCC of horn(Fig 15). Immunoexpression of EGFR was observed in the surrounding tissue excluding large cell nests and in periphery of distinct keratin pearls with absence of immunoexpression inside cell nests (Fig 16). BovHC4 revealed strong immunoexpression with IHS of 3+, which signified its highly malignant and invasive nature even after the absence of well differentiated cell nests. Both SCC of horn and eye showed positive immunoreaction towards EGFR although few samples were detected non-immunoreactive towards EGFR.
Immunoexpression of p53
Tumour suppressor p53 gene immunoexpression was more pronounced in nucleus of cancerous cells in well differentiated SCC of horn. SCC of horn exhibited p53 nuclear staining of tumour cells with a score of 4+ (BovHC3; Fig 19) scattered in connective tissue stroma and peripheral neoplastic cells of tumour islands showing intense nuclear staining of p53. BovEC4 revealed mild to moderate immuno-positive reaction towards p53 with 1+ score . p53 immunore- activity was not observed in lipoma (BovVC1; Fig 22).
Immunoexpression of p16
Negative reaction/non-immunoreactive towards p16 was observed in all SCC of horn including well differentiated (BovHC3; Fig 20), moderately differentiated (BovHC1) and poorly differentiated (BovHC4). BovEC6 exhibited mild immunoexpression for p16 with dark brown staining of nuclei and cytoplasm of tumour cells given the IHS of 1+ (Table 6).
Immunohistochemical findings were partially in accordance with the findings of
Sharma et al., (2020). They observed Strong cytoplasmic immunoexpression of Pan-CK in almost all neoplastic cells in poorly differentiated SCC but present study revealed highest immunoexpression of Pan-CK in well differentiated SCC of horn (BovHC3) although high and moderate immunoexpression of Pan- CK was observed in poorly and moderately differentiated SCC. Nuclear staining of tumour cells around periphery of epithelial pearls, sparing the region of keratinization with p53 tumour marker was also reported by
Carvalho et al., (2005), Fornazari et al., (2017) and
Sharma et al., (2020). High immunoexpression in well differentiated SCC of horn contradicts from the findings of
Sharma et al., (2020) where strong immunoexpresssion of p53 was observed in poorly differentiated SCC of horn and OSCC. Intense positive immunoexpression of p53 and expression mostly within outer epithelial layer of the cell nests was observed by
Fornazari et al., (2017). Findings of immunoexpression of EGFR were similar with
Lakshmi et al., (2020). The malignancy of tumour of epithelial origin was confirmed by high immunoexpression of EGFR. Findings of p16 immunoexpression were consistent with
Fornazari et al., (2017) as negative immunoexpression of p16 was reported in majority of cases of OSCC.
Marinescu et al., (2016) reported positive immunoexpression for p16 in poorly differentiated squamous cell carcinomas in the periphery as well as random in the islands of neoplastic cells. Further investigations are required for p16 immunoexpression as a prognostic factor for bovines with cutaneous SCC as has been previously established for humans with the same tumour in the skin
(Marinescu et al., 2016).
Grading and differentiation
Grading of SCC (horn and eye) revealed a total of 3 cases (2 of horn; 1 of eye) as Grade I -poorly differentiated SCC, 3 cases (2 of horn; 1 of eye) as Grade II -moderately differentiated SCC and 5 cases (4 of horn; 1 of eye) as Grade III -well differentiated SCC (Table 7) on the basis of histopathological and immunohistochemical examination.
