In this study host related factors such as weight, reproductive status and hormonal therapies were not significantly related to patient outcomes. Although these conclusions are in agreement with previous studies [4, 10, 19, 20], the lack of influence of the reproductive status and hormonal therapies must be regarded with caution since only six animals were spayed at the time of mastectomy and only eight had received oestrous control therapy.
Some prognostic studies suggested that there is an increasing malignancy from complex carcinomas to simple carcinomas to sarcomas [3, 6, 16, 21], although this fact was not demonstrated in other publications [4, 20, 22]. In this study, considering the most frequent histological types (number > 5), carcinosarcomas were the most aggressive tumours (33% local recurrences and/or distant metastases), while complex carcinomas were the less aggressive ones (20% local recurrences and/or distant metastases and significantly lower risk of death due to MMTs). The differences between simple carcinomas and the other groups were not significant, demonstrating that solid and tubulopapillary carcinomas are probably an heterogeneous group of neoplasms, with distinct invasive and metastatic capacities equally distributed among both groups, as demonstrated by the very similar percentages of local recurrences and/or distant metastases (26.7% for solid and 27.7% for tubulopapillary carcinomas).
Ulceration and necrosis, two features that have been suggested to be indicators of higher tumour aggressiveness [4, 6, 10], were not significantly related to prognosis in this study. Although ulceration may be caused by the tumour invasive growth, it must be highlighted that it may also be due to self-induced trauma, skin ischemia or infection, features that are not necessarily associated to an aggressive biological behaviour. In a previous study , necrosis was associated to poorer outcome but different assessment methodologies may explain the divergent results.
Although squamous metaplasia is often regarded as a sign of tumour aggressiveness in human breast cancer [23, 24], this is the first study in canine mammary tumours (CMTs) to address it as an independent variable and, contradicting the previous notion, squamous metaplasia failed to demonstrate a significant prognostic value.
Corroborating previous findings [2, 7, 10, 15], our study showed that histological grade might be helpful to predict survival time, although not time to recurrence or metastasis. It must be remembered, however, that several grading methods have been used to classify MMTs in dogs. The Nottingham method is, in the authors’ opinion, a well standardized method that, although developed for human breast cancer, is applicable to canine MMTs and, as these results demonstrate, associated to survival time.
High MIB-1 LI were strongly associated to the development of recurrences/distant metastases as well as with shorter DFS and OS intervals, both in univariate and multivariable analysis. Half of the animals bearing tumours with LI higher than 40% developed local recurrences and/or distant metastasis and 42% died within 2 years after surgery. Previous CMTs studies associated higher MIB-1 LI with other aggressive tumour features (larger size [5, 13], infiltrative growth , high histological grade ), higher risk of metastatic disease  and decreased DFS and OS [7, 9], strengthening our findings. Other multivariable studies, however, reported opposite results [9, 22, 25], which may be explained by the small number of cases studied in the Lee et al. (2004)  series and by the different assessment methodologies between studies. Our methodology consisted in the evaluation of the highest labelled areas. When compared to the previously described counting in 3–5 randomized fields , our method eases the work of the observer, reduces variability between samples, and allows a more rigorous comparison between large tumours, where it is easier to find suitable non-overlapping fields, and smaller ones, where the choice of fields is more limited. Mitotic index, frequently used to assess tumour proliferative activity, was not associated to patient prognosis in this series. Therefore, it seems that MIB-1 it is a more suitable proliferation-associated prognostic indicator than mitotic index.
The presence of RLN metastases was associated to an increased risk for the development of recurrence or distant metastases and tumour-associated death in univariate analysis. Unfortunately, it was not possible to demonstrate its value as an independent prognostic factor in multivariable analysis because RLN were not surgically removed in 19 (22%) cases. Apparently, as demonstrated by previous studies, this variable fails to maintain its prognostic significance when included in multivariable models [4, 6].
In human breast cancer studies, it has been proposed that the expression of VEGF by cancer cells is a poor prognostic factor for survival , but this hypothesis was contested by other studies [27, 28]. To the best of our knowledge, there is only one previous CMTs survival study that addressed VEGF expression  and reported it not to be associated with OS. Our findings demonstrate that VEGF expression by cancer cells is not associated with either OS or DFS, suggesting that this angiogenic factor is not useful as a prognosticator of canine MMTs. Recently, Al-Dissi et al. (2010)  verified that there were no correlations between the expression of VEGF, its receptor-2 and tumour microvascular density, suggesting that other factors are more important than VEGF in CMTs angiogenesis. Furthermore, it has been suggested that VEGF could be an early carcinogenic factor that declines with malignant progression , a hypothesis that may justify our results.
The protein TIMP-2, initially described as an inhibitor and regulator of the activity of matrix metalloproteinase-2 (MMP-2), has recently been proven to stimulate cell growth and angiogenesis, as well as inhibit apoptosis, hence contributing to tumour aggressiveness . Although no other CMTs study groups assessed the prognostic value of TIMP-2 expression, human breast cancer studies revealed that high levels of TIMP-2 mRNA and TIMP-2 protein are correlated with the development of distant metastasis and decreased DFS [33, 34]. In the present study, the quantitative expression of TIMP-2 (i.e. number of positive cells) was not related to outcome. Although this may seem in contradiction with our previous results  it must be noticed that the expression of the molecule was evaluated, in that study, using a score obtained by evaluating both number and intensity of expression. However, the reproducibility of intensity scales is very difficult in the routine setting. Therefore, we decided to use a more reproducible classification system in this study.
An increasing number of studies over the last decade identified the tumour stroma as a major player of the carcinogenic process reviewed by . During tumour invasion, cancer cells interact with their microenvironment to activate signalling pathways and to increase growth factors bioavailability, thus favouring tumour progression . Stromal proteases, such as MMPs and uPA, are frequently upregulated in tumour microenvironment and influence tumour behaviour by tissue architecture disruption and signalling interactions . We demonstrated that stromal expressions of both uPA and MMP-9 were associated to poorer outcomes in univariate study, although only MMP-9 was able to maintain its independent prognostic value in multivariable analysis. We also verified that stromal expressions of uPA and MMP-9 are highly associated, which may justify why uPA lost significance in the multivariable model that included MMP-9. To the best of our knowledge, this is the first multivariable survival study addressing these stromal markers in canine MMTs.