Pathological parameters
As shown in table 1, nine tubulopapillary carcinomas (34.6%), three complex carcinomas (11.6%), two papillary carcinomas (7.7%), two solid carcinomas (7.7%), one simple carcinoma (3.8%), one inflammatory carcinoma (3.8%), 2 cases of lobular hyperplasia (7.7%) and six benign mix tumors (23.1%) were diagnosed. Therefore, out of 26 observed mammary tumors, 18 (69.23%) were observed as malignant tumors and 8 (30.76%) as benign tumors.
In dogs with malignant tumors, 66.66% of cases showed M/R and 55.55% of them died. While37.5% of patients with benign tumors showed M/R but were still alive until the end of the study period.
As shown in table 2, in dogs with malignant mammary tumors, the presence of necrosis, infiltrative growth, and stromal invasion significantly reduced the DFS and OS. However, the presence of necrosis (HR: 12.46; 95% CI: 2.47-26.83; P = 0.002), infiltrative growth (HR: 21.82; 95% CI: 2.65-29.4; P = 0.004), and stromal invasion (HR: 21.82; 95% CI: 2.65-29.4; P = 0.004) were significantly considered the risk for M/R in malignant mammary tumors of the dogs. In addition, the presence of necrosis (HR: 12.55; 95% CI: 2.49-33.21; P = 0.002) was significantly associated with death.
Although, lymphocyte infiltration in malignant tumor decreased DFS and OS significantly and increased pulmonary metastasis and recurrence , only grade 2 lymphocyte infiltration was a significant risk factor compared to other grades for M/R (HR: 0.036; 95% CI: 0-0.38; P = 0.006) and death (HR: 0.1 ; 95% CI: 0.01-0.94; P = 0.045) in dogs with malignant mammary tumors. The histology grade of malignant mammary tumors and mitotic index in dogs did not significantly affect the DFS and OS (table 2).
Meanwhile, M/R and death were observed significantly faster in dogs with lymph nodes metastasis (LNM),but only LNM increase with DFS was a risk factor in the Cox regression analysis (HR: 21.82; 95% CI: 2.65-19.44; P = 0.004) but LNM increase with OS was not a risk factor (HR: 13.54; 95% CI: 0.9-13.94; P = 0.07). In addition, dogs with inguinal lymph node metastasis had significantly shorter OS than axillary cases, although it was not found to be a risk factor (HR: 3.61; 95% CI: 1.82-22.42; P = 0.242) . Two cases of metastasis to the lung are shown in Fig. 1.
Serum gelatin zymography
Semiquantitative analysis of zymograms from dog serums indicates that only Pro-MMP-2 (72 kDa) and Pro-MMP-9 (92 kDa) bands appeared in the control group. Furthermore, on the day of surgery, dogs with benign and malignant mammary tumors showed bands of Pro-MMP-2, Pro-MMP-9 and active MMP-9 (82 kDa), respectively. Fig. 2-A shows serum gelatin zymography of a dog with benign mixed tumor without M/R. It should be noted that similar to the control group, two patients with benign lobular hyperplasia tumor left the only Pro-MMP-2 and Pro-MMP-9 in the gel. In the preoperative samples, only one case of inflammatory carcinoma revealed the active form of MMP-2 with molecular weight of 66 kDa. The patient eventually showed pulmonary metastasis seven months after the surgery, and in the next year, the active form of MMP-2 with molecular weight of 62 kDa was detected in the gel. Fig. 2-B shows serum gelatin zymography of a dog with inflammatory carcinoma with M/R.
As shown in table 3 statistical analysis of zymograph data in preoperative samples showed a significant increase in Pro-MMP-2 levels in patients with malignant mammary tumors compared with patients with benign mammary tumors and the control group. The observed increase is 2 times compared to the control group. Also, a significant increase in Pro-MMP-9 levels was observed in patients with malignant tumors only compared to the control group. Moreover, the increase in active MMP-9 levels in patients with benign and malignant tumors compared to the control group was significant, but no significant difference was observed between the levels of Pro-MMP-9 and active MMP-9 in the group of benign and malignant tumors.
One month after surgical treatment, the active form of MMP-9 appeared in nine cases of malignant tumors, seven cases of which were recurred or metastasized during the period of study but two cases did not experience M/R by the end of the study (36 months after surgery). At the same time, the active band of MMP-2 was not observed in any of the samples .Also, in the four months after surgery, six cases of malignant tumors revealed the active MMP-9, all of which represented M/R. Furthermore, after one year of surgical treatment, 12 cases showed active MMP-9 and eight cases active MMP-2 bands with molecular weight of 62 kDa (five cases) and 66 kDa (three cases), all of which had M/R.
As shown in Fig. 3, in dogs with mammary tumors that were metastasized or recurred, serum concentration of Pro-MMP-2 was decreased significantly one month after surgery compared to the preoperative sample. On the other hand, a significant increase in the levels of Pro-MMP-2 was observed in serum samples of 4 and 12 months after surgery in comparison to one month after surgery (P≤0.05). The active form of MMP-2 showed a significant increase in the M/R group 12 months after surgery compared with the day of surgery, 1 and 4 months after surgery (P≤0.05). One and four months after surgery, there were no significant changes in serum levels of Pro-MMP-9 in M/R dogs compared to the day of surgery (P≥0.05), but a significant increase in its concentrations was observed 12 months after surgery than the other sampling times (P≤0.05). Serum active MMP-9 in the M/R group had a significant decrease in 1, 4, and 12 months after surgery compared to the day of surgery, while in the 12-month follow-up sampling, its activity showed significant increases than 1 and 4 months after surgery (P≤0.05).
However, in without M/R group, the changes in Pro –MMP-2 and Pro-MMP-9 levels on the day of surgery and sampling times were not significant and only the amounts of active MMP-9 observed in one month after surgery showed a significant decrease compared to the day of surgery. In addition, active MMP-9 band was not observed in other sampling times (Fig.3).
As shown in table 4, in dogs with serum Pro-MMP-2 levels of more than 1.5 ng/lane, M/R and death were seen 77.8 % and 66. 7 %, respectively. While for dogs whose serum Pro-MMP-2 was less than 1.5 ng/lane, 47 % M/R and 23.5 % death were observed. Regarding Pro-MMP-9, dogs with serum Pro-MMP-9 levels greater than 0.9 ng/lane, 66.7 % had M/R and died but in dogs with Pro-MMP-9 less than 0.9 ng/lane, M/R and death were observed 50 % and 14.3 % respectively. Meanwhile, 73.3 % M/R and 46.7 % death were seen in dogs with more than1.8 ng/lane serum active MMP-9 concentrations, but in the group with active MMP-9 less than 1.8 ng/lane, M/R and death were observed 36.4 % and 27.3 %, respectively. Also, dogs with mammary tumors that had a serum Pro-MMP-2 level of more than 1.5 ng/lane had a faster DFS and less OS. However, only their OS showed a significant difference with the group less than 1.5 ng/lane (HR: 3.62; 95 % CI: 1.01-12.97; P=0.048). Besides, Faster DFS and lower OS were observed in dogs with serum Pro-MMP-9 levels greater than 0.9 ng/lane, but only OS was statistically significant (HR: 6.08; 95 % CI: 1.29-28.72; P=0.023). Regarding serum active MMP-9, dogs whose serum active MMP-9 was more than 1.8 ng/lane had faster DFS and shorter OS than those with less than 1.8 ng/lane, however, the differences were not significant.