Masahiro Sakakibara

Cutting Edge: Negative Regulation of Immune Synapse Formation by Anchoring Lipid Raft to Cytoskeleton Through Cbp-EBP50-ERM Assembly

Ag recognition by T lymphocytes induces immune synapse formation and recruitment of signaling molecules into a lipid raft. Cbp/PAG is a Csk-associated membrane adapter protein exclusively localized in a lipid raft. We identified NHERF/EBP50 as a Cbp-binding molecule, which connects the membrane raft and cytoskeleton by binding to both Cbp through its PDZ domain and ezrin-radixin-moesin through the C terminus. Overexpression of Cbp reduced the mobility of the raft on the cell surface of unstimulated T cells and prevented synapse formation and subsequent T cell activation, whereas a mutant incapable of EBP50 binding restored both synapse formation and activation. These results suggest that anchoring of lipid raft to the cytoskeleton through Cbp-EBP50-ezrin-radixin-moesin assembly regulates membrane dynamism for synapse formation and T cell activation.

Accumulation of regulatory T cells in sentinel lymph nodes is a prognostic predictor in patients with node-negative breast cancer.

It has been revealed that sentinel lymph nodes (SLNs) from patients with node-negative breast cancer involve RT-PCR detected micrometastases and isolated tumour cells. However, the prognostic significance of the pathologically undetectable micrometastases is still controversial. In this study, we evaluated Foxp3 positive regulatory T cells (Treg) in SLNs as host-side immune marker that has the potential to detect these micrometastases. In the analyses of training set (n=30), elevated Treg was strongly associated with the pathologically undetectable micrometastases. In the analyses of validation set (n=129) in patients with node-negative, relapse-free survival in patients with elevated Treg was significantly shorter than those with lower Treg (p=0.005). Furthermore, in multivariate analyses, elevated Treg was correlated with relapse-free survival (p=0.012). Our data indicate that Treg may increase in the microenvironment of SLNs along with pathologically undetectable micrometastases and is a prognostic predictor in patients with node-negative breast cancer.

Breast-Conserving Surgery Using Projection and Reproduction Techniques of Surgical-Position Breast MRI in Patients with Ductal Carcinoma In Situ of the Breast

BACKGROUND In this study, we report a breast-conserving surgery (BCS) approach that uses projection and reproduction techniques of breast MRI obtained in the surgical position to the breast surface in patients with ductal carcinoma in situ (DCIS) of the breast. STUDY DESIGN Between February 2005 and January 2007, a total of 104 patients with operable breast cancer at our hospital had surgical-position breast MRI examinations. The 24 patients with relatively localized DCIS received BCS using the projection and reproduction techniques of the surgical-position breast MRI. During the same time period, 28 patients with relatively localized DCIS in whom prone-position breast MRI was performed, had conventional BCS using mammography-guided hookwires. In this study, we compared the surgical outcomes of our surgical approach with those of the conventional approach in a total of 52 patients with relatively localized DCIS. RESULTS Average volume of the pathologic specimens in the new technique group (27.5 cm(3)) was substantially smaller than that in the conventional BCS group (57.6 cm(3), p = 0.0007). In addition, the positive margin rate was substantially lower in the new technique group (12.5%) than in the conventional BCS group (39.3%; p = 0.029). CONCLUSIONS This study demonstrates that BCS can be done guided by the precise projection and reproduction techniques of the lesion obtained by surgical-position breast MRI. To the best of our knowledge, this is the first report of BCS technique for DCIS in this manner. Our surgical approach can be clinically useful in surgical planning and management in patients with DCIS.

Identification of vitronectin as a novel serum marker for early breast cancer detection using a new proteomic approach

PurposeBreast cancer is the most frequent malignancy in women. However, no useful serum markers with high sensitivity and specificity for the detection of early breast cancer have been identified. The search for biological markers of early breast cancer is of continual interest in experimental and clinical breast cancer research. We recently described a simple and highly reproducible three-step proteome analysis for identifying potential disease-marker candidates among the low-abundance serum proteins.MethodsSerum samples from breast ductal carcinoma in situ (DCIS) patients and normal controls were subjected to a three-step serum proteome analysis. The steps were the following: first, immunodepletion of most abundant proteins; second, fractionation using reverse-phase high-performance liquid chromatography; and third, separation using two-dimensional electrophoresis (2-DE). Differences revealed by protein staining were further confirmed by Western blotting, immunohistochemical staining, and enzyme-linked immunosorbent assays (ELISA).ResultsTwenty-two upregulated and 26 downregulated spots were detected on the 2-DE gels, and a total of 33 proteins were identified by liquid chromatography and tandem mass spectrometry. Western blotting confirmed that the level of vitronectin was significantly increased in DCIS patients compared with that of normal controls. Immunohistochemical staining of vitronectin in breast cancer tissue revealed high expression in small vessel walls surrounding cancer cells and the extracellular matrix of stroma. Moreover, vitronectin serum concentrations, as measured by ELISA, were significantly increased in patients with DCIS or more advanced breast cancer compared with those of normal controls.ConclusionsVitronectin could serve as a promising serum marker for the detection of primary breast cancer.

Dynamic enhanced MRI predicts chemosensitivity in breast cancer patients

BACKGROUND Primary chemotherapy for breast cancer is effective as postoperative adjuvant therapy. However, one of the critical disadvantages was a treatment delay for patients with progressive disease. The present study attempts to clarify quantitative parameters on MRI which can be used to predict the sensitivity to treatment in breast cancer patients. METHODS The subjects consisted of 26 patients with invasive ductal breast cancer who received primary chemotherapy before surgery. The mean maximum tumor dimension was 3.3cm, and 21 cases had nodal involvements. Three cases demonstrated histological grade 3. Dynamic enhanced MRI was evaluated at three different time periods; prior to, in the midst of preoperative chemotherapy, and just before the initial operation. The signal intensity ratio (SIR) and early contrast uptake (ECU) were calculated, as well as the correlation between these dynamic data and the tumor reduction rates were analyzed retrospectively. P-values of less than 0.05 were considered to indicate statistically significant. RESULTS Responders to chemotherapy had the significantly higher SIR and ECU values than non-responders (p=0.0454 and 0.0334, respectively). ECU value significantly decreased as tumor reduction by chemotherapy (p=0.0028). Pathological tumor dimension was significantly correlated with the tumor size estimated on presurgical MRI (p<0.0001). CONCLUSIONS Our current series demonstrated the significant correlation between pretreatment MRI data and tumor reduction by chemotherapy in breast cancer patients. With these results, it seems possible to define good and non-responders prior to treatment.

Aldehyde dehydrogenase 1-positive cells in axillary lymph node metastases after chemotherapy as a prognostic factor in patients with lymph node-positive breast cancer.

Aldehyde dehydrogenase 1 (ALDH1)‐positive cells exhibit stem‐like or progenitor ability and have been considered a clinically important diagnostic and therapeutic target in patients with breast cancer. In this study, the authors evaluated responsiveness to chemotherapy of ALDH1‐positive cells in primary and metastatic lesions and its relation to prognosis for patients with lymph node‐positive breast cancer.

Cancer-mediated adipose reversion promotes cancer cell migration via IL-6 and MCP-1

The objective of this study is to investigate interactions between adipocytes and breast cancer cells, and identify the responsible factors for the observed effects. In 27 breast cancer patients undergoing mastectomy, mammary adipose tissue was obtained from the breast quadrant bearing the tumor and corresponding non-tumoral quadrant. Isolated normal breast adipocytes (NBAs) and cancer-associated adipocytes (CAAs) were cultured in collagen gels to mimic the in vivo environment. Immunohistochemistry, qRT-PCR, and cell proliferation assays were performed to analyze adipocyte phenotypes. MCF7 and MDA-MB-231 breast cancer cell lines were co-cultured with adipocytes to detect phenotypic changes. Migration of MCF7 and MDA-MB-231 cells was assessed in NBA- and CAA-conditioned media. Cytokine levels in conditioned media were measured by cytokine array. Migration assays were repeated using conditioned media containing neutralizing antibodies. NBAs and CAAs lost their morphological phenotype in culture, acquiring a spindle-like shape, and CAAs showed higher cell proliferation, suggesting reversion to an immature phenotype. In co-cultures with MCF7 or MDA-MB-231 cells, NBAs exhibited increased cell proliferation, indicating acquisition of the immature phenotype of CAAs. MCF7 and MDA-MB-231 showed higher migration in a CAA-conditioned medium than in an NBA-conditioned medium. Cytokine array analysis of conditioned media revealed higher levels of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in the CAA-conditioned medium. Neutralization experiments using antibodies against IL-6 or MCP-1 showed abrogation of migration-enhancing effects of the CAA-conditioned medium. Adipocytes revert to an immature and proliferative phenotype in the presence of breast cancer cells, and promote cancer cell migration via adipokines including IL-6 and MCP-1.

CXCR4/CXCL12 expression profile is associated with tumor microenvironment and clinical outcome of liver metastases of colorectal cancer

Interaction between CXCR4 and CXCL12 plays a role in tumor progression. The present study examined CXCR4, CXCL12 and CD133 expression in liver metastases of colorectal cancer (CLM) and determined whether the expression profiles affect the tumor microenvironment and thus progression, and whether they could serve as a prognostic marker for survival. Liver metastases of colorectal cancer collected from 92 patients were evaluated by CXCR4, CXCL12 and CD133 immunohistochemistry and clinicopathological data were analyzed. The expression profile of CXCR4 was determined in the colorectal cancer cell line, SW48. The expression of cytoplasmic CXCR4 was higher in 36 (39%) patients than that indicated by CXCR4 staining intensity of hepatocytes. High levels of nuclear CXCR4 expression in 23 (25%) patients significantly correlated with CXCL12 expression in hepatocytes. Nuclear CXCR4 expression was increased in the cancer cells after exposure to CXCL12. Univariate and multivariate analyses demonstrated that the high levels of nuclear CXCR4 and CXCL12 expression in hepatocytes were significantly better prognostic factors for overall and hepatic disease-free survival in patients with CLM. The expression of CXCR4 and CXCL12 in CLM may have an interactive effect that could alter the tumor microenvironment. CXCR4 expression in metastatic liver tumors together with the upregulation of CXCL12 in hepatocytes may help to predict the clinical outcomes of patients with CLM after hepatectomy.

Surrounding rim formation and reduction in size after radiofrequency ablation for primary breast cancer

PurposeRadiofrequency ablation (RFA) has recently been used to treat small breast cancer. However, there are no data on the long-term morphological features after the procedure. The present study attempts to clarify the characteristics of and changes in the ablated lesion.Materials and methodsA total of 17 breast cancer patients underwent RFA using a single needle featuring an internally cooled electrode; this was followed by whole-breast irradiation and adjuvant systemic therapy. Magnetic resonance imaging (MRI) using a 1.5-T system was performed before and 1, 3, 6, and 12 months after ablation, and the morphological characteristics and the size of the ablated lesion were evaluated. Mammography was also performed for a comparison with the MRI measurement.ResultsMRI displayed no residual enhancement of the tumor after RFA; there was an altered signal intensity with peripheral enhancement, however, and the area decreased in size gradually at a rate of 3.3% per month. Mammography showed a ring surrounding a roundish area whose size was equal to that seen with MRI.ConclusionOur current series demonstrated the morphological characteristics on breast imaging after RFA plus radiation therapy. The size of the ablated area decreased over time. These findings are valuable for clinical follow-up of breast cancer patients undergoing RFA.

Impact of body fat distribution on neoadjuvant chemotherapy outcomes in advanced breast cancer patients

Obesity is known to decrease the efficacy of neoadjuvant chemotherapy (NAC) against breast cancer; however, the relationship between actual body composition and NAC outcomes remains unknown. Therefore, we determined the effect of body composition on NAC outcomes. A total of 172 advanced breast cancer patients who underwent surgery after NAC were retrospectively analyzed. Body composition parameters including abdominal circumference (AC), subcutaneous fat area (SFA), visceral fat area (VFA), and skeletal muscle area (SMA) were calculated using computed tomography volume‐analyzing software. VFA/SFA ratio was used to evaluate visceral obesity. The associations of body composition parameters with pathological complete remission (pCR) and survival were analyzed. AC, SFA, and VFA were significantly correlated with body mass index (BMI) (all P < 0.05; r = 0.82, r = 0.71, and r = 0.78, respectively). AC, SFA, and VFA increased significantly and SMA decreased significantly after menopause (all P < 0.05). VFA/SFA ratio increased significantly after menopause, even though BMI remained unchanged. Body composition parameters were not associated with pCR. Distant disease‐free survival (DDFS) was significantly worse in the high VFA group than in the low VFA group (P < 0.05). Furthermore, in the high VFA group, postmenopausal patients had significantly shorter DDFS than premenopausal patients (P < 0.05). VFA was independently associated with DDFS in the multivariate analysis (P < 0.05). High visceral fat is associated with worse NAC outcomes in breast cancer patients, especially postmenopausal patients. Interventions targeting visceral fat accumulation will likely improve NAC outcomes.