Tumors are stiff and data suggest that the extracellular matrix stiffening that correlates with experimental mammary malignancy drives tumor invasion and metastasis. Nevertheless, the relationship between tissue and extracellular matrix stiffness and human breast cancer progression and aggression remains unclear. We undertook a biophysical and biochemical assessment of stromal-epithelial interactions in noninvasive, invasive and normal adjacent human breast tissue and in breast cancers of increasingly aggressive subtype. Our analysis revealed that human breast cancer transformation is accompanied by an incremental increase in collagen deposition and a progressive linearization and thickening of interstitial collagen. The linearization of collagen was visualized as an overall increase in tissue birefringence and was most striking at the invasive front of the tumor where the stiffness of the stroma and cellular mechanosignaling were the highest. Amongst breast cancer subtypes we found that the stroma at the invasive region of the more aggressive Basal-like and Her2 tumor subtypes was the most heterogeneous and the stiffest when compared to the less aggressive Luminal A and B subtypes. Intriguingly, we quantified the greatest number of infiltrating macrophages and the highest level of TGF beta signaling within the cells at the invasive front. We also established that stroma stiffness and the level of cellular TGF beta signaling positively correlated with each other and with the number of infiltrating tumor-activated, macrophages, which was highest in the more aggressive tumor subtypes. These findings indicate that human breast cancer progression and aggression, collagen linearization and stromal stiffening are linked and implicate tissue inflammation and TGF beta.

Retrospective clinical studies have used immune-based biomarkers, alone or in combination, to predict survival outcomes for women with breast cancer (BC); however, the limitations inherent to immunohistochemical analyses prevent comprehensive descriptions of leukocytic infiltrates, as well as evaluation of the functional state of leukocytes in BC stroma. To more fully evaluate this complexity, and to gain insight into immune responses after chemotherapy (CTX), we prospectively evaluated tumor and nonadjacent normal breast tissue from women with BC, who either had or had not received neoadjuvant CTX before surgery. Tissues were evaluated by polychromatic flow cytometry in combination with confocal immunofluorescence and immunohistochemical analysis of tissue sections. These studies revealed that activated T lymphocytes predominate in tumor tissue, whereas myeloid lineage cells are more prominant in “normal” breast tissue. Notably, residual tumors from an unselected group of BC patients treated with neoadjuvant CTX contained increased percentages of infiltrating myeloid cells, accompanied by an increased CD8/CD4 T-cell ratio and higher numbers of granzyme B-expressing cells, compared with tumors removed from patients treated primarily by surgery alone. These data provide an initial evaluation of differences in the immune microenvironment of BC compared with nonadjacent normal tissue and reveal the degree to which CTX may alter the complexity and presence of selective subsets of immune cells in tumors previously treated in the neoadjuvant setting.

Neoadjuvant chemotherapy for breast cancer allows individual tumor response to be assessed depending on molecular subtype, and to judge the impact of response to therapy on recurrence-free survival (RFS). The multicenter I-SPY 1 TRIAL evaluated patients with ≥3 cm tumors by using early imaging and molecular signatures, with outcomes of pathologic complete response (pCR) and RFS. The current analysis was performed using data from patients who had molecular profiles and did not receive trastuzumab. The various molecular classifiers tested were highly correlated. Categorization of breast cancer by molecular signatures enhanced the ability of pCR to predict improvement in RFS compared to the population as a whole. In multivariate analysis, the molecular signatures that added to the ability of HR and HER2 receptors, clinical stage, and pCR in predicting RFS included 70-gene signature, wound healing signature, p53 mutation signature, and PAM50 risk of recurrence. The low risk signatures were associated with significantly better prognosis, and also identified additional patients with a good prognosis within the no pCR group, primarily in the hormone receptor positive, HER-2 negative subgroup. The I-SPY 1 population is enriched for tumors with a poor prognosis but is still heterogeneous in terms of rates of pCR and RFS. The ability of pCR to predict RFS is better by subset than it is for the whole group. Molecular markers improve prediction of RFS by identifying additional patients with excellent prognosis within the no pCR group.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-011-1895-2) contains supplementary material, which is available to authorized users.

The purpose of this study is to determine the biologic impact of short-term lipophilic statin exposure on in situ and invasive breast cancer through paired tissue, blood and imaging-based biomarkers. A perioperative window trial of fluvastatin was conducted in women with a diagnosis of DCIS or stage 1 breast cancer. Patients were randomized to high dose (80 mg/day) or low dose (20 mg/day) fluvastatin for 3–6 weeks before surgery. Tissue (diagnostic core biopsy/final surgical specimen), blood, and magnetic resonance images were obtained before/after treatment. The primary endpoint was Ki-67 (proliferation) reduction. Secondary endpoints were change in cleaved caspase-3 (CC3, apoptosis), MRI tumor volume, and serum C-reactive protein (CRP, inflammation). Planned subgroup analyses compared disease grade, statin dose, and estrogen-receptor status. Forty of 45 patients who enrolled completed the protocol; 29 had paired Ki-67 primary endpoint data. Proliferation of high grade tumors decreased by a median of 7.2% (P = 0.008), which was statistically greater than the 0.3% decrease for low grade tumors. Paired data for CC3 showed tumor apoptosis increased in 38%, remained stable in 41%, and decreased in 21% of subjects. More high grade tumors had an increase in apoptosis (60 vs. 13%; P = 0.015). Serum CRP did not change, but cholesterol levels were significantly lower post statin exposure (P<0.001). Fluvastatin showed measurable biologic changes by reducing tumor proliferation and increasing apoptotic activity in high-grade, stage 0/1 breast cancer. Effects were only evident in high grade tumors. These results support further evaluation of statins as chemoprevention for ER-negative high grade breast cancers.

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SUMMARY Early full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women, and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44+ progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44+p27+ cells in parous women, and showed using explant cultures that parity-related signaling pathways play a role in regulating the number of p27+ cells and their proliferation. Our results suggest that pathways controlling p27+ mammary epithelial cells and the numbers of these cells relate to breast cancer risk, and can be explored for cancer risk assessment and prevention.

Purpose The recent increase in the incidence of ductal carcinoma in situ (DCIS) has sparked debate over the classification and treatment of this disease. Although DCIS is considered a precursor lesion to invasive breast cancer, some DCIS may have more or less risk than is realized. In this study, we characterized the immune microenvironment in DCIS to determine if immune infiltrates are predictive of recurrence. Methods Fifty-two cases of high-grade DCIS (HG-DCIS), enriched for large lesions and a history of recurrence, were age matched with 65 cases of non-high-grade DCIS (nHG-DCIS). Immune infiltrates were characterized by single- or dual-color staining of FFPE sections for the following antigens: CD4, CD8, CD20, FoxP3, CD68, CD115, Mac387, MRC1, HLA-DR, and PCNA. Nuance multispectral imaging software was used for image acquisition. Protocols for automated image analysis were developed using CellProfiler. Immune cell populations associated with risk of recurrence were identified using classification and regression tree analysis. Results HG-DCIS had significantly higher percentages of FoxP3+ cells, CD68+ and CD68+PCNA+ macrophages, HLA-DR+ cells, CD4+ T cells, CD20+ B cells, and total tumor infiltrating lymphocytes (TILs) compared to nHG-DCIS. A classification tree, generated from 16 immune cell populations and 8 clinical parameters, identified three immune cell populations associated with risk of recurrence: CD8+HLADR+ T cells, CD8+HLADR− T cells, and CD115+ cells. Conclusion These findings suggest that the tumor immune microenvironment is an important factor in identifying DCIS cases with the highest risk for recurrence and that manipulating the immune microenvironment may be an efficacious strategy to alter or prevent disease progression.

IntroductionThe molecular biology involving neoadjuvant chemotherapy (NAC) response is poorly understood. To elucidate the impact of NAC on the breast cancer transcriptome and its association with clinical outcome, we analyzed gene expression data derived from serial tumor samples of patients with breast cancer who received NAC in the I-SPY 1 TRIAL.MethodsExpression data were collected before treatment (T1), 24–96 hours after initiation of chemotherapy (T2) and at surgery (TS). Expression levels between T1 and T2 (T1 vs. T2; n = 36) and between T1 and TS (T1 vs. TS; n = 39) were compared. Subtype was assigned using the PAM50 gene signature. Differences in early gene expression changes (T2 − T1) between responders and nonresponders, as defined by residual cancer burden, were evaluated. Cox proportional hazards modeling was used to identify genes in residual tumors associated with recurrence-free survival (RFS). Pathway analysis was performed with Ingenuity software.ResultsWhen we compared expression profiles at T1 vs. T2 and at T1 vs. TS, we detected significantly altered expression of 150 and 59 transcripts, respectively. We observed notable downregulation of proliferation and immune-related genes at T2. Lower concordance in subtype assignment was observed between T1 and TS (62 %) than between T1 and T2 (75 %). Analysis of early gene expression changes (T2 − T1) revealed that decreased expression of cell cycle inhibitors was associated with poor response. Increased interferon signaling (TS − T1) and high expression of cell proliferation genes in residual tumors (TS) were associated with reduced RFS.ConclusionsSerial gene expression analysis revealed candidate immune and proliferation pathways associated with response and recurrence. Larger studies incorporating the approach described here are warranted to identify predictive and prognostic biomarkers in the NAC setting for specific targeted therapies.Clinical trial registrationClinicalTrials.gov identifier: NCT00033397. Registered 9 Apr 2002.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0582-3) contains supplementary material, which is available to authorized users.