Mast cell tumor is the most common skin neoplasia in dogs (London & Seguin 2003), whose prognosis and treatment may be difficult to accomplish owing to the clinical behavior varying from extremely benign to extremely malignant (Macy 1986). The treatment may consist of surgery, radiotherapy, or a combination of both modalities (O'Keefe 1990). Chemotherapy with multiple drugs is used in cases of unsuccessful surgery or radiotherapy (Gerritsen et al. 1998). In some neoplasias failures in chemotherapy is ascribed to a phenomenon known as multidrug resistance (MDR) (Germann 1996, Persidis 1999, Munoz et al. 2007). In the last decade many cellular mechanisms explaining multiple drug resistance have been characterized. Such mechanisms include the activation of transmembrane proteins that reduce intracellular concentrations os several chemical substances; the activation of glutathione/glutatione-stransferase mechanisms; alterations in the target of the drug; alterations in the mechanisms of cellular repair; alterations of genes and proteins involved in the control of apoptosis (Munoz et al. 2007). Therefore, this study was aimed at evaluating the expression of multiple drug resistance protein 1 (MRP1), glutathione-S-transferase isoenzyme pi (GSTpi), as well as mutant p53 using immunohistochemistry in canine mast cell tumors.

Materials and Methods

Sixteen samples of canine cutaneous mast cell tumors stored in paraffin blocks, with no history of chemotherapy, were chosen at the archives of the Department of Veterinary Pathology of São Paulo State University, Campus of Jaboticabal, São Paulo, Brazil, which were then allocated in two groups, namely: MASTI, with cutaneous grade I mast cell tumors (n = 8); MASTIII, with grade III cutaneous mast cell tumors (n = 8). The immunohistochemistry assay was based on the method of streptoavidine-biotine complex (ABC). Primary antibodies were MRP1 (monoclonal antibody NCL-MRP1, Novocastra Laboratories Ltd, Newcastle, UK), mutant p53 (polyclonal antibody NCL-p53-CM1, Novocastra Laboratories Ltd, Newcastle, UK) and isoenzyme GSTpi (polyclonal antibody RB-050-A0, Labvision Corporation, Fremont CA, US), diluted to 1:25, 1:100, 1:50, respectively. Human liver and canine testis, human and canine colon were used as positive controls for the reactions with primary antibodies anti-GSTpi and anti-MRP1, respectively. Canine mammary carcinoma, which is known to express p53 (Moro et al. 2004) was used as a positive control for the reactions with antibody anti-p53. As a negative control all elements of the reactions were used in the absence of primary antibodies. To evaluate the frequency of immunostaining five fields per section were randomly chosen and, in each field 100 neoplastic cells, including stained and non-stained cells, were counted with a reticulated micrometer (Nikon, Inc.--Japan) under a x400 magnification (x10 ocular and x40 objective). Therefore, the percentages of marked neoplastic cells (PCNM) were calculated in relation to the total. Samples were considered positive for the expression of MRP1, GSTpi and p53 when more than 10% of the cells were marked, as proposed by other investigators (Miyoshi et al. 2002, Sueiro et al. 2004, Sklodowska et al. 2005, Yonemaru et al. 2007). Fisher's exact test was used to analyze the results, considering P < 0.05 indicative of significant differences (Prisma 5.00 for Windows, GraphPad Software, San Diego California USA).

Results

The expression of proteins MRP1 and p53 were positive in 100% of the samples of group MAST I and in 87.5% of MAST III (P > 0.05). For GSTpi the expression was positive in 75% of the samples of group MAST I and in 37.5% of MAST III (P > 0.05). The expression of the three was documented in 75% of the samples of MAST I and in 37.5% of MAST III. The co-expression of proteins MRP1 and p53 was observed in 100% of MAST I and in 75% of MAST III, whereas GSTpi and p53, and MRP1 and GSTpi were seen in 75% of MAST I and 37.5% of MAST III. Cytoplasmic staining with a diffuse granular aspect was observed in neoplastic cells that expressed MRP1, whereas staining was cytoplasmic and/or nuclear in those who expressed GSTpi and p53.

Discussion and Conclusions

According to literature grade III mast cell tumors have a worst prognosis in comparison to grade I (Gerritsen et al. 1998). However, this study could not associate histological grading of mast cell tumors to the reactivity of MRP1, GSTpi and p53. On the contrary, a tendency of increased expression of proteins was observed in grade I mast cell tumors as compared to the grade III ones, although it did not attain statistical significance. This could indicate, in theory, that grade III mast cell tumors were likely to present a better response to chemotherapy (Miyoshi et al. 2002). Nonetheless, experience shows that little differentiated mast cell tumors (grade III) present the worst response to chemotherapy even in the face of a combination of agents (Gerritsen et al. 1998). These results reinforce the necessity of establishing other methods of classification for mast cell tumors because histological criteria alone may incur in errors owing to its subjectivity (Pinczowski et al. 2008). Similar results were documented by Miyoshi et al. (2002) with MRP1 in canine grade III mast cell tumor that, as seen in this study, did not find a significant association between the number of positive cases and histological grading. The same was observed by Ginn et al. (2000) and Wu et al. (2006) with p53.

The co-expression of proteins MRP1, GSTpi and p53 was seen in 75% of grade I mast cell tumors and in 37.5% of grade III ones. This might suggest that the concomitant expression of these three proteins is interrelated and acting in a synergic way in some histological types of tumors. Nevertheless, the co-expression of only MRP1 and p53 was higher than that of the three proteins together, which indicated that GSTpi might be less important in the development of MDR in mast cell tumors. The cytoplasmic staining observed for antibody anti-MRP1 was similar to that described for some human and canine tumors (Fukushima et al. 1999, Miyoshi et al. 2002, Kamata et al. 2008). Moreover, this investigation documented a cytoplasmic staining with granular aspect, which was also described by Larkin et al. (2004) and Calatozzolo et al. (2005). Investigators have suggested that MRP1 accumulated in vesicles in the cytoplasm (Loe et al. 1996, Renes et al. 2000) and the staining granular pattern could be attributed to such distribution. The observed cytoplasmic and nuclear staining of GSTpi followed the same pattern described for human neoplastic tissues (Ali-Osman et al. 1997, Howells et al. 2004). Although controversy does exist, this staining pattern has been related to prognosis in human beings (Ali-Osman et al. 1997, Howells et al. 2004). In two studies the concomitant nuclear and cytoplasmic expression of GSTpi were associated to a shorter survival of patient and resistance to chemotherapy (Ali-Osman et al. 1997), whereas another investigation associated the same staining pattern of the enzyme to more favorable features (Howells et al. 2004). In this study, concomitant staining of cytoplasm and nucleus was mainly observed in group MAST I, which does not allow to propose any correlation with prognosis. Also for p53, studies indicate that the cytoplasmic pattern might be related to a worse prognosis in human colorectal tumors (Bosari et al. 1994; Flamini et al. 1996), although the rabbit antihuman polyclonal antibody clone CM1 (Novocastra Laboratories Ltd, Newcastle, UK) indicated a nuclear staining pattern. In dogs, cytoplasmic staining was reported in mammary (Inoue & Shiramizu 1999; Haga et al. 2001; De Nardi 2007), intestinal (Gamblin et al. 1997, Wolf et al. 1997) and bone neoplasias (Loukopoulos et al. 2003) using monoclonal and polyclonal anti-p53 antibodies. However, Keller et al. (2007) considered unspecific the cytoplasmic staining with antibody CM1 in canine lymphomas, osteosarcomas and mammary carcinomas. Although nuclear, nuclear and cytoplasmic, or only cytoplasmic staining have been found in this study, only neoplastic cells with marked nucleus were considered positive.

This study showed that the majority of the studied canine cutaneous mast cell tumors expressed and co-expressed MRP1, GSTpi and p53, although in the future it will be necessary to evaluate whether their expression alone or in conjunction would have any implication in the development of clinical resistance to chemotherapy, as well as their utility as predictive indicators of prognosis.

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