Dog, hair follicle tumor. AgNOR stain. A) Malign trichoepithelioma with a higher count of NORs when compared with benign hair follicle neoplasms. Figure shows smaller sized NORs dispersed throughout the nucleus (Bar= 16 μ m). B) Benign trichoepithelioma with a higher count of NORs (Bar= 13 μ m) when compared with the infundibular keratinizing acanthoma (C), pilomatricoma (D), and trichoblastoma (E) (Bar= 16 μ m). 

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... a ruler, diameters (longitudinal and transversal) of 30 cellular nuclei were measured using the immersion objective (1000 × magnification). In the end, values were converted to micrometers by using a micrometric slide. Histological sections were impregnated with silver in order to individualize nucleolar organizer regions (NORs), in accordance with a described technique (Ploton, 1994) with some changes (Aubele et al., 1994). NORs counting was performed with an immersion objective (1000 × magnification). NORs present in 50 nuclei of neoplastic cells were counted. The counting of the NORs was manually performed by the same operator. Only those NORs, which were individualized with black or brown dots, were counted. When clustered, they were considered as a single NOR. The number of cells and fields was determined using the technique of studying mean value instability variation. Standard deviation and variation coefficient of the studied variables were stable in ten fields, 30 cells, and 50 nuclei for determining the number of mitoses, the mean nuclear diameter, and the number of NORs, respectively. Histological sections of 4μm were also submitted to immunohistochemical analysis, using anti- MIB-1. 1 Streptavidin-biotin-peroxidase 2 and antigenic recovery techniques with the use of Retrieval solution 3 were used for 15 minutes. Histological sections were incubated with primary antibody (anti-MIB-1, 1:10 dilution) for one hour in humidified chamber and for 15 minutes in blockade phases of endogenous peroxidase, blockade serum 2 , secondary antibody 2 , and streptavidin peroxidase. DAB 2 was the chromogen used, with 10-minute incubation. Sections were stained with Harris hematoxylin. Dog skin was used as a positive control. Negative control was obtained by replacing primary antibody by PBS. All histological sections were stained with HE and were also analyzed regarding clean surgical margins of tumors. Follow-up monitoring was obtained by reevaluation or telephone conversations with referring veterinarians or dog owners. Animals were followed between one and five years to evaluate neoplasm recurrence rate. However, only 57 dogs could be evaluated because the remaining 25 dogs did not complete the minimum one-year post-tumor-removal period. Mean and standard deviation were calculated for the following variables: size of neoplastic nodules, number of mitoses/field, mean nuclear diameter, number of NORs/nucleus, and percentage of cells with Ki-67 expression. Data were submitted to analyses of variance using Instat (Graph Pad Software, Version 3.00, 32 Win 95/NT, Created December 23, 1997) and means were compared by the Student-Newman- Keuls test. Chi-square test was employed in order to evaluate the macroscopic presentation as for the number of nodules (simple or multicentric). Differences were considered significant if P<0.05. From January 1 2000 to July 31 2006, 2,678 canine neoplasms were diagnosed. Skin tumors corresponded to 36.1% (n=968). Hair follicle neoplasms represented 3.1% (n=82) of all neoplasms (n=2678) and 8.5% of all skin neoplasms, corroborating with the results obtained by Bostock (1986), Walder and Gross (1992), and Goorman and Dobson (1995). Trichoblastoma was the most common neoplasia, affecting 32 animals, followed by benign trichoepithelioma (n=30), benign pilomatricoma (n=7), malignant trichoepithelioma (n=6), infundibular keratinizing acanthoma (n=5), and tricholemmoma (n=2). No case of malignant pilomatricoma was diagnosed. Tricholemmoma was not included in the statistical analysis due to its low occurrence. No significant differences were observed when the types of tumor were compared between males (n=33) and females (n=44) and between animals from different age groups. Head, dorsum, and limbs were the most affected sites. The average age of animals was 7.08±2.14 years. Poodle dogs were the most frequently affected (n=21), followed by dogs with undefined breed (n=15), and cocker spaniels (n=10). The running time from when the tumoral mass was first noticed to its surgical removal was equivalent to 146±226.87 days. Mean tumor diameter corresponded to 2.09±1.64cm; and there was no significant difference among tumor types. Regarding the number of nodules, trichoblastoma presented a significant higher frequency in the simple form (P<0.05). The other tumors presented no significant difference between simple and multicentric forms (P>0.05). There was a significant difference in the number of NORs in cells of malignant trichoepithelioma when compared with the others types of neoplasms. Among benign neoplasms, trichoepithelioma presented a significantly higher number of NORs when compared with the other benign neoplasms. In addition to the number and the size of NORs, they also differed between benign and malignant neoplasms (Table 1). Malignant trichoepithelioma presented smaller NORs which were dispersed all around the nucleus, while the other neoplasms presented larger and fewer NORs (Fig. 1). Ki-67 expression was higher in the malignant trichoepithelioma when compared to benign neoplasms. Among benign tumors, there was no significant difference regarding Ki-67 expression (Fig. 2). The percentage of cells with Ki-67 expression in malignant trichoepithelioma was equivalent to 49.1%, significantly larger than those of infundibular keratinizing acanthoma, pilomatricoma, trichoblastoma, and trichoepithelioma (Tab 1). Malignant trichoepithelioma presented a significant higher number of mitoses/field when compared with benign trichoepithelioma and with the other benign tumors of the hair follicle. Trichoblastoma presented a larger number of mitoses/field when compared with the other benign neoplasms. However, the mean diameter of nuclei did not differ between benign and malignant neoplasms (Table 1). Two out of 57 evaluated dogs presented tumor recurrence. One of these presented a benign trichoepithelioma and the other a malignant trichoepithelioma. These two cases were considered recurrences, and at the second biopsy revealed that the site and type of neoplasm found was the same encountered in the first biopsy. No metastasis was found and no animals died. These two cases were the only tumors removed with dirty surgical margins. The number of NORs was significantly higher in malignant trichoepithelioma cells when compared with the others types of neoplasms. In mammary malignant tumors, the number of NORs is also higher than that in benign neoplasms (Underwood and Giri, 1988; Treré, 1993; Derenzini and Treré, 1994). Malignant trichoepithelioma presented smaller NORs dispersed all around the nucleus, in contrast with benign neoplasms that presented larger NORs, which is in accordance with previous results (Crocker et al., 1989). Ki-67 expression was nuclear. Those cells, whose nuclei presented a brown color, regardless of intensity, were considered positive for immunohistochemistry with anti-MIB-1, while negative cells presented basophilic nucleus. In this study, Ki-67 expression was higher in the malignant trichoepithelioma, when compared to the other hair follicle tumors analyzed, similar to what was observed in mastocytomas of grade III (Seguin et al., 2006), lymphosarcomas (Scase et al., 2006), malignant melanomas (Millanta et al., 2002), spinocellular carcinomas (Della Salda et al., 2002), and malignant tumors of mammary gland (Lohr et al., 1997). There was no significant correlation between the number of mitoses/field and Ki-67 expression and between the number of mitoses/field and the number of NORs. Results similar to this one were found in other neoplasms (Simões et al., 1994; Greattu et al., 2004; Brunetti et al., 2005). NOR and Ki-67 did not have significant statistic correlation (r=-0.5506). No significant correlation was observed between NOR and Ki- 67 in neoplasms of mammary (Ceccarelli et al., 2000), lung (Soomro and Whimster et al., 1990), intestines (Nanashima et al., 1999), and female genital tract (Chung et al., 1994). This may be due to the fact that these methods quantified different phases of cell proliferative activity (Ceccarelli et al., 2000). The number of NORs has been related to cell proliferation (Derenzini and Treré, 1994; Underwood and Giri, 1988). The higher number of NORs in proliferating cells may be explained by the fact that in G1 phase of the cell cycle, NORs proteins are synthesized. Newly synthesized proteins accumulate in the nucleolus, where they associate with ribosome genes to originate new NORs. The association with NORs proteins induces decondensation of ribosome chromatin, which is required for the next phase of gene duplication (Derenzini and Ploton, 1991). Since NORs are associated with cell proliferation, the technique plays an important role in the prognosis of neoplasms (Treré, 1993). Nuclear Ki-67 antigen is present in the following phases of the cell cycle: late G1, S, G2, and M, and it is absent in the early G1 phase and G0. It is known that cell cycle is composed of G1, S, G2, and, M phases. G0 phase is when cells are quiescent but at any time they can start their cycle and therefore start their multiplication process. Phases G1 and G2 are transitional phases, phase S is when DNA is synthesized and duplicated, and phase M is when mitosis occurs (Gerdes et al., 1984; Brown and Gatter, 1990). In humans, several studies have shown the relationship between Ki-67 expression and neoplasms malignity grade (Gasparini et al., 1989; Keshgegian and Canaan, 1995). High Ki- 67 immunolabeling has been correlated with unfavorable prognosis of several tumors (Veronese et al., 1993; Kerns et al., 1994; Lam et al., 1996). In the present study, recurrence rate of hair follicle tumors might be more related to surgical margins during surgical removal than to Ki-67 expression, number of NORs or mitotic index. In this study it was observed that the frequency of hair follicle tumor, considering breed, gender, age, and anatomical site, was similar to other ...

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... higher number of NORs when compared with the other benign neoplasms. In addition to the number and the size of NORs, they also differed between benign and malignant neoplasms (Table 1). Malignant trichoepithelioma presented smaller NORs which were dispersed all around the nucleus, while the other neoplasms presented larger and fewer NORs (Fig. ...