A phenotype from tumor stroma based on the expression of metalloproteases and their inhibitors, associated with prognosis in breast cancer.

The objective of the present work was to evaluate the impact of the phenotype of both mononuclear inflammatory cells (MICs) and cancer-associated fibroblast (CAFs) in early breast cancer patients, specifically assessed as to their expression of MMP/TIMP relative to their position within the tumor (i.e., localization at the tumor center or invasive front) and the occurrence of distant metastases.. An immunohistochemical study was performed using tissue arrays and specific antibodies against matrix metalloproteinase (MMP)-1, -2, -7, -9, -11, -13 and -14, tissue inhibitors of metalloproteinase (TIMP)-1, -2 and -3, both at tumor center and at invasive front, in 107 patients with primary ductal invasive breast tumors. Data were analyzed by unsupervised hierarchical clustering analysis. Our results indicated that MMP-11 expression by MICs, and TIMP-2 expression by CAFs at either the tumor center or the invasive front, were the most potent independent prognostic factors for predicting the clinical outcome of patients. Using the unsupervised hierarchical clustering analysis, we found well-defined clusters of cases identifying subgroups of tumors showing a high molecular profile of MMPs/TIMPs expression by stromal cells (CAFs and MICs), both at the tumor center and at the invasive front, which were strongly associated with a higher prevalence of distant metastasis. In addition, we found combinations of these clusters defining subpopulations of breast carcinomas differing widely in their clinical outcome. The results presented here identify biologic markers useful to categorize patients into different subgroups based on their tumor stroma, which may contribute to improved understanding of the prognosis of breast cancer patients.

Clinical significance of toll-like receptor 3, 4, and 9 in gastric cancer.

Toll-like receptors (TLRs) have raised an extraordinary interest in cancer research due to their role in tumor progression. By activating the production of several biological factors, TLRs drive an inflammatory response and activate the adaptive immune system. The aim of this study was to investigate the expression and clinical relevance of TLR3, TLR4, and TLR9 in gastric cancer. For this purpose, an immunohistochemical study on cancer specimens from 106 patients with gastric cancer was performed using tissue arrays and specific antibodies against TLR3, TLR4, and TLR9. The results indicate that gastric carcinomas samples show high expression of TLR3, TLR4, and TLR9 by cancer cells. The expression of TLR3 by cancer cells was significantly associated with a poor overall survival in patients with resectable tumors. Moreover, in patients with resectable tumors and lymph node invasion, a high TLR3 expression defines a population with even worse prognosis. Therefore, TLR3 may have clinical interest as indicator of tumor aggressiveness and as a prognostic indicator in gastric cancer.

Low microvascular density at the tumor center is related to the expression of metalloproteases and their inhibitors and with the occurrence of distant metastasis in breast carcinomas.

BACKGROUND: The aims of this study were to evaluate the microvascular density (MVD) at the center of breast carcinomas, its relationship with the expression of metalloproteases (MMPs) and their inhibitors (TIMPs), and its connection with the distant metastasis rate. METHODS: An immunohistochemical study of four MMPs and two TIMPs was performed on cancer specimens from 97 women with a histological confirmed diagnosis of early invasive breast cancer. RESULTS: Expressions of MMP-9 by cancerous cells, or MMP-11 and TIMP-2 by stromal cells, were all negative and significantly associated with MVD, whereas MMP-7 score values were positive and also significantly associated with MVD. However, positive expression of MMP-1 by mononuclear inflammatory cells was significantly associated with MVD. Multivariate analysis demonstrated a significant and inverse relationship between MVD and the occurrence of distant metastasis. CONCLUSIONS: Our data point out the clinical importance of low MVD at the tumor center as an independent prognostic factor of distant metastasis development in breast cancer.

Impact of CD68/(CD3+CD20) ratio at the invasive front of primary tumors on distant metastasis development in breast cancer.

Tumors are infiltrated by macrophages, T and B-lymphocytes, which may favor tumor development by promoting angiogenesis, growth and invasion. The aim of this study was to investigate the clinical relevance of the relative amount of macrophages (CD68⁺), T-cells (CD3⁺ and B-cells (CD20⁺) at the invasive front of breast carcinomas, and the expression of matrix metalloproteases (MMPs) and their inhibitors (TIMPs) either at the invasive front or at the tumor center. We performed an immunohistochemical study counting CD3, CD20 and CD68 positive cells at the invasive front, in 102 breast carcinomas. Also, tissue sections were stained with MMP-2, -9, -11, -14 and TIMP-2 antibodies, and immunoreactivity location, percentage of reactive area and intensity were determined at the invasive front and at the tumor center. The results showed that an increased CD68 count and CD68/(CD3+CD20) ratio were directly associated with both MMP-11 and TIMP-2 expression by mononuclear inflammatory cells at the tumor center (p = 0.041 and p = 0.025 for CD68 count and p = 0.001 and p = 0.045 for ratio, respectively for MMP-11 and TIMP-2). In addition, a high CD68/(CD3+CD20) ratio (>0.05) was directly associated with a higher probability of shortened relapse-free survival. Multivariate analysis revealed that CD68/(CD3+CD20) ratio was an independent factor associated with distant relapse-free survival (RR: 2.54, CI: (1.23-5.24), p<0.01). Therefore, CD68/(CD3+CD20) ratio at the invasive front could be used as an important prognostic marker.

Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis.

BACKGROUND: Toll-like receptors (TLRs) have garnered an extraordinary amount of interest in cancer research due to their role in tumor progression. By activating the production of several biological factors, TLRs induce type I interferons and other cytokines, which drive an inflammatory response and activate the adaptive immune system. The aim of this study was to investigate the expression and clinical relevance of TLR3, 4 and 9 in breast cancer. METHODS: The expression levels of TLR3, TLR4 and TLR9 were analyzed on tumors from 74 patients with breast cancer. The analysis was performed by immunohistochemistry. RESULTS: Samples of carcinomas with recurrence exhibited a significant increase in the mRNA levels of TLR3, TLR4 and TLR9. Tumors showed high expression of TLRs expression levels by cancer cells, especially TLR4 and 9. Nevertheless, a significant percentage of tumors also showed TLR4 expression by mononuclear inflammatory cells (21.6%) and TLR9 expression by fibroblast-like cells (57.5%). Tumors with high TLR3 expression by tumor cell or with high TLR4 expression by mononuclear inflammatory cells were significantly associated with higher probability of metastasis. However, tumours with high TLR9 expression by fibroblast-like cells were associated with low probability of metastasis. CONCLUSIONS: The expression levels of TLR3, TLR4 and TLR9 have clinical interest as indicators of tumor aggressiveness in breast cancer. TLRs may represent therapeutic targets in breast cancer.

Comparative analysis and clinical value of the expression of metalloproteases and their inhibitors by intratumour stromal mononuclear inflammatory cells and those at the invasive front of breast carcinomas.

AIMS: Matrix metalloproteases (MMPs) and their inhibitors (TIMPs) play an essential role in the degradation of stromal connective tissue and basement membrane components. The aim of this study was to determine whether the dynamic analysis of these components can help to predict tumour aggressiveness. METHODS AND RESULTS: An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs -1, -2, -7, -9, -11, -13 and -14 and TIMPs -1, -2 and -3. More than 5000 determinations on cancer specimens from 124 patients with invasive breast cancer were performed on the tumour centre core as well as on the invasive front. Immunostaining for MMPs/TIMPs on mononuclear inflammatory cells (MICs) was evaluated. To identify specific groups of tumours with distinct expression profiles, data obtained from both MICs populations were analysed by unsupervised hierarchical cluster analysis. When compared with MICs at the invasive front, intratumour MICs more frequently showed expression of MMP-7 and -1 and TIMP-3, but less frequently expression of MMP-9 and -11 and TIMP-2. CONCLUSIONS: Our data led us to consider the need of further studies in order to identify subsets of MICs and other protein elements of the microenvironment as attractive targets for new therapeutic strategies against cancer.

Immunohistochemical study of matrix metalloproteinases and their inhibitors in pure and mixed invasive and in situ ductal carcinomas of the breast.

We assessed differences in the patterns of expression of matrix metalloproteases and their inhibitors (tissue inhibitors of metalloproteases) in ductal carcinoma in situ alone and admixed with invasive ductal carcinomas (n = 40), as well as in pure invasive ductal carcinomas (n = 40), immunohistochemically and using tissue arrays. The invasive ductal carcinoma components showed higher expression of matrix metalloprotease-9 and -13 than did the admixed ductal carcinoma in situ, whereas stromal fibroblasts of the invasive components showed higher expression of matrix metalloprotease-2, -7, -9, -13, and -14 and tissue inhibitor of metalloprotease-1 and -3 than did fibroblasts around the neoplastic ducts of the admixed ductal carcinoma in situ. Expression of matrix metalloprotease-14 and tissue inhibitor of metalloprotease-3 was significantly higher in the mononuclear inflammatory cells of the invasive components. By contrast, matrix metalloprotease-1 expression was significantly higher in stromal cells of the ductal carcinoma in situ admixed with invasive ductal carcinoma. The pure invasive ductal carcinomas had significantly higher expression of matrix metalloprotease-1, -9, -11, and -14 and tissue inhibitor of metalloprotease-1 and -3 than the invasive ductal carcinomas admixed with ductal carcinoma in situ. Our findings indicate a significant association of matrix metalloprotease expression by the periductal stromal cells of the ductal carcinoma in situ component of mixed tumors and the occurrence of distant metastasis. Our data suggest that the molecular matrix metalloprotease/tissue inhibitor of metalloprotease profile can contribute to better characterization of early breast carcinomas.

Expression of metalloproteases and their inhibitors in primary tumors and in local recurrences after mastectomy for breast cancer.

AIMS: To investigate the expression of matrix metalloproteases (MMPs) and their inhibitors (TIMPs) in patients who develop local recurrence (LR) after mastectomy. METHODS: We analyzed the expressions of MMP-1, -2, -7, -9, -11, -13, -14, TIMP-1, -2, and -3, using immunohistochemical techniques, in primary tumors from patients without tumoral recurrence (n = 50), patients who developed distant metastasis (n = 50), and from patients who develop LRs (n = 25). LRs of the latter group were also analyzed for MMPs expression. All the patients underwent mastectomy. RESULTS: Score values for all MMPs and TIMPs were significantly higher in primary tumors of patients with distant metastasis. Primary tumors from patients with LR have lower expressions of MMPs and TIMPs compared with those from patients who developed distant metastasis, and with patients without recurrence for some MMPs. Remarkably, however, primary tumors from patients with LR showed significantly higher percentage of TIMP-1 and 2 expression in stromal cells compared to primary tumors from patients with distant metastasis or primary tumors from patients without tumoral progression. Furthermore, LRs had significantly higher MMP-9 expression than their corresponding primary tumors. CONCLUSIONS: Our data indicate differences in MMPs/TIMPs expression between primary tumors of patients with LRs and of those with distant metastasis, both after mastectomy for breast cancer.

Expression and prognostic significance of metalloproteases and their inhibitors in luminal A and basal-like phenotypes of breast carcinoma.

To analyze the expression and prognostic value of matrix metalloproteases and their tissue inhibitors in luminal A and basal-like breast carcinomas, an immunohistochemical study was performed on cancer specimens from 93 randomly selected patients with invasive primary ductal tumors of the breast (46 with and 47 without distant metastasis) and with luminal A (n = 48) (ER+, HER2-) or basal-like (HER2-, ER-, PgR-) (n = 45) lesions. Luminal B cases were too few to analyze. Specimens were also studied using tissue microarrays and specific antibodies against matrix metalloproteases 1, 2, 7, 9, 11, 13, and 14 and tissue inhibitors 1, 2, and 3. There were no significant differences in matrix metalloprotease or tissue inhibitor expression in the 2 phenotypes of tumors. In basal-like carcinomas, high scores for matrix metalloproteases 9 and 11 were significantly associated with a high distant metastasis rate. Likewise, data showed associations between matrix metalloprotease/tissue inhibitor expression by either stromal fibroblasts or mononuclear inflammatory cells and distant relapse-free survival in both tumor phenotypes. In addition, in infiltrating luminal A and basal-like tumors, we identified a prometastatic phenotype of mononuclear inflammatory cells, showing a high matrix metalloprotease/tissue inhibitor molecular profile. Expression of matrix metalloproteases and tissue inhibitors is related to the characteristics of breast tumor cells. As prognostic factors in breast carcinomas of both luminal A and basal-like phenotypes, our results point to the importance of the expression of matrix metalloproteases and tissue inhibitors by the stromal cells.

Lymphatic and/or blood vessel invasion in gastric cancer: relationship with clinicopathological parameters, biological factors and prognostic significance.

BACKGROUND: Lymphatic and/or blood vessel tumoral invasion (LBVI) is a common histopathologic finding of gastric carcinomas, which could make it an additional cost efficient marker and help in the detection of patients at risk for recurrence. MATERIALS AND METHODS: The subjects of this study were 144 patients with primary gastric adenocarcinoma, who consecutively underwent surgery. LBVI was evaluated by H&E staining and complementary with immunohistochemical staining with anti-CD34. Intratumoral levels of EGFR were analyzed with a radioligand technique, whereas c-erbB-2 and tPA were determined by ELISA methods; pS2, cathepsin D and hyaluronic acid by immunoradiometric assays; and VEGFR-1 and -2 by immunohistochemical assays. The mean follow-up period for these patients was 33.1 months. RESULTS: LBVI was present in 46 patients (31.9%). The presence of LBVI correlated significantly with tumor stage, lymph node involvement, surgical resectability, histological type and histological grade, being present in a higher percentage among II-IV tumor stage (P = 0.0001), poorly differentiated (P = 0.01), diffuse type (P = 0.009), R1-R2 (P = 0.002) and lymph node-positive (P = 0.005) tumors. In addition, statistical analysis demonstrated that LBVI was significantly associated with a poorer overall patients' survival in the univariate analysis (P = 0.0001) as well as in the multivariate analysis (P = 0.009). However, our results failed to show any significant relationship between LBVI and any of the intratumoral biological parameters studied. CONCLUSION: LBVI provides additional useful information that could be applied to identify gastric cancer patients at risk for recurrence, who might be candidates for further adjuvant therapies.

Local recurrence after mastectomy for breast cancer: analysis of clinicopathological, biological and prognostic characteristics.

BACKGROUND: Despite the increasing use of breast-conserving therapy, modified radical mastectomy retains an important role in primary as well as in salvage treatment of breast cancer. Nevertheless, a significant number of patients will eventually develop a local recurrence (LR). AIMS: To identify the potential prognostic factors at the time of the first isolated LR, and to compare the expression of several parameters of the molecular biology of breast carcinomas by primary tumors and paired isolated LRs. METHODS: We analyzed the medical records from 1,087 women who underwent mastectomy for breast cancer, out of which 98 developed LRs as the first manifestation of tumor progression. We investigated the prognostic value of various classical prognostic factors, at the time of mastectomy as well as when the diagnosis of LR was made. In addition, by using tissue microarrays and immunohistochemical techniques, we analyzed the expression of estrogen (ER), progesterone (PR) and androgen receptors (AR), ki67, p53, c-erbB-2 and apolipoprotein D in primary tumors and paired isolated LRs from a subset of patients (n = 25). RESULTS: Patients who developed distant metastases as well as patients with local recurrent disease showed a significantly higher percentage of larger tumors, node-positive status and higher tumoral grade than patients without evidence of tumoral recurrence. Furthermore, patients with LR had a better outcome compared with those with distant metastases, although the former received less frequently adjuvant systemic therapy and/or radiotherapy. Tumor size, histological grade, ER and PR status, and a shorter disease-free interval (<12 months) were significantly associated with overall survival amongst mastectomized patients that developed isolated LR. There was a significant concordance between primary tumors and LRs regarding the expression of the following factors: ER, PR and p53. However, we were not able to demonstrate similar findings for AR, c-erbB-2 and ki67. In addition, ER, PR and p53 status in the LRs were significantly associated with a poorer overall survival. CONCLUSIONS: Based on classical clinicopathological factors as well as on some new biological parameters we have been able to identify subgroups of mastectomized patients with LR differing in their prognosis. Thus, at the present time it would be possible to select group of patients candidates for further and individualized therapeutic strategies.