A standardized method for quantifying proliferation by Ki-67 and cyclin A immunohistochemistry in breast cancer.

Immunohistochemical analysis of proliferation markers such as Ki-67 and cyclin A is widely used in clinical evaluation as a prognostic factor in breast cancer. The proliferation status of tumors is guiding the decision of whether or not a patient should be treated with chemotherapy because low-proliferative tumors are less sensitive by such treatment. However, the lack of optimal cutoff points and selection of tumor areas hamper its use in clinical practice. This study was performed to compare the Ki-67 and cyclin A expression counted in hot-spot vs average counting based on 5 to 14 random tumor areas in 613 breast carcinomas. We correlated the findings with 10-year follow-up in order to standardize the evaluation of proliferation markers in clinical practice. A significant correlation was found between the percentage of positive cells estimated by Ki-67 and cyclin A both by hot-spot and by average counting. Both methods showed that high expression of Ki-67 and cyclin A is associated with more adverse tumor stage. The cutoff value for Ki-67 for distant metastases was set to 22% and to 15%, using hot-spot and average counting, respectively. For cyclin A, the values were set to 14% and 8% using the respective methods. Survival curves revealed that patients with a high hot-spot proliferation index had a significantly greater risk of shorter tumor-free survival. Our findings suggest that the determination of proliferation markers in breast cancer should be standardized to hot-spot counting and that specific cutoff values for proliferation could be useful as prognostic markers in clinical practice. Moreover, we suggest that expression levels of cyclin A could be used as a complementary marker to estimate the proliferation status in tumors, especially those with "borderline" expression levels of Ki-67, in order to more accurately estimate the proliferations status of the tumors.

SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Up-regulation of Survivin in Glioblastoma.

Stromal cell-derived factor 1 (SDF-1)/CXCR4 ligand-receptor axis is widely recommended as an attractive target for cancer therapy. Meanwhile, epithelial-mesenchymal transition (EMT) process is linked to disease pathophysiology. As one of inhibitors of apoptosis proteins, survivin is implicated in the onset and development of cancer. In the present study, we tried to determine the cause-effect associations between SDF-1/CXCR4 axis and survivin expression in glioblastoma U-251 cell line. Survivin activation and inhibition were induced with exogenous SDF-1 and survivin small interfering RNA (survivin siRNA), respectively. Western blot was used to detect relevant proteins in SDF-1/CXCR4 axis. Western blot analysis revealed that survivin expression in U-251 increased in a dose- and time-dependent manner in response to SDF-1 treatment. However, the interference with MEK/ERK and PI3K/AKT pathway prohibited SDF-1-induced survivin up-regulation. Importantly, survivin knockdown abrogated cell cycle progression and the expression of snail and N-cadherin, compared with non-transfectants. In conclusion, the present study shows that SDF-1 up-regulates survivin via MEK/ERK and PI3K/AKT pathway, leading to cell cycle progression and EMT occurrence dependent on survivin. The blockade of survivin will allow for the treatment of glioblastoma.

Alterations of biomarker profiles after neoadjuvant chemotherapy in breast cancer: tumor heterogeneity should be taken into consideration.

Tumor biomarkers including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 are routinely tested in breast cancer patients and their status guides clinical management and predicts prognosis. A few retrospective studies have suggested that neoadjuvant chemotherapy (NAC) in breast cancer may change the status of biomarker expression, which in turn will affect further management of these patients. In this study we take advantage of a relatively large cohort and aim to study the effect of NAC on biomarker expression and explore the impact of tumor size and lymph node involvement on biomarker status changes. We collected 107 patients with invasive breast cancer who received at least three cycles of NAC. We retrospectively performed and scored the immunohistochemistry (IHC) of ER, PR, HER2 and Ki-67 using both the diagnostic core biopsies before NAC and excisional specimens following NAC. HER2 gene status was assessed by fluorescence in situ hybridization for cases with IHC result of 2+. We demonstrated that there was a significant decrease in expression of PR (P = 0.013) and Ki-67 (P = 0.000) in post-NAC specimens compared to pre-NAC core biopsies. In addition, cases with large tumor size (≥ 2 cm) and cases with lymph node metastasis were more frequently to have biomarker changes. Finally we studied cases with HER2 status changes after NAC treatments in detail and emphasized the nature of tumor heterogeneity.

Over expression of hRad9 protein correlates with reduced chemosensitivity in breast cancer with administration of neoadjuvant chemotherapy.

Human Rad 9 (hRad9), part of the Rad9-Hus1-Rad1 complex plays an important role in DNA damage repair as an up-stream regulator of checkpoint signaling, however little is known about its role in response to chemotherapy of breast cancer and whether hRad9 inhibition can potentiate the cytotoxic effects of chemotherapy on breast cancer cells remains to be elucidated. Fifty cases of breast cancer receiving neoadjuvant therapy were collected. All these cases were revised and classified into chemotherapy sensitive (CS) or chemotherapy resistant (CR) group according to the Miller and Payne (MP) grading system. Immunohistochemically, hRad9 positive tumours showed nuclear and/or cytoplasmic staining. hRad9 over-expression was associated with an impaired neoadjuvant chemotherapy response. A significant correlation was found between expression of hRad9 and Cyclin D1. In vitro, hRad9 was knocked down using siRNA in breast cancer cell line MCF-7 and MDA-MB-231. Deregulated expression of Rad9 accompanied by down expression of chk1 enhanced the sensitivity of human breast cancer cells to doxorubicin. Our work suggests that hRad9 might be a potential predictor for the response to chemotherapy in patients with breast cancer and its clinical value as a target for improving chemosensitivity needs further exploration.