Maspin influences response to doxorubicin by changing the tumor microenvironment organization.

Altered degradation and deposition of extracellular matrix are hallmarks of tumor progression and response to therapy. From a microarray supervised analysis on a dataset of chemotherapy-treated breast carcinoma patients, maspin, a member of the serpin protease inhibitor family, has been the foremost variable identified in non-responsive versus responsive tumors. Accordingly, in a series of 52 human breast carcinomas, we detected high maspin expression in tumors that progressed under doxorubicin (DXR)-based chemotherapy. Our analysis of the role of maspin in response to chemotherapy in human MCF7 and MDAMB231 breast and SKOV3 ovarian carcinoma cells transfected to overexpress maspin and injected into mice showed that maspin overexpression led to DXR resistance through the maspin-induced collagen-enriched microenvironment and that an anti-maspin neutralizing monoclonal antibody reversed the collagen-dependent DXR resistance. Impaired diffusion and decreased DXR activity were also found in tumors derived from Matrigel-embedded cells, where abundant collagen fibers characterize the tumor matrix. Conversely, liposome-based DXR reached maspin-overexpressing tumor cells despite the abundant extracellular matrix and was more efficient in reducing tumor growth. Our results identify maspin-induced accumulation of collagen fibers as a cause of disease progression under DXR chemotherapy for breast cancer. Use of a more hydrophilic DXR formulation or of a maspin inhibitor in combination with chemotherapy holds the promise of more consistent responses to maspin-overexpressing tumors and dense-matrix tumors in general.

Neoplastic and stromal cells contribute to an extracellular matrix gene expression profile defining a breast cancer subtype likely to progress.

We recently showed that differential expression of extracellular matrix (ECM) genes delineates four subgroups of breast carcinomas (ECM1, -2, -3- and -4) with different clinical outcome. To further investigate the characteristics of ECM signature and its impact on tumor progression, we conducted unsupervised clustering analyses in 6 additional independent datasets of invasive breast tumors from different platforms for a total of 643 samples. Use of four different clustering algorithms identified ECM3 tumors as an independent group in all datasets tested. ECM3 showed a homogeneous gene pattern, consisting of 58 genes encoding 43 structural ECM proteins. From 26 to 41% of the cases were ECM3-enriched, and analysis of datasets relevant to gene expression in neoplastic or corresponding stromal cells showed that both stromal and breast carcinoma cells can coordinately express ECM3 genes. In in vitro experiments, ß-estradiol induced ECM3 gene production in ER-positive breast carcinoma cell lines, whereas TGFß induced upregulation of the genes leading to ECM3 gene classification, especially in ER-negative breast carcinoma cells and in fibroblasts. Multivariate analysis of distant metastasis-free survival in untreated breast tumor patients revealed a significant interaction between ECM3 and histological grade (p = 0.001). Cox models, estimated separately in grade I-II and grade III tumors, indicated a highly significant association between ECM3 and worse survival probability only in grade III tumors (HR = 3.0, 95% CI = 1.3-7.0, p = 0.0098). Gene Set Enrichment analysis of ECM3 compared to non-ECM3 tumors revealed significant enrichment of epithelial-mesenchymal transition (EMT) genes in both grade I-II and grade III subsets of ECM3 tumors. Thus, ECM3 is a robust cluster that identifies breast carcinomas with EMT features but with accelerated metastatic potential only in the undifferentiated (grade III) phenotype. These findings support the key relevance of neoplastic and stroma interaction in breast cancer progression.

ELISPOT-IFN-gamma assay instead of tuberculin skin test for detecting latent Mycobacterium tuberculosis infection in rheumatic patients candidate to anti-TNF-alpha treatment.

In rheumatic patients candidate to anti-TNF-alpha treatment, there is an increased risk of developing tuberculosis (TB). The tuberculin skin test (TST), the standard diagnostic test for latent tuberculosis infection (LTBI), suffers low specificity and sensitivity. Here, we compared the performance characteristics of an in-house ELISPOT-IFN-gamma assay (using a restricted pool of Mycobacterium tuberculosis-specific peptides or MTP) to TST for the diagnosis of LTBI in 69 rheumatic patients candidate to anti-TNF-alpha treatment and in 60 healthy LTBI individuals. Among the 69 patients enrolled, 17 (25%) had a positive TST response and 15 (22%) a positive ELISPOT-MTP response. Among the patients with a positive TST result, eight had a positive and nine a negative ELISPOT-MTP response, whereas among the 49 patients with a negative TST result, 42 were ELISPOT-MTP negative, but seven (14%) were ELISPOT-MTP positive, with three indeterminate results. The agreement between the two tests was poor (k = 0.341, 95% CI = 0.060 to 0.622) and the test of symmetry was not significant (P = 0.8). Considering the ELISPOT assay, rheumatic patients had a reduced number of spot-forming cells after stimulation of lymphocytes with PHA or PPD when compared with healthy LTBI individuals. Thus, the ELISPOT-IFN-gamma assay performs better than the TST in recognizing patients with LTBI, on one hand reducing the number of patients submitted to isoniazid prophylaxis, and on the other hand, since the assay is less biased by immunosuppressive regimens than TST, recognizing LTBI patients among those with a negative TST response.