Peripheral cytotoxic T lymphocyte predicts first-line progression free survival in HER2-positive advanced breast cancer.

BACKGROUND: The role of peripheral blood lymphocyte (pBL) in breast cancer has long been studied. However, the predictive role of pBL in advanced breast cancer (ABC) is poorly understood. METHODS: A total of 303 patients with ABC were consecutively recruited at our center between January 2015 and September 2019. At baseline, pBL subtypes were detected in all patients with 229 blood samples available for circulating tumor DNA (ctDNA) detection. pBL was analyzed through flow cytometry. ctDNA-based gene mutations were detected using next generation sequencing. The cutoff value of pCTL was estimated by X-tile software. Progression free survival (PFS) was estimated by Kaplan-Meier curve and Cox hazard proportion regression model, with difference detection by log-rank test. RESULTS: Median follow-up time of the study was 21.0 months. The median age of diagnosis was 52.0 years. Among the pBL subtypes, only pCTL level was found predictive for PFS in the HER2+ patients whom received anti-HER2 therapy (13.1 vs. 5.6 months, P = 0.001). However, the predictive role of pCTL was not found in HR-positive (P = 0.716) and TNBC (P = 0.202). pCTL high associated with suppressive immune indictors including lower CD4/CD8 ratio (P = 0.004) and high level of Treg cell (P = 0.004). High occurrence of FGFR1 amplification which has been reported as immune suppressor was also found in HER2+ patients with pCTL high (22.2% vs. 4.3%, P = 0.048). CONCLUSIONS: Higher pCTLs level associated with shorter PFS and FGFR1 mutation in HER2+ ABC patients.

Methylome Variation Predicts Exemestane Resistance in Advanced ER+ Breast Cancer.

BACKGROUND: More than 30% of estrogen receptor-positive breast cancers are resistant to primary hormone therapy, and about 40% that initially respond to hormone therapy eventually acquire resistance. Although the mechanisms of hormone therapy resistance remain unclear, aberrant DNA methylation has been implicated in oncogenesis and drug resistance. PURPOSE: We investigated the relationship between methylome variations in circulating tumor DNA and exemestane resistance, to track hormone therapy efficacy. METHODS: We prospectively recruited 16 patients who were receiving first-line therapy in our center. All patients received exemestane-based hormone therapy after enrollment. We collected blood samples at baseline, first follow-up (after 2 therapeutic cycles) and at detection of disease progression. Disease that progressed within 6 months under exemestane treatment was considered exemestane resistance but was considered relatively exemestane-sensitive otherwise. We obtained circulating tumor DNA-derived methylomes using the whole-genome bisulfide sequencing method. Methylation calling was done by BISMARK software; differentially methylated regions for exemestane resistance were calculated afterward. RESULTS: Median follow-up for the 16 patients was 19.0 months. We found 7 exemestane resistance-related differentially methylated regions, located in different chromosomes, with both significantly different methylation density and methylation ratio. Baseline methylation density and methylation ratio of chromosome 6 [32400000-32599999] were both high in exemestane resistance. High baseline methylation ratios of chromosome 3 [67800000-67999999] (P = .013), chromosome 3 [140200000-140399999] (P = .037), and chromosome 12 [101200000-101399999] (P = .026) could also predict exemestane resistance. During exemestane treatment, synchronized changes in methylation density and methylation ratio in chromosome 6 [32400000-32599999] could accurately stratify patients in terms of progression-free survival (P = .000033). Cutoff values of methylation density and methylation ratio for chromosome 6 [149600000-149799999] were 0.066 and 0.076, respectively. CONCLUSION: Methylation change in chromosome 6 [149600000-149799999] is an ideal predictor of exemestane resistance with great clinical potential.

Identification of high independent prognostic value of nanotechnology based circulating tumor cell enumeration in first-line chemotherapy for metastatic breast cancer patients.

Enumeration of circulating tumor cells (CTCs) is a promising tool in the management of metastatic breast cancer (MBC). This study investigated the capturing efficiency and prognostic value of our previously reported peptide-based nanomagnetic CTC isolation system (Pep@MNPs). We counted CTCs in blood samples taken at baseline (n = 102) and later at patients' first clinical evaluation after starting firstline chemotherapy (n = 72) in a cohort of women treated for MBC. Their median follow-up was 16.3 months (range: 9.0-31.0 months). The CTC detection rate was 69.6 % for the baseline samples. Patients with ≤2 CTC/2 ml at baseline had longer median progression-free survival (PFS) than did those with >2 CTC/2 ml (17.0 months vs. 8.0 months; P = 0.002). Patients with ≤2 CTC/2 ml both at baseline and first clinical evaluation had longest PFS (18.2 months) among all patient groups (P = 0.004). Particularly, among patients with stable disease (SD; per imaging evaluation) our assay could identify those with longer PFS (P < 0.001). Patients with >2 CTC/2 ml at baseline were also significantly more likely to suffer liver metastasis (P = 0.010). This study confirmed the prognostic value of Pep@MNPs assays for MBC patients who undergo firstline chemotherapy, and offered extra stratification regarding PFS for patients with SD, and a possible indicator for patients at risk for liver metastasis.

[Bushen Daozhuo Granules for type â ¢ prostatitis: A multicenter randomized controlled clinical trial].

OBJECTIVE: To study the safety and efficacy of Bushen Daozhuo Granules (BDG) in the treatment of type Ⅲ prostatitis. METHODS: This multicenter randomized controlled clinical trial included 478 patients with type Ⅲ prostatitis, 290 in the trial group and 188 as controls, the former treated with BDG at 200 ml bid and the latter with tamsulosin hydrochloride sustainedrelease capsules at 0.2 mg qd, both for 4 weeks. Before treatment, after 4 weeks of medication, and at 4 weeks after drug withdrawal, we obtained the NIH Chronic Prostatitis Symptom Index (NIHCPSI) scores and compared the safety and effectiveness rate between the two groups of patients. RESULTS: Compared with the baseline, the NIHCPSI score was markedly decreased in the control group after 4 weeks of medication (21.42 ± 4.02 vs 15.67 ± 3.65, P < 0.05) but showed no statistically significant difference from that at 4 weeks after drug withdrawal (19.03 ± 3.86) (P>0.05), while the NIHCPSI score in the trial group was remarkably lower than the baseline both after 4 weeks of medication and at 4 weeks after drug withdrawal (10.92 ± 2.06 and 12.91 ± 2.64 vs 21.58 ± 3.67, P < 0.05). The trial group exhibited both a higher rate of total effectiveness and safety than the control (P < 0.05). CONCLUSIONS: BDG is safe and effective for the treatment of type Ⅲ prostatitis.

The prognostic value of peripheral CD4+CD25+ T lymphocytes among early stage and triple negative breast cancer patients receiving dendritic cells-cytokine induced killer cells infusion.

OBJECTIVE: This study aimed to assess the prognostic value of CD4+CD25+ T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy. METHODS: 217 patients participated in the follow-up study. CD4+CD25+ proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD4+CD25+ proportion more than 5% and less than or equal to 5% in peripheral T cells. RESULTS: Peripheral CD4+CD25+ T lymphocytes had not a relationship with progression-free survival. It was featured that above 5% peripheral CD4+CD25+ proportion of T cells was related with the median overall survival by a shorten of 51 months (p < 0.05) with the HR 1.65 (95%CI 1.04, 2.62). Above 5% CD4+CD25+proportion of T cells produced the HR to be 1.76 (95%CI 1.07, 2.87) In stage 0-II patients, and 3.59 (95%CI 1.05, 12.29) in triple negative breast cancer patients. CONCLUSIONS: Cellular immunity restoration recovered by adoptive T cell infusions which resulted in less proportion of peripheral CD4+CD25+T lymphocytes could be a potential prognostic indicator among early stage and triple negative patients.

[Prostatic inflammation-induced chronic pelvic pain: Roles of substance P and c-fos in the spinal cord].

OBJECTIVE: To explore the possible pain mechanism of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). METHODS: The models of CP/CPPS were established in male Wistar rats by the autoimmune method. The paw withdrawal threshold (PWT) was detected using Von Frey filament. The expressions of the substance P and c-fos in the prostate and spinal L5-S2 segments were determined by immunohistochemistry followed by analysis of their correlation with CP/CPPS. RESULTS: Compared with the control rats, the CP/CPPS models showed significantly decreased PWT (P < 0.05), remarkable prostatic inflammation, enlarged scope of lesions, and obvious interstitial lymphocytic infiltration (P < 0.05). Both the expressions of substance P and c-fos were markedly elevated in the prostate and spinal dorsal horn (L5-S2) of the rat models (P < 0.05), but the expression of substance P in the prostate exhibited no correlation with that in the spinal cord (r = 0.099, P = 0.338), nor did that of c-fos (r = 0.027, P = 0.454). CONCLUSION: The upregulated expressions of substance P and c-fos in the spinal cord L5-S2 sections may be associated with the pain mechanism of CP/CPPS.

[Roles of substance P and transient receptor potential vanilloid 1 in neuralgia in rats with chronic nonbacterial prostatitis].

OBJECTIVE: To study the possible mechanisms of chronic nonbacterial prostatitis (CNP) pain. METHODS: CNP models were established in male Wistar rats by the autoimmune method. Then the paw withdrawal threshold (PWT) was detected using the Von Frey filament, prostate pathological examination was conducted, the expressions of substance P (SP) and transient receptor potential vanilloid 1 (TRPV1) in the prostate tissue and L5-S2 spinal segments were determined by immunohistochemistry and their correlations were analyzed. RESULTS: Compared with the control group, the CNP model rats showed markedly decreased PWT (P < 0.05) and obvious inflammation in the prostate tissue, with significant differences in the scope of lesion and interstitial lymphocyte infiltration (P < 0.05). The expressions of SP and TRPV1 in the prostate and spinal cord dorsal horn L5-S2 were remarkably upregulated in the models as compared with the control rats (P < 0.05). However, the expression of SP in the prostate was not correlated with that in the spinal cord (r = 0.099, P = 0.338), nor was that of TRPV1 (r = 0.000, P = 0.5). CONCLUSION: SP and TRPV1 were involved in the formation and persistence of pain in CNP rats through their upregulated expressions in the L5-S2 spinal segments.

[Changes of mechanical pain threshold in rats with experimental autoimmune prostatitis].

OBJECTIVE: To observe the changes of the mechanical pain threshold in the rat model of autoimmune prostatitis, explore the mechanism of autoimmune prostatitis pain and offer some animal experimental evidence for the drug therapy of the condition. METHODS: Twenty male Wistar rats weighing 180 - 220 g were divided into a model and a control group. The autoimmune prostatitis model was established by subcutaneous injection of an extract of male rat prostate glands (RPG) at 60 mg/ml in Freund's complete adjuvant (FCA) and pertussis-diphtheria-tetanus vaccine at 0 and 30 days, respectively. Mechanical tactile hyperalgesia was measured once a week using Von Frey Filaments from the beginning of the study. At 8 weeks after modeling, the rats were sacrificed and the prostate tissues harvested for observation of histomorphological changes by HE staining. RESULTS: HE staining revealed different degrees of benign prostatitis in the model rats. Compared with the controls, the mechanical pain threshold in the model rats was significantly decreased with the increased time of modeling, from (65.52 +/- 6.27) g at 0 week to (23.67 +/- 4.09) g at 8 weeks (P < 0.01). Statistically significant differences were found in the variation trend at different time points between the two groups (P < 0.01). CONCLUSION: Autoimmune prostatitis models were successfully established in rats and hyperalgesia was induced after modeling.

[Erectile function of male rats in different age groups: an experimental study].

OBJECTIVE: To explore the relationship between aging and erectile function changes in rats in order to establish a rat model of aging-related erectile dysfunction (ED). METHODS: Eighty male Wistar rats were equally divided into four age groups (3-, 6-, 12- and 18-month) and treated with intragastric administration of sildenafil citrate (Sn) for penile erection tests. Twenty 3-month-old female Wistar rats were randomized to four groups as oestrous rat models. We recorded the rate and frequency of penile erections of the male rats in different age groups. RESULTS: The rates of penile erection were 85%, 75%, 40% and 30% and erectile frequencies were 2.27 +/- 0.80, 2.00 +/- 0.61, 1.40 +/- 0.51 and 1.29 +/- 0.49 in the 3-, 6-, 12- and 18-month rats, respectively, with statistically significant differences among different age groups (P < 0.01). And their erectile function exhibited a tendency to decrease with the increase of age. Besides, comparison of the 3-month with the 6-, 12- and 18-month groups showed significantly reduced erectile function in the 18-month rats (P < 0.05) but no remarkable difference between the 3-month and the 6- and 12-month groups (P > 0.05). CONCLUSION: Aging is one of the main risk factors of rat erectile dysfunction, and 18-month-old male rats are qualified for the establishment of the rat model of aging-related erectile dysfunction.

[Detection and clinical value of epithelial cellular adhesion molecule (EpCAM) mRNA positive circulating tumor cells in metastatic breast cancer].

OBJECTIVE: To test for circulating tumor cells (CTCs) relying on epithelial cellular adhesion molecule (EpCAM) expression in metastatic breast cancer by quantitative real-time reverse transcription-PCR. METHODS: In the study,47 metastatic breast cancer patients were evaluated by quantitative real-time PCR for detecting EpCAM mRNA. In addition, analyses were carried out for their correlation with patients' clinicopathologic features, response, and the time to progression (TTP). RESULTS: The sensitivity of EpCAM mRNA in the metastatic breast cancer patients was about 40%. However, the specificity of EpCAM mRNA for 20 healthy controls was 100%. TTP was calculated, and compared with that between EpCAM mRNA-positive and EpCAM mRNA-negative groups. For the retrospective study, the median TTP was 7.1 months and 11.1 months (P=0.013) for patients with EpCAM mRNA-positive and EpCAM mRNA-negative, respectively, after the first cycle chemotherapy. Moreover, a statistically significant correlation was demonstrated between EpCAM mRNA and TTP in patients who underwent the first or the second-line chemotherapy (P=0.018), but there was no significance in the patients pretreated with two or more previous chemotherapy lines (P=0.471). CONCLUSION: This study provides evidence of the presence of EpCAM mRNA in approximately 40% of patients with metastatic breast cancer. There is a strong correlation between EpCAM mRNA results after the first cycle therapy and TTP in metastatic breast cancer patients, and EpCAM mRNA positive after chemotherapy may predict shorter TTP.

[Establishment of a nonbacterial prostatitis model in rats using the autoimmune method: a dose-effect study].

OBJECTIVE: To establish an animal model of chronic nonbacterial prostatitis (CNP) using different doses of purified prostate protein with Freund's complete adjuvant (FCA), and to investigate the relationship of the doses with the success of the model construction. METHODS: Thirty male Wistar rats were divided into a control (A) and 4 experimental groups (B, C, D and E) of equal number. The latter 4 groups were given multi-loci intracutaneous injection of 1.0 ml of a 1:1 mixture of purified prostate protein at 20, 40, 60 and 80 mg/ml with Freund's complete adjuvant (FCA), and meanwhile intraperitoneally injected with 0.5 ml of pertussis-diphtheria-tetanus vaccine at 0 and 30 days. On the 45th day, the rats were sacrificed for observation of the pathomorphological changes in the prostate glands with the naked eyes and microscope. RESULTS: Different degrees of chronic inflammation were observed with different degrees of lymphocyte infiltration and interstitial hyperplasia in the experimental rats. More obvious changes were found in Groups C and D than in A, and even more significant in Group E (P < 0.05). CONCLUSION: The rat model of CNP can be successfully established by multi-loci intracutaneous injection of 1.0 ml of a 1: 1 mixture of purified prostate protein at 40 - 60 mg/ml with FCA, and simultaneously intraperitoneal injection of 0.5 ml of pertussis-diphtheria-tetanus vaccine twice within 30 days.

[Dendritic cells infected by recombinant adeno-associated virus with CEA gene to induce antigen-specific CTL response to CD44(+)CD24(-/low) cells from MCF-7 breast cancer cell line].

OBJECTIVE: To infect dendritic cells (DC) by recombinant human adeno-associated virus (rh-AAV) vector with CEA gene to generate antigen-specific CTL cells in vitro and to assay the CEA specific cytotoxic T lymphocyte(CTL), response to CD44(+)CD24(-/low) breast cancer stem cells. METHODS: Peripheral blood mononuclear cells were induced to generate DCs by cytokines interleukin-4(IL-4), granulocyte-macrophage colony-stimulating factor(GM-CSF)and tumor necrosis factor-alpha(TNF-α) while T lymphocytes were cultured with cytokines interleukin-2(IL-2). DCs were infected by CEA gene containing rh-AAV. After being matured, DCs were co-cultured with T cells to generate CTL cells. CD44(+)CD24(-/low) breast cancer stem cells were sorted out from MCF-7 and MDA-MB-231 cells. CEA specific CTL response to CD44(+)CD24(-/low) breast cancer stem cells was assayed by MTT method. RESULTS: The percentages of CD44(+)CD24(-/low) breast cancer stem cells subsets in MCF-7 and MDA-MB-231 were 5.1% and 76.3% respectively. DCs transfected with CEA gene could induce CEA antigen CTLs response. The killing ratio of MCF-7 cells in CEA gene transfection group was 46.5% plusmn; 15.0% with significant difference (P=0.009) as compared with that of CEA gene untransfection group. As for CD44(+)CD24(-/low) breast cancer stem cells subset from MCF-7 cells, CEA gene transfection group resulted in a higher killing ratio of 44.7% ± 28.2% as compared with that of CEA untransfection group. While the inhibitory rate of non-breast cancer stem cells subset from MCF-7 cells in CEA gene transfection group was 50.6% ± 22.2% (P=0.05). CTL cells generated by DCs transfected with CEA gene did not show inhibitory activity to MDA-MB-231 breast cancer cells (without CEA expression) as compared with CEA gene untransfection group. It was the same in CD44(+)CD24(-/low) breast cancer stem cells subset and non-breast cancer stem cells subset from MDA-MB-231 breast cancer cells (P>0.05). CONCLUSION: DCs infected by rh-AAV with CEA gene could induce antigen-specific CTL response to kill CEA expressing breast cancer cells involved in CD44(+)CD24(-/low) breast cancer stem cells subset. It suggests that immune therapy might be a potential treatment of breast cancer stem cells.

[Randomized clinical case-control trial for the comparison of docetaxel plus thiotepa versus docetaxel plus capecitabine in patients with metastatic breast cancer].

OBJECTIVE: To evaluate the efficacy and safety of docetaxel plus thiotepa(TXT/TSPA) and docetaxel plus capecitabine(TXT/CAPE) in patients with metastatic breast cancer. METHODS: The patients were randomized to give intravenous TXT 35 mg/m2 on days 1 and 8 plus intravenous TSPA 60-65 mg/m(2) on day 1 every 3 weeks, or intravenous TXT 35 mg/m(2) on days 1 and 8 plus oral CAPE 1 000 mg/m(2) twice daily on days 1 to 14 every 3 weeks, at least 2 cycles applied. RESULTS: TXT/TSPA group (22 patients) and TXT/CAPE group (24 patients) had consistent baseline. Docetaxel thiotepa group (21 cases) and docetaxel combined with capecitabine group (22 cases) were evaluated for their clinical responses, which showed that 2 of the 21 (9.52%) from TXT/TSPA group and 6 of the 22 (27.27%) from TXT/CAPE group had achieved partial remission; 11 of the 21 (52.38%) from TXT/TSPA group versus 7 of the 22 (31.82%) from TXT/CAPE group for stable diseases; 8 of the 21 (38.10%) from TXT/TSPA group versus and 9 of the 22 (40.91%) from TXT/CAPE group for progressive diseases, respectively. The disease control rate was 61.90% (13/21) and 59.09% (13/22) for TXT/TSPA and TXT/CAPE groups, the median progression-free survival(PFS) was 7.9 months [95% confidence interval(CI) 0.77 to 15.03] from TXT/TSPA group versus 8.3 months (95% CI 4.01 to 11.79) from TXT/CAPE group. One year survival rate was 88.20% for TXT/TSPA versus 81.00% for TXT/CAPE group, respectively. P values all exceeded 0.05, and the two groups showed no difference. No chemotherapy-related deaths occurred. Myelosuppression was the major side effect. The adverse events of grades 3 to 4 respectively occurred in TXT/TSPA and TXT/CAPE groups:leucocytopenia was 45.45% vs. 26.09%; neutropenia 45.45% vs. 21.74%; thrombocytopenia 9.09% vs. 0%; hand-foot syndrome 0% vs. 13.04%. P values all exceeded 0.05, and the two groups showed no difference. CONCLUSION: Combination of docetaxel and thiotepa in the treatment of metastatic breast cancer has some curative effect and adverse reactions can be tolerated. It can be used as an economical and effective rescue plan.

Multicenter clinical study for evaluation of efficacy and safety of transdermal fentanyl matrix patch in treatment of moderate to severe cancer pain in 474 chinese cancer patients.

OBJECTIVE: Although a new matrix formulation fentanyl has been used throughout the world for cancer pain management, few data about its efficacy and clinical outcomes associated with its use in Chinese patients have been obtained. This study aimed to assess the efficacy and safety of the new system in Chinese patients with moderate to severe cancer pain. METHODS: A total of 474 patients with moderate to severe cancer pain were enrolled in this study and were treated with the new transdermal fentanyl matrix patch (TDF) up to 2 weeks. All the patients were asked to record pain intensity, side effects, quality of life (QOL), adherence and global satisfaction. The initial dose of fentanyl was 25 µg/h titrated with opioid or according to National Comprehensive Cancer Network (NCCN) guidelines. Transdermal fentanyl was changed every three days. RESULTS: After 2 weeks. The mean pain intensity of the 459 evaluated patients decreased significantly from 5.63±1.26 to 2.03±1.46 (P<0.0001). The total remission rate was 91.29%, of which moderate remission rate 53.16%, obvious remission rate 25.49% and complete remission rate 12.64%. The rate of adverse events was 33.75%, 18.78% of which were moderate and 3.80% were severe. The most frequent adverse events were constipation and nausea. No fatal events were observed. The quality of life was remarkably improved after the treatment (P<0.0001). CONCLUSION: The new TDF is effective and safe in treating patients with moderate to severe cancer pain, and can significantly improve the quality of life.

Mobilization of Peripheral Blood Stem Cells Using Regimen Combining Docetaxel with Granulocyte Colony-stimulating Factor in Breast Cancer Patients.

OBJECTIVE: To evaluate the effectiveness and safety of the mobilization of peripheral blood hematopoietic stem cells by combining docetaxel with granulocyte colony-stimulating factor (G-CSF) in breast cancer patients. METHODS: A total of 57 breast cancer patients were treated with docetaxel 120 mg/m(2). When the white blood cell (WBC) count decreased to 1.0×10(9)/L, patients were given G-CSF 5 µg/kg daily by subcutaneous injection until the end of apheresis. Peripheral blood mononuclear cells (MNC) were isolated by Cobe Spectra Apheresis System. The percentage of CD34(+) cell was assayed by flow cytometry. RESULTS: At a median 6 of days (range 3-8) after the administration of docetaxel, the median WBC count decreased to 1.08×10(9)/L (range 0.20-2.31). The median duration of G-CSF mobilization was 3 days (range 2-7). The MNC collection was conducted 8-12 days (median 10 days) after docetaxel treatment. The median MNC was 5.35×10(8)/kg (range 0.59-14.07), the median CD34(+) cell count was 2.43×10(6)/kg (range 0.16-16.69). The CD34(+) cell count was higher than 1.00×10(6)/kg in 47 of 57 cases (82.46%) and higher than 2.00×10(6)/kg in 36 cases (63.16%). The CD34(+) cell count was higher than 2.00×10(6)/kg in 27 collections (23.68%). The MNC count and the CD34(+) cell count were correlated with the bottom of WBC after docetaxel chemotherapy (r=0.364, 0.502, P=0.005, 0.000). The CD34(+) cell count was correlated with the MNC count (r=0.597, P=0.000). The mobilization and apheresis were well tolerated in all patients. Mild perioral numbness and numbness of hand or feet were observed in 3 cases. No serious adverse events were reported. CONCLUSION: Mobilization of peripheral blood hematopoietic stem cell by combining docetaxel with G-CSF was effective and safety in breast cancer patients.

[Expression of genes related to Sonic Hedgehog signaling in human hepatocellular carcinomas].

OBJECTIVE: To investigate the expression status of Sonic Hedgehog signaling genes and molecules in human hepatocellular carcinomas(HCC), and to explore the relationship between these genes and clinical prognosis. METHODS: HCC tissue and adjacent normal tissue from 29 HCC patients were assayed for the expression of hedgehog signaling genes by reverse transcription-polymerase chain reaction chain reaction (RT-PCR) techniques and for the expression of hedgehog signaling molecules by immunohistochemistry. The expressions of Shh, Ptch, Smoh, Gli-1 mRNA were assayed as well as Shh, Ptch proteins in 29 cases of HCC and in 29 liver tissues adjacent to the tumor. RESULTS: Expression of Shh mRNA was detectable in about 51% of HCCs examined. Consistent with this, hedgehog target genes Ptch, Smoh and Gli-1 mRNA were expressed in over 68%, 48% and 62% of the tumors, respectively, and the expressions of Shh and Ptch proteins in HCC tumor tissues correlated with those of Shh and Ptch mRNA in tumor tissues (P=0.041 and P=0.035). This suggested that the hedgehog pathway was frequently activated in HCCs. The simultaneous expression of Gli-1 in HCC and liver tissues adjacent to the tumor had significantly relationship with poor prognosis. CONCLUSION: Hedgehog signaling activation is an important event for development of human HCCs. It also suggests that markers for hedgehog signaling activation may be useful for the determination of prognosis.

[Pilot study on the correlation between high incidence of CD44+/CD24 -/low/ABCG2- cells and poor prognosis in breast cancer].

OBJECTIVE: To explore whether the CD44+/CD24(-/low)/ABCG2(-) (ATP binding cassette superfamily G member 2) cells are associated with prognosis and clinical response in breast cancer patients. METHODS: We investigated the paraffin-embedded tissues of 43 breast cancer patients with (23 cases) and without (20 cases) recurrences. Double-staining immunohistochemistry (IHC) was applied for the detection of CD44+/CD24(-/low) cells and single-staining IHC for ABCG2. Flow cytometry was used to analyze the CD45(-)/CD44+/CD24(-/low)/ABCG2(-) cells in 4 mL peripheral blood of patients with metastasis breast cancer and 11 healthy female volunteers as controls. RESULTS: The positive rate of ABCG2 in recurrence-group was higher but with no difference compared with controls (78.3 % vs 60.0%, P = 0.32). Double-staining IHC revealed that the percentage of CD44(+)/CD24(-/low) cells was higher in recurrence-group than non-recurrence group(65.2% vs 35.0%, P = 0.048)and higher percentage of CD44+/CD24(-/low) cells was significant associated with poor overall survival(P = 0.031). Patients with higher percentage of CD44+/CD24(-/low) cells have shorter disease free survival (DFS), but have no statistical significance. Flow cytometry revealed that the CD45(-)/CD44+/CD24(- /low)/ABCG2(-) cells were higher in breast cancer patients than those of the volunteers (median 679/10(5) cells vs 12/10(5) cells). The cell number of this subset was affected by chemotherapy but was not statistically consistent with clinical response. CONCLUSION: Our study suggests that CD44+/CD24(-/low) breast cancer stem cells in tumor tissue may be associated with poor prognosis. The incidence of CD44+/CD24(-/low)/ABCG2(-) cells in peripheral blood is more frequent in breast cancer patients but further investigation should be made to explore the relationship of this subset and disease prognosis.

[Detection of breast cancer stem cells and the expression of key molecules in Hedgehog signaling pathway].

OBJECTIVE: To isolate and identify breast cancer initiating cells (CD44(+)/CD24(-/low) cells) in breast cancer cell lines MCF-7 and MDA-MB-231, and to evaluate the activity of the Hedgehog signaling pathway in different subpopulations. METHODS: CD44+/CD24(-/low) subpopulation and non-CD44+/CD24(-/low) subpopulation from breast cancer cell lines of MCF-7 and MDA-MB-231 were separated by fluorescence-activated cell sorting (FACS). Laser Scanning Confocal Microscope was used to identify CD44+/CD24(-/low) cells. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to analyze the expression of Human Patched gene (PTCH), Sonic Hedgehog (SHH), glioma-associated oncogene homoglog-1(GIi-1), smoothened homolog (SMOH) and GAPDH of CD44+/CD24(-/low) subpopulation and non-CD44+/CD24(-/low) subpopulation from MCF-7 and MDA-MB-231. RESULTS: The CD44+/CD24(-/low) subpopulation in MCF-7 and MDA-MB-231 cell lines were (1.70+/-1.43)% and (94.2+/-1.2)% respectively. The Hedgehog signaling pathways were active in CD44+/CD24(-/low) subpopulation of MCF-7 and MDA-MB-231 cell lines. The expression of transcription factor GIi-1, which was downstream components of the hedgehog pathway, was assayed and the results showed that the level of GIi-1 mRNA of CD44+/CD24(-/low) cells was much higher than that of non-CD44+/CD24(-/low) cells (P = 0.007 in MCF-7, and 0.005 in MDA-MB-231). CONCLUSION: Breast cancer initiating cells (CD44+/CD24(-/low) subpopulation) exist in MCF-7 and MDA-MB-231 cell lines. The Hedgehog signaling pathway is active in the subpopulation of MCF-7 and MDA-MB-231 cell lines.

[Treatment of malignant effusions with the injection of dendritic cells derived from autologous peripheral CD34+ stem cells].

OBJECTIVE: To evaluate the effect and side effects of CD34+ originated dendritic cells (DCs) infusion to treat malignant effusions. METHODS: Twenty-three patients with 26 malignant effusions received chemotherapy and granulocyte colony-stimulating factor (G-CSF) or G-CSF alone to mobilize peripheral blood hematopoietic stem cells. CD34+ stem cells were induced to differentiate into DCs by cytokines interleukin 4 (IL-4), granulocyte-macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). After being cultured for 10 to 14 days to stimulate proliferation and grow mature, DCs were refused into the body cavity by drainage tube weekly for 3 weeks. RESULTS: The response rate was 54% (14/26) and the benefit rate 81%. The median duration of response was 20 weeks (range:4-45 weeks). Karnofsky performance score (KPS) was improved in 15 patients. The response rate was 50% for the naive patients and 57% for the refractory patients. There were no severe side-effects. CONCLUSION: This pilot study demonstrates that DCs derived in vitro can exist viably after intropleural injection and can mediate biologic activity to malignant effusions. DCs immunotherapy is promising to treat malignant effusions with good tolerance.

Clinical study on the treatment of premature ejaculation by Uighur medicine gu-jing-mai-si-ha tablet.

OBJECTIVE: To observe the effect of Uighur medicine gu-jing-mai-si-ha tablet (GJMSHT) for treatment of premature ejaculation (PE) and to explore part of its mechanism. METHODS: The condition of patients was scored by related questionnaire, and the intravaginal ejaculation latency time (IELT) was observed before and after GJMSHT treatment, with the blood levels of nitric oxide (NO) and prostaglandin F2alpha (PGF2alpha) detected in PE patients as well. The results were compared with those in the control group. RESULTS: After treatment, the scores of PE and IELT, as well as the levels of NO and PGF2alpha, all increased significantly compared to those before treatment in the treated group (P<0.01), while in the control group, all the parameters were insignificantly changed (P>0.05). Therefore, the difference of these parameters between the two groups after treatment all showed statistical significance (P<0.01). CONCLUSION: GJMSHT could treat PE effectively, its mechanism is possibly by strengthening the coordination of the related smooth muscles through increasing the blood levels of NO and PGF2alpha, and the endurance of patients to the cavitary effect of prostatico-urethral pressure, thus postponing the arrival of urgent ejaculatory feeling.