Replication-selective Oncolytic Adenovirus CNHK300 in the Treatment of Breast Cancer Cell Lines in vitro

Objective： To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells. Methods： RT-PCR was used to detect the hTERT mRNA activity in various breast cancer and normal fibroblast cell lines. Virus proliferation assay, cell viability assay and Western blot were applied to evaluate the proliferation and cytolysis selectivity of CNHK300. Results： The telomerase activity of MCF-7, BT-549 and SK-BR-3 was positive, while telomerase in MRC-5 and BJ was negative. The progeny virus titers in MCF-7, BT-549 and SK-BR-3 after 48 h of CNHK300 exposure was 40 625, 1 265 and 20 000 fold higher than those of 0 h, even slightly higher than those of wtAd5 （except in SK-BR-3）. ONYX-015 virus proliferation ability was weaker than that of CNHK300 in cancer cells. However, CNHK300 exhibited attenuated replicative ability as compared with wtAd5 in MRC-5 and BJ. The CNHK300 replicatative multiple was 63 and 192 fold at 48 h respectively, while the wtAd5 still multiplied 3 160-4 846 fold. CNHK300 could cause about half of breast cancer cells to die within 7 days at MOI 10 pfu/cell and below, whereas the IC50 in BJ and MRC-5 was as high as MOI 100 pfu/cell. CNHK300 E1A protein could be detected in breast cancer cells and 293 cells but not in normal fibroblast cells. Conclusion： hTERT promoter can successfully modulate the CNHK300 to be selectively replicated in breast cancer cells positive for telomerase, which may be a potential treatment strategy in breast cancer.

Measurement of Serum Total and Free Prostate-Specific Antigen for Breast Cancer Diagnosis in Women

Objective: To investigate the diagnostic value and the relationship between the clinicopathological features and the levels of total and free prostate-specific antigen (PSA) in women with breast cancer.Methods: Using the microparticle enzyme immunoassay system, we measured the concentrations of these markers in the sera of 85 women with breast cancer and in 30 healthy women.Rseults: The lowest detection level for both markers was 0.01 ng/ml. Free PSA levels were significantly higher in women with breast cancer than that in healthy women (P<0.05). The percentage of free PSA predominant subjects was 37.6% in breast cancer patients and 3.3% in healthy women. Cut-off values were 0.36 ng/ml for total PSA and 0.02 ng/ml for free PSA. In women with breast cancer, total PSA positivity was 23.5% and free PSA positivity was 27.1%. Compared to negatives, total PSA positive patients had a higher percentage of lymph node involvement tumours (P>0.05). However, patients with predominant free PSA had a higher percentage of early stage than patients with predominant PSA-ACT.Conclusion: Although the sensitivity of free PSA predominance is low (37.6%) in distinguishing women with breast cancer from healthy women, its specificity is high (97.0%).Free PSA predominance tends to be present in early stage tumours. These findings may indicate clinical significance of preoperative measurement of serum total and free PSA in women with breast cancer.

CORRELATION BETWEEN SERUM HER-2 ONCOPROTEIN AND PATIENTS WITH BREAST CANCER

To detect serum HER-2 oncoprotein levels in patients with operable and metastatic breast cancers, and to study the correlations between serum HER-2 level and lymph node status as well as other clinical parameters. Methods A total of 120 women were studied consisting of 10 healthy volunteers, 31 benign breast disease, 53 operable breast cancer, and 26 metastatic breast cancer patients. The levels of serum HER-2 were measured using an enzyme-liked im-munosorbent assay (ELISA). Results The mean serum HER-2 levels were 9.6 ± 1.5 ng/mL in healthy volunteers, 11.9 ± 1.6 ng/mL in benign breast disease, 13.2 ± 4.2 ng/mL in operable breast cancer, and 30.5 ± 30.8 ng/mL in metastatic breast cancer patients. The former is much lower than the latter three (P = 0.02, 0.001, 0.03, respectively). If using 15 ng/mL as a normal baseline, elevated serum HER-2 levels were observed in none of the healthy volunteers as well as patients with benign disease, but in 18.9% (10/53) operable breast cancer patients and 61.5% (16/26) metastatic patients. In patients with operable breast cancer, there was a positive correlation between serum concentrations of HER-2 and the size of primary tumor (P < 0.05), whereas there was no correlation between serum concentration and axillary lymph node or estrogen receptor status. In patients with metastatic dise-ase, there was no correlation with site of metastases (P > 0.05). Conclusion Serum HER-2 level was strongly correlated with tumor loads and clinical stages, thus acting as a promising predictor of cancer recurrence in breast cancer patients.

KNOCKDOWN OF SURVIVIN EXPRESSION BY SMALL INTERFERING RNA SUPPRESSES PROLIFERATION OF TWO HUMAN CANCER CELL LINES

Objective To construct an expression vector of small interfering RNA （siRNA） against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. Methods Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine^TM 2000. The expression of survivin was detected by semi-quanfifive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. Results The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79. 72% at protein level The proliferation of PC-2 and MCF-7 cells was also suppressed, and24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28. 00% and 33. 38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. Conclusions The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.

Study on interleukin-18 gene transfer into human breast cancer cells to prevent tumorigenicity

To study the effect of interleukin-18 gene transfection on the tumorigenesis of breast cancer cell line Bacp37,human breast cancer cell line Bcap37 were transfected with Lipofectamine and selected by G418.The biological expression of rhIL-18 was tested by RT-PCR and ELISA method;nude mice were injected with Bcap37 cell with or without the hIL-18 gene.The hIL-18 cDNA was successfully integrated into Bcap37 cell; 126.3±4.5pg hIL-18 secreted by one million transduced cells in 24 hours. Nude mice injected with IL-18 gene engineered Bcap37 cell had no tumor growth.These findings indicated that human breast cancer cells were successfully modified by the gene of IL-18 cytokine;the IL-18 gene engineered Bcap37 cells secreted hIL-18 and lost their tumorigenicity.The Bcap37 cells transduced with IL-18 gene may be used as breast cancer vaccine.

CLINICOPATHOLOGICAL SIGNIFICANCE OF PTEN AND CASPASE-3 EXPRESSIONS IN BREAST CANCER

Objective To investigate the expressions of PTEN and Caspase-3 proteins in human breast carcinoma,and to evaluate their clinicopathological implications during the tumorigenesis and progression of breast cancer.Methods The expressions of PTEN and Caspase-3 proteins in 95 cases of breast cancer and 15 cases of benign breast diseases were investigated immunohistochemically.Correlations between the expression of PTEN protein,Caspase-3 protein,and clinicopathological features of breast cancers were analyzed.Results The loss expression rate of PTEN protein in tumor tissues was significantly higher than that in benign breast diseases(33.7% vs.0,P<0.01).Analysis of the clinicopathological features showed that PTEN expression level was negatively correlated with TNM stage,histological grade,axillary lymph node status,recurrence,and metastasis(P<0.05).The positive expression level of Caspase-3 was negatively correlated with TNM stage(P<0.01),but not related with histological grade,axillary lymph node status,recurrence,or metastasis(P>0.05).In addition,the expression of PTEN protein had significantly positive correlation with the expression of Caspase-3 protein in breast cancer(P<0.01).Conclusion The combination detection of PTEN and Caspase-3 may serve as an important index to estimate the pathobiological behavior and prognosis of breast cancer.

18β-glycyrrhetinic Acid-induced Apoptosis and Relation with Intracellular Ca^2＋ Release in Human Breast Carcinoma Cells

Objective:To study the effects of 18β-glycyrrhetinic acid (GA) on proliferation inhibition, apop totic induction, and the relationship between GA-induced apoptosis and intracellular Ca2+ concentration in human breast carcinoma (MCF-7) cells. Methods: After MCF-7 cells were treated with GA at the concentrations from 50 μmol/L to 250 μmol/L for 24 h, cell viability of proliferation was assessed by MTT assay. After the cells were treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L GA for 24 h, the rates of cell apoptosis were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method and flow cytometry with Annexin V/propidium iodide fluorescent stain. After the cells treated with 150 μmol/L GA for 24 h, intracellular Ca2+ concentration was measured by Fure-2 fluorescein load method. Results: After the cells were treated with GA at the concentrations from 100 μmol/L to 250 μmol/L, the rates of proliferative inhibition were increased significantly (P<0.05 and P<0.01) in a dose dependent fashion. IC50 of the proliferation inhibition was 234.33 μmol/L. Treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L, the rates of cell apoptosis were increased significantly (P<0.01). Intracellular Ca2+ concentration after treatment with GA was higher evidently than that of control (P<0.05). Conclusion: 18β-glycyrrhetinic acid has the effects of the proliferation inhibition and the apoptotic induction on MCF-7 cells. The rise of intracellular Ca2+ level may be depended on apoptosis induced by GA in MCF-7 cells.

Percutaneous Removal of Benign Breast Lesions with an Ultrasound-guided Vacuum-assisted System:Influence Factors in the Hematoma Formation

Objective To explore the influence factors in hematoma formation after removing benign breast lesions with an ultrasound-guided vacuum-assisted system.Methods A total of 232 females with 312 benign breast masses received excisional biopsy with ultrasoundguided vacuum-assisted system.The pathology of patients,results of hematoma development and outcome,influence factors for hematoma occurrence(nodule size,nodule location,number of nodule,breast shape,menstrual period,efficacy time of bandage,and application of hemostatic agents during the procedure) were recorded.Results Pathologic examination revealed fibroadenomas in 138 lesions,fibroadenosis in 127 lesions,intraductal papillomas in 39 lesions,inflammatory change in 4 lesions,retention cyst of the breast in 3 lesions,and benign phyllodes tumor in 1 lesion.Thirty hematomas were observed in patients(9.6%).Finally,97.0%hematomas were absorbed completely within 6 months follow-up.The incidence rates of hematoma were increased by 24.7%,10.0%,63.2%,13.9%in the nodule diameter larger or equal to 25 mm group,removal of larger or equal to two nodules once time from one patient group,menstrual period group,and larger and loose breast group,respectively(all P<0.05).However,the incidences were decreased by 60.6%in the bandage performed for 12-24 hours or beyond 24 hours group(P<0.05).The multiple logistic regression models revealed that nodule size(x^2=15.227,P<0.001),number of nodule(x^2=7.767,P=0.005),menstrual period(x^2=24.530,P<0.001),and breast shape(x^2=9.559,P=0.002) were independent risk factors associated with hematoma occurrence,but efficacy time of bandage was a protective factor associated with hematoma occurrence.Conclusion The occurrence of hematoma after the minimally invasive operation was associated with nodule size,number of nodule,menstrual period,breast shape,and efficacy time of bandage.

The potential for liquid biopsies in the precision medical treatment of breast cancer

Currently the clinical management of breast cancer relies on relatively few prognostic/predictive clinical markers(estrogen receptor, progesterone receptor, HER2), based on primary tumor biology. Circulating biomarkers, such as circulating tumor DNA(ctDNA) or circulating tumor cells(CTCs) may enhance our treatment options by focusing on the very cells that are the direct precursors of distant metastatic disease, and probably inherently different than the primary tumor's biology. To shift the current clinical paradigm, assessing tumor biology in real time by molecularly profiling CTCs or ctDNA may serve to discover therapeutic targets, detect minimal residual disease and predict response to treatment. This review serves to elucidate the detection,characterization, and clinical application of CTCs and ctDNA with the goal of precision treatment of breast cancer.

Levovist ultrasonography imaging in intracystic hemorrhage of simple liver cyst

The differential diagnosis between intracystic hemorrhage and cystadenocarcinoma of the liver is often difficult even with the use of various imaging modalities. A 73-year-old woman was admitted to our hospital with the complaint of right upper quadrant pain. Ultrasonography (US) demonstrated a heterogeneous echogenic cyst measuring 11 cm × 8 cm in size in S2 of the liver, indicated intracystic hemorrhage of simple liver cyst or cystadenocarcinoma, but the differential diagnosis was considerably difficult. Levovist (Schering, Berlin, Germany) US revealed no enhancement of the intracystic structures, suggesting a clot in the case of intracystic hemorrhage. An operation was performed and the resected lesion showed a solitary benign liver cyst, measuring 5.5 cm × 4.7 cm × 8.5 cm containing a large blood clot. The patient had an uneventful recovery after the surgery. Levovist US may play an important role in discrimination between intracystic hemorrhage of simple hepatic cysts and cystadenocarcinoma of the liver.

The 10-Year Local Recurrence and Partial Breast Radiotherapy for Early Breast Cancer Treated by Conservative Surgery

To study the local recurrence and the role of whole breast radiotherapy for early breast cancer treated by conservative surgery. METHODS From April 1990 to December 2000, 49 patients with early primary breast cancer were treated by conservative surgery in our hospital. The cases were comprised of Stage 0, 1; Stage Ⅰ, 31; and Stage Ⅱa, 17. Forty cases underwent quadrantectomy plus axillary lymph node dissection, and the other 9 cases had lumpectomy alone. Irradiation, which was received by 39 patients, was administered by using low tangential half fields with 6 MV X-ray to decrease the pulmonary irradiative volume. The dose to the whole breast was 45 Gy/22 ～23f/4.5W, then a 15 Gy boost dose was delivered to the tumor bed by an electron beam. The other patients underwent an irradiated regional field according to postoperative pathology. RESULTS All patients were followed-up for 10 years or more. The 10- year local recurrence rates, distant metastasis rates and survival rates were 6.1%, 4.1% and 98.0% respectively. All of the 3 patients who had a local recurrence had infiltrative carcinomas and negative lymph nodes. The 10-year local recurrence rate was higher （2.6% vs. 20.0%） with nonpostoperative whole breast radootherapy, but the statistical difference was not marked because of the low number of cases. All of the recurrent lesions localized within 3 cm of the primary lesion. CONCLUSION Original recurrence of the tumor was the main type of local recurrence. Radiotherapy after conservative surgery is very essential. After conservative surgery it is feasible that irradiation can be delivered alone to the neighboring region of the tumor bed. Partial breast radiotherapy can substitute for whole breast radiotherapy.

Effect of vitamin E succinate on the proliferation of human breast cancer cells

Objective：Tn investigate the growth inhibition and apoptosis induced by vitamin E suceinate （VES） on human breast cancer cells and to analyze the possible mechanism in this process. Methods： Human breast cancer cell line Bcap-37 was treated with VES for 12, 24 and 48 h at the concentrations of 5, 10and 20 μg/ml. Then MTT assay was employed to detect the inhibitory effect of VES on the growth of breast cancer cells. Flow cytometry was used to analyze the cell cycle and apoptosis. To find out whether the Fas/FasL pathway was involved in this process, RT-PCR and flow cytometry assay were used to detect the Fas expression at the mRNA and protein level. Results： VESexhibited a significant inhibitory effect on the growth of human breast cancer cells, presenting in a dose- and time-dependant manner. The apoptotic rate of Bcap 37 cells was 0.6%, rose to 21.0% and 37.5% after treated with VES for 24 and 48 h at the concentration of 20 μg/ml. Fas mRNA transcription was upregulated after VES treatment and cell surface Fas expression increased according to the flow cytometry assay. Concluslon：Significant growth inhibition and apoptosis are induced in human breast cancer cells after treated with VES. The modulation of Fas/FasL pathway may related to the upregulation of Fas molecule on the cancer cell surface.

Cancer stem cells and early stage basal-like breast cancer

Ductal carcinoma in situ （DCIS） is a category of early stage, non-invasive breast tumor defined by the intraductal proliferation of malignant breast epithelial cells. DCIS is a heterogeneous disease composed of multiple molecular subtypes including luminal, HER2 and basal-like types, which are characterized by immuno-histochemical analyses and gene expression profiling. Following surgical and radiation therapies, patients with luminal-type, estrogen receptor-positive DCIS breast tumors can benefit from adjuvant endocrine-based treatment. However, there are no available targeted therapies for patients with basal-like DCIS （BL-DCIS） tumors due to their frequent lack of endocrine receptors and HER2 amplification, rendering them potentially susceptible to recurrence. Moreover, multiple lines of evidence suggest that DCIS is a non-obligate precursor of invasive breast carcinoma. This raises the possibility that targeting precursor BL-DCIS is a promising strategy to prevent BL-DCIS patients from the development of invasive basal-like breast cancer. An accumulating body of evidence demonstrates the existence of cancer stem-like cells （CSCs） in BL-DCIS, which potentially determine the features of BL-DCIS and their ability to progress into invasive cancer. This review encompasses the current knowledge in regard to the characteristics of BL-DCIS, identifcation of CSCs, and their biological properties in BL-DCIS. We summarize recently discovered relevant molecular signaling alterations that promote the generation of CSCs in BL-DCIS and the progression of BL-DCIS to invasive breast cancer, as well as the infuence of the tissue microenvironment on CSCs and the invasive transition. Finally, we discuss the translational implic-ations of these fndings for the prognosis and prevention of BL-DCIS relapse and progression.

Effect of breast-cancer metastasis suppressor 1 (BRMS1) on growth and metastasis of human gastric cancer cells in vivo

Objective： The aim of the study was to investigate inhibitory effects of breast-cancer metastasis suppressor 1 protein （BRMS1） on primary tumor growth and metastasis of human gastric cancer （GC） cells in nude mice. Methods： We compared the expression of BRMS1 in the primary gastric tumor and metastatic gastric tumor by immunohistochemistry. Expression of BRMS1 also was detected in the GC cells by RT-PCR and Western blot. Three groups of cultured human GC cell line SGC-7901, were maintained： transfected cells with pcDNA3.1（-）B/myc-BRMS1; negative control cells with pcD- NA3.1/myc-his（-）B; and blank control ceils without any transfection. Histologically intact samples of the cells, maintained by passage in the subcutis of nude mice, were transplanted orthotopically into stomach walls of nude mice to establish a nude mouse model of human gastric carcinoma. Their primary tumor growth and metastasis were then observed. Results： The expression of BRMS1 was markedly stronger in the primary gastric tumor compared with metastatic gastric tumor. We also detected BRMS1 gene and protein in the gastric cancer cell tines. Numbers of metastasis tumors significantly differed among mice infected with transfected cells, with negative controls and with blank controls （4.38 ± 0.60, 7.75 ± 0.59, and 7.63± 0.65, respectively; P 〈 0.05）. However, there were no significant differences in the size of orthotopic tumors among mice infected with transfected, negative control and blank control cells [（12.02 ± 0.70）, （12.71 ± 0.63） and （12.89 ± 0.71） mm, respectively; P 〉 0.05]. Conclusion： BRMS1 suppresses metastasis of GC cells, but does not inhibit growth of gastric tumors.

Intraductal papillary neoplasm of the bile duct in liver cirrhosis with hepatocellular carcinoma

A case of intraductal papillary neoplasm of the bile duct (IPNB) arising in a patient with hepatitis B-related liver cirrhosis with hepatocellular carcinoma (HCC) is reported. A 76-year-old man was admitted to our hospital with recurrent HCC. Laboratory data showed that levels of carcinoembryonic antigen and carbohydrate antigen 19-9 were elevated. He died of progressive hepatic failure. At autopsy,in addition to HCCs,an intraductal papillary proliferation of malignant cholangiocytes with fibrovascular cores was found in the dilated large bile ducts in the left lobe,and this papillary carcinoma was associated with an invasive mucinous carcinoma (invasive IPNB). Interestingly,extensive intraductal spread of the cholangiocarcinoma was found from the reactive bile ductular level to the interlobular bile ducts and septal bile ducts and to the large bile ducts in the left lobe. Neural cell adhesion molecule,a hepatic progenitor cell marker,was detected in IPNB cells. It seems possible in this case that hepatic progenitor cells located in reactive bile ductules in liver cirrhosis may have been responsible for the development of the cholangiocarcinoma and HCC,and that the former could have spread in the intrahepatic bile ducts and eventually formed grossly visible IPNB.

Small invasive ductal carcinoma of the pancreas distinct from branch duct intraductal papillary mucinous neoplasm

Endoscopic ultrasonography （EUS） is a highly sensitive diagnostic method for the detection of small pancreatic carcinomas. Recently, there have been some reports describing the utility of contrast-enhanced harmonic EUS （CEH-EUS） which uses sonographic contrast agent for differentiation of a pancreatic mass. This report describes a case of small adenocarcinoma of the pancreas distinct from branch duct intraductal papillary mucinous neoplasm （IPMN） in which investigation by EUS took place every 6 mo and diagnosis was made accurately by additional CEH-EUS during the follow-up of the branch duct IPMN. A 68-year-old female was admitted to our hospital because of a branch duct IPMN in the pancreatic body. She had been followed-up by EUS every 6 too. However, after 2 years EUS demonstrated a low echoic area distinct from the branch duct IPMN which was vaguely discernible by EUS, and accurate sizing and differential diagnosis were considered difficult on the EUS imaging. CHEUS with Sonazoid revealed a hypovascular tumor and we suspected small pancreatic carcinoma. The histopathological diagnosis was adenocarcinoma （10 mm） in the pancreatic tail, distinct from the branch duct IPMN of the pancreatic body. EUS and CEH-EUS may play an important role in the correct diagnosis of small pancreatic tumors, including synchronous and metachronous occurrence of IPMN and ductal adenocarcinoma of the pancreas.

Correlation between Expression of P38 MAPK-Signaling and uPA in Breast Cancer

OBJECTIVE To study the expression of phosphorylated p38 mitogen-activated protein kinase （p-p38） and uPA and the correlation of their expression with breast cancer clinicopathological characteristics, and to investigate the role of the p38MAPK-signaling pathway in regulating uPA expression in breast cancer cells.METHODS Immunohistochemistry （S-P） was used to test the expression of p-p38 and uPA in 60 specimens of breast cancer tissues. Western blots were adopted to detect expression of the p-p38 and uPA proteins in MDA-MB-231 and MCF-7 breast cancer cells, and uPA expression after treatment with SB203580, a specific inhibitor of p38 MAPK.RESULTS The positive rate of the p-p38 protein and uPA protein expression in the breast cancer tissues was 56.7% and 60.0%,respectively. The expression of p-p38 was positively related to the expression of uPA （r = 0.316, P 〈 0.05）. The expression of p-p38 and uPA was related to lymph node metastasis and the TNM stage （P 〈 0.05）, but it was not related to the patient＇s age or tumor size （P 〉 0.05）. The expression of p-p38 and uPA in MDA- MB-231 cells was higher than that in MCF-7 cells. SB203580 inhibited the p38 MAPK pathway and reduced uPA protein expression.CONCLUSION The p38 MAPK-signaling pathway promotes breast cancer malignant progression by up-regulating uPA expression ,and it may be an important process in breast cancer invasion and metastasis.

Effects of postmastectomy radiotherapy on prognosis in different tumor stages of breast cancer patients with positive axillary lymph nodes

Objective: To explore the effects of postmastectomy radiotherapy(PMRT) on the locoregional failure-free survival(LRFFS) and overall survival(OS) of breast cancer patients under different tumor stages and with one to three positive axillary lymph nodes(ALNs). Methods: We conducted a retrospective review of 527 patients with one to three positive lymph nodes who underwent modified radical or partial mastectomy and axillary dissection from January 2000 to December 2002. The patients were divided into the T1-T2 N1 and T3-T4 N1 groups. The effects of PMRT on the LRFFS and OS of these two patient groups were analyzed using SPSS 19.0, Pearson's χ2-test, Kaplan-Meier method, and Cox proportional hazard model. Results: For T1-T2 N1 patients, no statistical significance was observed in the effects of PMRT on LRFFS [hazard ratio(HR)=0.726; 95% confidence interval(CI): 0.233-2.265; P=0.582] and OS(HR=0.914; 95% CI: 0.478-1.745; P=0.784) of the general patients. Extracapsular extension(ECE) and high histological grade were the risk factors for LRFFS and OS with statistical significance in multivariate analysis. Stratification analysis showed that PMRT statistically improved the clinical outcomes in high-risk patients [ECE(+), LRFFS: P=0.026, OS: P=0.007; histological grade III, LRFFS: P<0.001, OS: P=0.007] but not in low-risk patients [ECE(–), LRFFS: P=0.987, OS: P=0.502; histological grade I-II, LRFFS: P=0.816, OS: P=0.296]. For T3-T4 N1 patients, PMRT effectively improved the local control(HR=0.089; 95% CI: 0.210-0.378; P=0.001) of the general patients, whereas no statistical effect was observed on OS(HR=1.251; 95% CI: 0.597-2.622; P=0.552). Absence of estrogen receptors and progesterone receptors(ER/PR)(–) was an independent risk factor. Further stratification analysis indicated a statistical difference in LRFFS and OS between the high-risk patients with ER/PR(–) receiving PMRT and not receiving PMRT [ER/PR(–), LRFFS: P=0.046, OS: P=0.039]. However, PMRT had a beneficial effect on the reduction of locoregional recurrence(LRR) but not in total mortality [ER/PR(+), LRFFS: P<0.001, OS: P= 0.695] in T3-T4 N1 patients with ER/PR(+) who received endocrine therapy. Conclusion: PMRT could reduce ECE(+), histological grade III-related LRR, and total mortality of T1-T2 N1 patients. T3-T4 N1 patients with ER/PR(–) could benefit from PMRT by improving LRFFS and OS. However, PMRT could only reduce LRR but failed to improve OS for T3-T4 N1 patients with ER/PR(+) who received endocrine therapy.

Phase Ⅱ study of mitomycin C and cisplatin in heavily pretreated advanced breast cancer

Objective： The mitomycin C and cisplatin combination was investigated in patients with advanced breast cancer who had been exposed to anthracyclines, vinorelbine and taxanes. Methods： Three-weekly regimen consisted of mitomycin, 6 mg/m^2 administered intravenously on day 1, and cisplatin, 25 mg/m^2 intravenously on day 1-3. Results： Thirty-eight patients aged 25-75 years （median, 46 years） were treated with an overall response rate of 31.6%. The median time to progression （TTP） was 4.0 months. Median TTP for 12 patients with a complete or partial response was 9.0 months, while stable disease and progression of disease 4.0 months, P=0.002. Grade 3/4 side effects of neutropenia, thrombocytopenia and nausea/vomiting were documented in 4 （10.5%）, 4 （10.5%） and 3 （7.9%） patients, respectively. The median overall survival was 13＊ months. Conclusion： Mitomydn C/cisplatin doublet showed antitumor activity for anthracydine-, vinorelbine- and taxane-resistant breast cancer comparable to other regimens. This well-tolerated regimen provides an affordable option for patients in China.

Expression of Preprotachykinin-I (PPT-I), Neurokinin-1 (NK-1) and Neurokinin-2 (NK-2) in Breast Cancer Cells Improves Tumor Cell Survival in Bone Marrow in the Early Stage of Metastasis

OBJECTIVE To study the potential relationship between the expression of PPT-I, NK-1, NK2 and the development of breast cancer cells in bone marrow stroma and to provide evidence of potential molecular mechanisms of breast cancer patients. of bone metastasis in early stage METHODS The cocultures of breast cancer cell line T-47D and marrow-derived mesenchymal stem cells （MSC） were established with equal numbers. T-47D cells were separated from the coculture system at 48 h and 96 h after coculture by MACS magnetic cell sorting （MicroBeads）. The expression of PPT-I, NK-1, NK-2 in T-47D was then examined before and after coculture by real-time PCR and by Western blot. Alterations in cellular ultrastructure of T-47D cells were detected before and after coculture under electron microscope. Finally, changes in cell cycle distribution were examined by flow cytometry, and growth curves from before and after coculture were drawn and analyzed. RESULTS Following coculture of T-47D and MSC, the expression of PPT-I mRNA and protein was significantly upregulated, while the expression of NK-1 and NK-2 mRNA and protein was greatly downregulated. The analysis of cell cycle distribution by flow cytometry showed that the proportion of T-47D during S phase was increased, and the duration of the G2/M phase was sharply decreased. Under electron microscope, we observed that the synthesis of hereditary material was increased, but the hepatin granules were shown prominent stacking in T-47D cells. These results suggest that although the synthesis of DNA was increased, the proliferation of cells was inhibited after coculture. The cell growth curve confirmed the findings from the observation under the electron microscope and flow cytometry. CONCLUSION Tumor cells could survive through the upregulation in expression of preprotachykinin-I gene during early bone metastasis in breast cancer. The phenomenon of growth suppression in breast cancer cells after coculture in the current study could be induced by downregulation in expression of NK-1 and NK-2.