Clever-1 positive macrophages in breast cancer.

PURPOSE: Common Lymphatic Endothelial and Vascular Endothelial Receptor 1 (Clever-1) is expressed by a subset of immunosuppressive macrophages and targeting the receptor with therapeutic antibodies has been shown to activate T-cell-mediated anti-cancer immunity. The aim of this research was to study Clever-1 expression in breast cancer. Specifically, how Clever-1 + macrophages correlate with clinicopathologic factors, Tumor Infiltrating Lymphocytes (TILs) and prognosis. METHODS: Tissue microarray blocks were made from 373 primary breast cancer operation specimens. Hematoxylin and Eosin (H&E-staining) and immunohistochemical staining with Clever-1, CD3, CD4 and CD8 antibodies were performed. Differences in quantities of Clever-1 + macrophages and TILs were analyzed. Clever-1 + cell numbers were correlated with 25-year follow-up survival data and with breast cancer clinicopathologic parameters. RESULTS: Low numbers of intratumoral Clever-1 + cells were found to be an independent adverse prognostic sign. Increased numbers of Clever-1 + cells were found in high grade tumors and hormone receptor negative tumors. Tumors that had higher amounts of Clever-1 + cells also tended to have higher amounts of TILs. CONCLUSION: The association of intratumoral Clever-1 + macrophages with better prognosis might stem from the function of Clever as a scavenger receptor that modulates tumor stroma. The association of Clever-1 + macrophages with high number of TILs and better prognosis indicates that immunosuppression by M2 macrophages is not necessarily dampening adaptive immune responses but instead keeping them in control to avoid excess inflammation.

Maintenance of hormone responsiveness in luminal breast cancers by suppression of Notch.

Luminal breast cancers express estrogen (ER) and/or progesterone (PR) receptors and respond to hormone therapies. Basal-like "triple negative" cancers lack steroid receptors but are cytokeratin (CK) 5-positive and require chemotherapy. Here we show that more than half of primary ER(+)PR(+) breast cancers contain an ER(-)PR(-)CK5(+) "luminobasal" subpopulation exceeding 1% of cells. Starting from ER(+)PR(+) luminal cell lines, we generated lines with varying luminal to luminobasal cell ratios and studied their molecular and biological properties. In luminal disease, luminobasal cells expand in response to antiestrogen or estrogen withdrawal therapies. The phenotype and gene signature of the hormone-resistant cells matches that of clinical triple negative basal-like and claudin-low disease. Luminobasal cell expansion in response to hormone therapies is regulated by Notch1 signaling and can be blocked by γ-secretase inhibitors. Our data establish a previously unrecognized plasticity of ER(+)PR(+) luminal breast cancers that, without genetic manipulation, mobilizes outgrowth of hormone-resistant basal-like disease in response to treatment. This undesirable outcome can be prevented by combining endocrine therapies with Notch inhibition.

Luminal breast cancer metastases and tumor arousal from dormancy are promoted by direct actions of estradiol and progesterone on the malignant cells.

INTRODUCTION: Luminal, estrogen receptor-positive (ER(+)) breast cancers can metastasize but lie dormant for years before recurrences prove lethal. Understanding the roles of estrogen (E) or progestin (P) in development of luminal metastases or in arousal from dormancy is hindered by few preclinical models. We have developed such models. METHODS: Immunocompromised, ovariectomized (ovx'd) mice were intracardiac-injected with luminal or basal human breast cancer cells. Four lines were tested: luminal ER(+)PR(+) cytokeratin 5-negative (CK5(-)) E3 and MCF-7 cells, basal ER(-)PR(-)CK5(+) estrogen withdrawn-line 8 (EWD8) cells, and basal ER(-)PR(-)CK5(-) MDA-MB-231 cells. Development of micrometastases or macrometastases was quantified in ovx'd mice and in mice supplemented with E or P or both. Metastatic deposits were analyzed by immunohistochemistry for luminal, basal, and proliferation markers. RESULTS: ER(-)PR(-) cells generated macrometastases in multiple organs in the absence or presence of hormones. By contrast, ovx'd mice injected with ER(+)PR(+) cells appeared to be metastases-free until they were supplemented with E or E+P. Furthermore, unlike parental ER(+)PR(+)CK5(-) cells, luminal metastases were heterogeneous, containing a significant (6% to 30%) proportion of non-proliferative ER(-)PR(-)CK5(+) cells that would be chemotherapy-resistant. Additionally, because these cells lack receptors, they would also be endocrine therapy-resistant. With regard to ovx'd control mice injected with ER(+)PR(+) cells that appeared to be metastases-free, systematic pathologic analysis of organs showed that some harbor a reservoir of dormant micrometastases that are ER(+) but PR(-). Such cells may also be endocrine therapy- and chemotherapy-resistant. Their emergence as macrometastases can be triggered by E or E+P restoration. CONCLUSIONS: We conclude that hormones promote development of multi-organ macrometastases in luminal disease. The metastases display a disturbing heterogeneity, containing newly emergent ER(-)PR(-) subpopulations that would be resistant to endocrine therapy and chemotherapy. Similar cells are found in luminal metastases of patients. Furthermore, lack of hormones is not protective. While no overt metastases form in ovx'd mice, luminal tumor cells can seed distant organs, where they remain dormant as micrometastases and sheltered from therapies but arousable by hormone repletion. This has implications for breast cancer survivors or women with occult disease who are prescribed hormones for contraception or replacement purposes.

Core needle biopsy changes the expression of TGFß1 and TGFßRII at protein level, and the distribution of CD4 and CD8 positive T cells in primary breast cancer.

Core needle biopsy (CNB) has become a paradigm in preoperative breast cancer (BC) diagnosis. Although considered safe, it is an invasive procedure, which changes the tumor microenvironment. It facilitates a tumor supportive immune response, induces epithelial-mesenchymal transition (EMT), and enables the release of circulating tumor cells. The cytokine Transforming Growth Factor ß (TGFß) with its pleiotropic immunologic functions has an important role in this process. The aim of this study was to clarify the specific impact of CNB on the activity of the TGFß pathway in early BC. We compared formalin fixed paraffin embedded samples from CNBs to the corresponding surgical resection specimens (SRSs) of 49 patients with BC. We found that the expression of TGFß1 at protein level was significantly higher in both tumor epithelial and benign stromal cells in the SRSs (p=0.001), whereas the expression of TGFßRII in tumor cells was lower (p=0.001). The frequency of intra tumoral CD8 and CD4 positive T lymphocytes was lower in SRSs (p=0081 and p=0001, respectively), while in the peripheral stroma their prevalence was increased (p=0001 and p=0012, respectively). Our results show that CNB changes the hallmarks of the TGFß path way in early BC. These CNB-induced changes in the tumor and in its microenvironment suggest that the procedure may change the immunological anti-tumor response of the host.

[Dormancy of tumor cells]. / Kasvainsolujen uinuminen.

Whereas tumor dormancy occurs in melanomas, renal carcinomas and non-Hodgkin lymphomas, late recurrence is characteristic of breast cancer in particular. In the early stage, cancer cells break away from the primary tumor into the circulation, some of which are able to attach themselves to a new target tissue and avoid apoptosis. This condition represents dormancy of tumor cells, in which the cell cycle has either stopped or cell division and cell death are in mutual balance. Cessation of the dormancy state and the growth spurt that follows may result from the initiation of angiogenesis (angiogenic switch).

Quantities of CD3+, CD8+ and CD56+ lymphocytes decline in breast cancer recurrences while CD4+ remain similar.

BACKGROUND: Much is known about tumor infiltrating lymphocytes (Tils) in primary breast cancer, as this has been the focus of much research in recent years, but regarding recurrent breast cancer, only few studies have been done. Our aim was to compare the quantities of Tils in primary breast carcinomas and their corresponding recurrences and to analyze the differences in the tumor Tils compositions in correlations with recurrence-free times and the clinicopathology of the tumor. METHODS: One hundred thirty-seven breast cancer patients self-paired for primary- tumor-recurrence were divided into three groups based on the length of the recurrence-free interval. H&E-staining and immunohistochemical staining with antiCD3, antiCD4, antiCD8 and antiCD56 were performed. Differences in Tils between primaries and recurrences, between the recurrence-free interval groups, and between different clinicopathologic parameters were statistically analyzed. RESULTS: Fewer stromal CD3+, CD8+ and CD56+ lymphocytes were found at recurrences compared to the primaries. No significant change in the percentage of CD4+ stromal lymphocytes. ER-negative primaries, PR-negative or HER2-positive tumors had more Tils in some subgroups. Ductal primaries had more Tils than lobular primaries and G3 tumors had more Tils than lower-grade tumors. The corresponding differences at recurrences could either not be detected or they were reversed. The fastest recurring group had generally more Tils than the slower groups. CONCLUSIONS: CD4+ cell numbers did not decline from primary to recurrence in contrast to all other subclasses of lymphocytes. The proportion of CD4+ cells was higher in recurrences than in primaries. Tumors with a higher grade and proliferation rate had higher percentages of Tils. HER2+ and hormone receptor negative tumors tended to have higher Tils scores. In recurrences these differences were not seen or they were reversed.

Core needle biopsies alter the amounts of CCR5, Siglec-15, and PD-L1 positivities in breast carcinoma.

Core needle biopsies (CNB) are widely used to diagnose breast cancer, but the procedure is invasive and thus, it changes the tumor microenvironment. The purpose of this study is to see how the expression of three potentially anti-inflammatory molecules, namely, programmed death-ligand 1 (PD-L1), sialic acid-binding immunoglobulin-like lectin-15 (Siglec-15), and C-C chemokine receptor-5 (CCR-5), are expressed in CNB and surgical resection specimens (SRS). To do this, we compared the amounts of tumor-infiltrating lymphocytes and the levels of CCR5, Siglec-15, and PD-L1 in tumor cells and inflammatory cells as assessed by immunohistochemistry in CNB and the corresponding SRS of 22 invasive breast carcinomas of no special type and 22 invasive lobular carcinomas. The Siglec-15 H-score was higher in tumor cells in the SRS than in the CNB groups. There was no change in tumor cells CCR5 or PD-L1 between CNB and SRS. The positive inflammatory cell numbers for all markers rose between CNB and SRS, as did the amount of Tils. Furthermore, higher grade tumors and tumors with a high proliferation rate had more inflammatory cells that were positive for the markers and also more PD-L1+ tumor cells. Although changes in inflammatory cells can partly be attributed to the larger sample size of operation specimens, the differences also mirror a true change in the tumor microenvironment. The changes in inflammatory cells could be partly due to the need to restrict excess inflammation at the site of the biopsy.

High expression of CCL2 in tumor cells and abundant infiltration with CD14 positive macrophages predict early relapse in breast cancer.

Macrophages are important for the function of the innate immune system, and in solid tumors, they represent a significant proportion of the tumor mass. Tumor-associated macrophages (TAM) have a M2 phenotype and show a multitude of pro-tumoral functions, promoting tumor cell survival, proliferation, and dissemination. CCL2, synthesized by tumor and stromal cells, initiates a chemokine cascade inducing these processes. We studied by immunohistochemistry (IHC) the frequency of TAMs and CCL2 expressing cells in three groups of primary tumor (PT)-recurrence (R) pairs, where relapse was recorded within 2 years (group 1), between 5 and 10 years (group 2), and after 10 years (group 3). In our study all established breast cancers were heavily infiltrated by CD68 positive cells. Both in PTs and in R lesions the infiltration was more abundant in the peritumoral than in the intratumoral stroma. The mean frequency of M2 marker and CD14 positive cells in the intratumoral stroma and CCL2 expressing tumor cells was higher in the Rs as compared to the corresponding PTs. In PTs, a high frequency of CD14 positive cells and a high expression of CCL2 by tumor cells was associated with an early recurrence. The findings support the current understanding of immune cell orchestrated development, progression and metastatic spread of breast cancer. Our study showed that a high frequency of CCL2 positive tumor cells and CD14 positive TAMs are significant risk factors for rapid tumor recurrence. Potential targets for intervention are discussed.

Tumor dormancy: elevated expression of stanniocalcins in late relapsing breast cancer.

BACKGROUND: Breast cancer is known for its propensity to recur even after decades. The biology behind this phenomenon of tumor dormancy is poorly understood. The stanniocalcins (stanniocalcin-1, STC-1 and stanniocalcin-2, STC-2) are 56kDa homodimeric proteins. They act as pro-survival factors and contribute to the endurance of terminally differentiated cells such as neurons and adipocytes. We investigated whether elevated expression of stanniocalcins also plays a part in the tumor dormancy of breast cancer. METHODS: The expression of STC-1, STC-2 and estrogen receptor (ER) was studied by immunohistochemistry in 72 primary breast cancers and in their metastatic relapses detected before two years, or after 5 or 10 years from primary surgery. RESULTS: When compared to primary tumors with early relapse and their metastases, the expression of STC-1 and STC-2 was significantly higher in relapses occurring after five year (STC-1 p=0.0012, STC-2 p=0.004) and even higher in very late relapses occurring 10 years after surgery (STC-1 p=0.0017, STC-2 p=0.0001). Moreover, primary tumors with a propensity of very late relapse displayed a higher initial expression of STC-2 (p=0.0001). A significantly increased frequency of ER expression was found in the very late relapses. CONCLUSION: These findings suggest that elevated expression of STC-1 or STC-2 act as survival factors also for breast cancer cells and thereby contribute to tumor dormancy.

Different Expression Patterns of CXCR4, CCR7, Maspin and FOXP3 in Luminal Breast Cancers and Their Sentinel Node Metastases.

AIM: Luminal A breast cancers (BC) represent low-risk tumors conferring better outcome than luminal B and human epidermal growth factor 2 (HER2)-positive or triple-negative tumors. One reason for the heterogeneous outcome among patients with luminal BC is the variation in cell proliferation. As chemokine receptors and tumor suppressors show potential for estimation of infiltration to regional lymph nodes, we aimed to compare differently sized sentinel node metastases with their primary tumors (PT). MATERIALS AND METHODS: We compared 29 BCs of luminal subtype A and 23 of subtype B (Ki-67 cut off at 14%) by immunohistochemistry for the chemokine receptors C-X-C chemokine receptor 4 (CXCR4), C-C-chemokine receptor 7 (CCR7), the tumor suppressor Maspin and the regulatory T-cell immunosuppressor forkhead box protein 3 (FOXP3) between PTs and their metastases of different size. RESULTS: Expression of CXCR4 was low in luminal A type tumors, and CCR7 and FOXP3 expression were high in luminal B type cancer. CXCR4 expression significantly positively correlated with CCR7 both in PTs and metastases. Most Maspin-positive PTs became negative in the metastases. The PTs for all Maspin-positive metastases were luminal B type. CONCLUSION: High CXCR4 expression in PTs was found to be associated with luminal A type tumor, suggesting more favorable outcome. In contrast, CCR7 and FOXP3 expressions in PTs represented luminal B tumors, pointing to more aggressive tumor behavior. Maspin expression did not differ between luminal types.

ER, PR, HER2, Ki-67 and CK5 in Early and Late Relapsing Breast Cancer-Reduced CK5 Expression in Metastases.

Breast cancer can recur even decades after the primary therapy. Markers are needed to predict cancer progression and the risk of late recurrence. The estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER2), proliferation marker Ki-67, and cytokeratin CK5 were studied to find out whether their expression or occurrence in subgroups of breast cancers correlated with the time of recurrence. The expression of HER2, ER, PR, Ki-67, and CK5 was studied by IHC in 72 primary breast cancers and their corresponding recurrent/metastatic lesions. The patients were divided into three groups according to the time of the recurrence/metastasis: before two years, after 5 years, and after 10 years. Based on their IHC profiles, the tumors were divided into surrogates of the genetically defined subgroups of breast cancers and the subtype definitions were as follows: luminal A (ER or PR+HER2-), luminal B (ER or PR+HER2+), HER2 overexpressing (ER-PR-HER2+), triple-negative (ER-PR-HER2-), basal-like (ER-PR-HER2-CK5+), non-classified (ER-PR-HER2-CK5-) and luminobasal (ER or PR+CK5+). In multivariate analysis, tumor size and HER2 positivity were a significant risk of early cancer relapse. The metastases showed a significantly lower CK5 expression. CK5 positivity distinguished triple negative tumors into rapidly and slowly recurring cancers. The IHC subtype ER or PR+HER2- luminal A presented a significantly lower risk of early tumor recurrence. Ki-67 expression denoted early-relapsing tumors and correlated linearly with tumor progression, since Ki-67 positivity declined gradually from early-relapsing toward late-recurring cancers.

Bmi-1, c-myc, and Snail expression in primary breast cancers and their metastases--elevated Bmi-1 expression in late breast cancer relapses.

Breast cancer is known for its propensity to recur decades after treatment. The biology behind the phenomenon of tumor dormancy is still poorly understood. Bmi-1, c-myc, and Snail are transcription factors that have prognostic roles in several malignancies. In order to reveal whether any of these markers has impact on late relapses, we used immunohistochemistry to study the expression of Bmi-1, c-myc, Snail, and estrogen receptor in 73 primary breast cancers and in their metastatic relapses detected within 2 years, or 5 or 10 years after primary surgery. The expression of Bmi-1 was higher in the metastases than in their corresponding primary tumors in both early and late relapses. The highest expression of Bmi-1 was seen in the very late relapsing tumors (first tumor relapse after 10 years). Previously, Bmi-1 has been reported to function as a marker of tumor stem cells in breast cancer. Our results indicate that metastases, when compared to primary tumors, arise from tumor cells that have retained stem cell properties. We also analyzed the relationship between the expression of these markers and clinical parameters. A significant association between the expression of Bmi-1 and estrogen receptor was found. Nuclear expression of c-myc in primary tumors correlated with an increased risk for axillary lymph node metastasis.

High expression of maspin is associated with early tumor relapse in breast cancer.

Maspin is a serine protease inhibitor with tumor suppressor activity. Maspin can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. Maspin also modulates apoptosis of tumor cells, possibly by modulating the expression of the B-cell lymphoma-2 family member. p53 regulates the expression of the tumor suppressor gene maspin. Breast cancer is known for its propensity to recur even after decades. The biology behind this phenomenon of tumor dormancy is poorly understood. This study was conducted to clarify the role of maspin and B-cell lymphoma-2 in early and late recurring breast cancer. The expression of maspin, B-cell lymphoma-2, p53, and estrogen receptor was studied by immunohistochemistry in 73 primary breast cancers and in their metastatic relapses detected within 2 years, or 5 or 10 years after primary surgery. The cytoplasmic expression of maspin was significantly higher in the primary tumors of the early metastasizing breast cancers (first tumor relapse within 2 years) and also in their metastases compared to late metastasizing cancers. An opposite activity was observed in the expression of B-cell lymphoma-2. The level of B-cell lymphoma-2 staining was lower in the early relapsing cancers and significantly lower in their metastases, compared to tumors which metastasized 5 or 10 years after primary surgery. High cytoplasmic expression of maspin and low expression of B-cell lymphoma-2 in primary breast cancer predict early tumor relapse.