Blood microbiota diversity determines response of advanced colorectal cancer to chemotherapy combined with adoptive T cell immunotherapy.

Human microbiota influence the response of malignancies to treatment with immune checkpoint blockade; however, their impact on other forms of immunotherapy is poorly understood. This study explored the effect of blood microbiota on clinical efficacy, represented by progression-free survival (PFS) and overall survival (OS), of combined chemotherapy and adoptive cellular therapy (ACT) in advanced colon cancer patients. Plasma was collected from colorectal cancer patients (CRC) treated with either chemotherapy alone (oxaliplatin and capecitabine) (XELOX CT alone group, n = 19), or ACT with a mixed dendritic cell/cytokine-induced killer cell product (DC-CIK) + XELOX (ICT group, n = 20). Circulating microbiota analysis was performed by PCR amplification and next-generation sequencing of variable regions V3~V4 of bacterial 16S rRNA genes. The association of the blood microbial diversity with clinical response to the therapy as measured by RECIST1.1 and OS was evaluated. The baseline Chao index of blood microbial diversity predicted prolonged PFS and OS of DC/CIK immunotherapy. More diverse blood microbiota that included Bifidobacterium, Lactobacillus, and Enterococcus were identified among responders to DC/CIK compared with non-responders. The plasma bacterial DNA copy number is inversely correlated with the CD3-/CD16+/CD56+ NK cells in circulation and decreased following DC-CIK; however, the Chao index of plasma microbiota significantly increased after administration of the DC-CIK product and this subsequent change was correlated with the number of CD3-/CD16+/CD56+ and CD8+/CD28+ cells infused. The diversity of the blood microbiome is a promising predictive marker for clinical responses to chemotherapy combined with DC-CIK. Cellular immunotherapy can affect the plasma microbiota's diversity in a manner favorable to clinical responses.

The role of prostate-specific antigen and multiparametric magnetic resonance imaging in the diagnosis of granulomatous prostatitis induced by intravesical Bacillus Calmette-Guérin vaccine therapy in patients with nonmuscle invasive bladder cancer.

AIMS: This study aimed to evaluate the role of serum prostate-specific antigen (PSA) levels and multiparametric magnetic resonance imaging (mpMRI) in the diagnosis of granulomatous prostatitis (GP) induced by intravesical Bacillus Calmette-Guérin vaccine (BCG) therapy in patients with nonmuscle invasive bladder cancer (NMIBC). SUBJECTS AND METHODS: We retrospectively analyzed eight patients with bladder cancer who underwent intravesical BCG therapy after transurethral resection of bladder tumor (TURBt) cancer. All these eight patients received 12-core transrectal ultrasound-guided prostate systemic biopsies. Clinical data on PSA with T1-weighted imaging (T1WI), T2WI, diffusion-weighted imaging (DWI), and apparent diffusion coefficient (ADC) on mpMRI were enrolled in the study. H and E and acid-fast staining was performed to pathologically prove GP. RESULTS: Four of all eight cases were above 4 ng/ml total PSA (tPSA) levels and four cases were within normal ranges, while free PSA/tPSA levels decreased to lower than 16% in all patients. Every patient had hard prostatic nodules through digital rectal examination (DRE). All characters of prostate mpMRI did not show signal intensity (SI) of prostate cancer before BCG therapy but showed abnormal signals after BCG therapy. All nodular lesions showed equal SI on T1WI, lower SI on T2WI, higher SI on DWI, and lower SI on ADC after BCG therapy. Pathologic results were GP and acid-fast staining outcomes were positive in all biopsies. CONCLUSIONS: Perioperative serum PSA levels, prostate magnetic resonance imaging, and DRE may help in the diagnosis of GP induced by intravesical BCG therapy. In general, male patients with middle- and high-risk NMIBC are recommended to undertake DRE, PSA, and prostate mpMRI, if possible, before and after TURBt."

Serial assessment of circulating T lymphocyte phenotype and receptor repertoire during treatment of non-muscle invasive bladder cancer with adoptive T cell immunotherapy.

Recurrence and progression of non-muscle-invasive bladder cancer (NMIBC), frequent despite the availability of multiple treatment modalities, may be partly explained by the presence of immunosuppressive cell populations. We hypothesized that progression of disease could be prevented by the administration of an activated T cell immunotherapy (ACT) at time points when immunosuppressive populations increased in peripheral blood. In an N-of-1 study, a patient with multiple primary bladder high grade urothelial carcinomas, previously treated with standard local resection and chemotherapy but with evidence of progression, received ACT consisting of dendritic cells mixed with cytokine induced killer cells (DC/CIK), intravenously 18 times over a 6 year period at indicated time of observed increases in peripheral blood immunosuppressive CD8+/CD28- cells. Peripheral blood was analyzed for T cell phenotype by flow cytometry, T cell receptor (TCR) repertoire, and circulating tumor DNA (ctDNA) by next generation sequencing (NGS) at the time of each infusion. Cystoscopy and pelvic CT scans were performed at routine intervals to assess clinical status of disease. There has been no recurrence or metastasis of urothelial carcinoma. Peripheral blood cytotoxic T cells and unique TCR clones increased and suppressive T cell populations decreased after DC/CIK infusions evidenced by the two more proof-of concept cases. ctDNA analysis detected mutations in six genes (ARID1B, MYCN, CDH23, SETD2, NOTCH4 and FAT1) which appeared at different times, but all of them disappeared after the DC-CIK infusions. These data suggest that DC/CIK infusions may be associated with beneficial changes in T cell phenotype, TCR repertoire, decreases in circulating tumor DNA and sustained recurrence-free survival.

Clinical efficacy of intra-cavitary infusions of autologous dendritic cell/cytokine-induced killer cell products for the treatment of refractory malignant pleural effusions and ascites.

To explore the safety and efficacy of intra-cavitary infusions of autologous mixed dendritic cell (DC)-cytokine-induced killer (CIK) cell products in advanced cancer patients with malignant pleural effusions or ascites. DC-CIKs were expanded ex vivo (mean yield of 1.36×109 cells (range, 0.74~4.98×109)) from peripheral blood mononuclear cells obtained by repeated venipuncture or apheresis. Patients received at least 1 cycle of 3 infusions of the DC-CIKs administered by indwelling catheter into the pleural or peritoneal cavity every other day. The volume of malignant effusions was assessed radiologically. Peripheral blood lymphocyte populations were enumerated by flow cytometry. Quality of life (QoL) during the DC-CIK infusions was assessed by the EORTC QLQ-30 instrument. ctDNA sequencing was performed to analyze gene clonal load and molecular tumor burden during the infusion treatment. Thirty-seven patients with breast, lung and other malignancies were enrolled. The results showed that intra-cavitary DC-CIK infusions (16 intrapleural and 21 intraperitoneal) were well-tolerated with no grade 3/4 adverse events. There was one complete response with effusion disappearance (CR) (3%), 13 partial responses (PR) (35%), 12 with stable disease (SD) (32%) and 11 with progressive disease (PD) (30%), resulting in a clinical effusion control rate (CCR) of 70% (26/37). The total number of infused CIKs and the CD3+/CD8+ and CD8+/CD28+ T cell frequencies within the CIKs were associated with effusion control (P=0.013). Moreover, increased peripheral blood CD3+/CD8+ (P=0.035) and decreased CD4+/CD25+ T cell frequencies (P=0.041) following the DC-CIK infusions were associated with malignant effusion and ascites control. Reductions in ctDNA correlated with clinical benefit. In conclusion, intra-cavitary autologous cellular immunotherapy is an alternative method to effectively control malignant pleural effusions and ascites. The overall effusion control rate was associated with higher peripheral blood effector T cell frequencies.

C/N0 Estimator Based on the Adaptive Strong Tracking Kalman Filter for GNSS Vector Receivers.

The carrier-to-noise ratio (C/N0) is an important indicator of the signal quality of global navigation satellite system receivers. In a vector receiver, estimating C/N0 using a signal amplitude Kalman filter is a typical method. However, the classical Kalman filter (CKF) has a significant estimation delay if the signal power levels change suddenly. In a weak signal environment, it is difficult to estimate the measurement noise for CKF correctly. This article proposes the use of the adaptive strong tracking Kalman filter (ASTKF) to estimate C/N0. The estimator was evaluated via simulation experiments and a static field test. The results demonstrate that the ASTKF C/N0 estimator can track abrupt variations in C/N0 and the method can estimate the weak signal C/N0 correctly. When C/N0 jumps, the ASTKF estimation method shows a significant advantage over the adaptive Kalman filter (AKF) method in terms of the time delay. Compared with the popular C/N0 algorithms, the narrow-to-wideband power ratio (NWPR) method, and the variance summing method (VSM), the ASTKF C/N0 estimator can adopt a shorter averaging time, which reduces the hysteresis of the estimation results.

Adoptive immunotherapy with autologous T-cell infusions reduces opioid requirements in advanced cancer patients.

Relief of cancer-related pain remains challenging despite the availability of a range of opioid and nonopioid medications. Animal models demonstrate that T lymphocytes may mediate analgesia by producing endogenous opioids, but definitive clinical data are limited. Transfer of ex vivo adoptive cellular therapy (ACT) is being tested as an anticancer therapy. We retrospectively reviewed the medical charts of 357 patients with various malignancies who received 3 intravenous infusions of autologous cytokine-activated T-cell-enriched products. Among these were 55 patients who required opioids for moderate or severe cancer-related pain. Opioid dosage and cancer pain score were recorded daily for 2 consecutive weeks before and 2 weeks after the ACT infusions. The average oral morphine equivalent doses and cancer pain scores were significantly decreased after the ACT infusions. The proportion of patients with breakthrough pain also declined. Moreover, higher frequencies of expanded CD3, CD3/CD4, and CD3/CD8 T cells within the ACT product were associated with favorable analgesic effects. Transient elevations in CD3 and CD3/CD8T-cell subpopulations and decreases in CD4CD25 Treg were observed in patients' blood after the ACT. In conclusion, ACT was capable of reducing cancer pain severity and opioid consumption and favorably modulating peripheral blood T-cell populations.

Synergistic association of FOXP3+ tumor infiltrating lymphocytes with CCL20 expressions with poor prognosis of primary breast cancer: A retrospective cohort study.

Studies have shown that forkhead/winged helix transcription factor P3 (FOXP3) tumor infiltrating lymphocytes (TILs) are intimately associated with invasion and survival of many invasive tumors. The inflammatory chemokine ligand 20 (CCL20) and its receptor CCR6 were found to be associated with tumor prognosis in some studies. Although increases in FOXP3 TILs infiltration and CCL20 expression have been revealed in several malignancies, their correlation in human breast tumors is as yet unclear.Surgically resected samples from 156 patients with invasive breast cancer (BC) were assessed for the expression of FOXP3 and CCL20 by immunohistochemistry. Correlation between their expressions and the association with clinicopathological characteristics and patient's prognosis were studied. Forty pairs of fresh BC and their nontumor adjacent tissues (NATs) in BC were carried out by real-time quantitative PCR (qRT-PCR) to evaluate the correlation between FOXP3 and CCL20 mRNA expression.CCL20 and FOXP3 TILs mRNA expression in tumor tissue demonstrated a high correlation (rs = 0.359, P < .001) in this cohort of breast cancer patients. Both elevated CCL20 expression and FOXP3 TILs infiltration were significantly correlated with high histological grade, positive human epidermal growth factor receptor-2 (HER2), high Ki67 index, and axillary lymph node metastases. Tumors with concomitant high expressions of both markers had the worst prognosis. Multivariate analysis showed that these 2 markers were independent predictors of overall survival. The patients with axillary lymph node metastases with the concomitant CCL20 high expression and increased FOXP3 TILs infiltration had the worst overall survival (OS) (P < .001), In lymph node-negative breast cancer patients, the status of CCL20 and FOXP3 was not related to OS (P = .22).The results suggest that CCL20 and FOXP3 TILs may have synergistic effects, and their upregulated expressions may lead to immune evasion in breast cancer. Combinatorial immunotherapeutic approaches aiming at blocking CCL20 and depleting FOXP3 might improve therapeutic efficacy in breast cancer patients.

Circulating CD8+CD28- suppressor T cells tied to poorer prognosis among metastatic breast cancer patients receiving adoptive T-cell therapy: A cohort study.

BACKGROUND AIMS: This study aimed to determine the prognostic value of circulating CD8+CD28- T lymphocytes among breast cancer patients treated with adoptive T-lymphocyte immunotherapy after chemotherapy. METHODS: Two hundred and thirty-two breast cancer patients underwent adoptive T-cell immunotherapy. Circulating CD8+CD28- proportion was measured by flow cytometry. Median proportion of CD8+CD28- was 24.2% and set as the categorical cutoff value for further analysis. The median survival was estimated by Kaplan-Meier curve, with difference detection and hazard ratio estimation by log-rank test and Cox hazard proportion regression model. RESULTS: With adoptive T-cell therapy, patients with higher CD8+CD28- levels experienced median progression-free and overall survival of 7.1 months and 26.9 months, respectively-significantly shorter than patients with lower levels (11.8 and 36.2 months). CD8+CD28- proportion >24.2% demonstrated a hazard ratio (HR) of 2.06 (95% confidence interval [CI] 1.31-3.12) for progression and an HR of 1.97 (95% CI 1.06-3.67) for death. Among patients who had received previous first-line chemotherapy, CD8+CD28- proportion >24.2% demonstrated an HR of 2.66 (95% CI 1.45-4.88) for progression. Among patients exposed to previous second-line or higher chemotherapy, CD8+CD28- proportion >24.2% demonstrated a 486% higher risk for death (HR = 5.86, 95% CI 1.77-19.39). A 1% increase in suppressive T cells was associated with a 5% increased risk of death. DISCUSSION: Elevated peripheral blood CD8+CD28- was associated with poorer prognosis for metastatic breast cancer, especially for higher risk of progression among patients with first-line chemotherapy and higher risk of death among patients with more than second-line chemotherapy.

Enhanced antitumor effects and improved immune status of dendritic cell and cytokine-induced killer cell infusion in advanced cancer patients.

Little progress has been made in the treatment of advanced cancer. Dendritic cells (DCs) plus cytokine-induced killer (CIK) cells have exhibited antitumor effects. Thus, the aim of the present study was to evaluate the clinical efficacy of DC-CIK cell treatment in patients with advanced cancer. A paired study including 57 patients treated with DC-CIK cells (DC-CIK group) and 33 patients treated with best supportive care alone (BSC group) was performed. The patients in the DC-CIK group were matched to those in the control group in terms of sex, age, tumor type and clinical stage. T-cell subsets were detected and overall survival (OS) was compared between the two groups. The results demonstrated that CD4+/CD25+ and CD8+/CD28- subsets significantly decreased following DC-CIK immunotherapy (P<0.05). The CD3+, CD3+/CD8+, CD8+/CD28+ and CD3+/CD56+ T-cell subsets were significantly increased in the DC-CIK group compared with the BSC group, while the CD8+/CD28- subset was significantly decreased. Univariate analysis demonstrated that a lower CD8+/CD28- and a higher CD8+/CD28+ ratio were associated with prolonged OS in advanced cancer patients. In addition, DC-CIK treatment administration, age (>60 vs. <60 years), clinical stage and the frequency of CIK treatment significantly affected the OS of patients in the DC-CIK group. A CD8+/CD28- ratio of <21.12 was found to decrease the hazard ratio (HR) of OS to 0.50 [95% confidence interval (CI): 0.29-0.87] and a CD8+/CD28+ ratio >9.04 was found to decrease the HR of OS to 0.45 (95% CI: 0.21-0.98). No serious side effects were observed in the DC-CIK group. Taken together, these data indicate that DC-CIK infusions were able to change the ratios of the T-cell subsets, which increased the T helper cell and cytotoxic T lymphocyte subsets, while it decreased regulatory T lymphocyte subsets. Thus, this method of immunotherapy was found to improve the imbalance in the immune system and prolong the OS in patients with advanced cancer.

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.

Elevated level of peripheral CD8(+)CD28(-) T lymphocytes are an independent predictor of progression-free survival in patients with metastatic breast cancer during the course of chemotherapy.

PURPOSE: Suppression of cellular immunity resulting from tumorigenesis and/or therapy might promote cancer cells' growth, progression and invasion. Here, we explored whether T lymphocyte subtypes from peripheral blood of metastatic breast cancer (MBC) female patients could be used as alternative surrogate markers for cancer progress. Additionally, plasma levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ, and transforming growth factor-ß1 were quantitated from MBC and healthy volunteers. EXPERIMENTAL DESIGN: This study included 89 female MBC patients during the post-salvage chemotherapy follow-up and 50 age- and sex-matched healthy volunteers as control. The percentages of T lymphocyte subpopulations from peripheral blood and plasma levels of cytokines were measured. RESULTS: Both CD8(+)CD28(-) and CD4(+)CD25(+) were elevated in MBC patients compared to the control cohort (P < 0.05). In contrast, CD3(+) and CD8(+)CD28(+)cells were significantly lower in MBC patients (P < 0.0001, P = 0.045, respectively). MBC patients had elevated levels of immunosuppressive cytokines IL-6 and IL-10. Patients with elevated CD8(+)CD28(-) and CD4(+)CD25(+) cells showed increased levels of IL-6, and only patients with elevated CD8(+)CD28(-) had decreased interferon-γ. Univariate analysis indicated increased CD3(+)CD4(+) or CD8(+)CD28(+)correlated with prolonged progression-free survival (PFS), while elevated CD8(+)CD28(-)associated with shorten PFS. The percent of CD8(+)CD28(-) T lymphocytes is an independent predictor for PFS through multivariate analysis. CONCLUSIONS: This study suggests that progressive elevated levels of CD8(+)CD28(-) suppressor T lymphocytes represent a novel independent predictor of PFS during post-chemotherapy follow-up.

Recombinant human endostatin could eliminate the pro-angiogenesis priority of SP cells sorted from non-small cell lung cancer cells.

PURPOSE: To ascertain the biologic significance of lung cancer Side population (SP) cells, which represent putative cancer stem cells (CSC) in the absence of consensus biomarkers for tumor-specific CSC. MATERIALS AND METHODS: We sorted and analyzed the angiogenic features of SP cells, isolated from tumor cell lines based on the exclusion of the DNA dye Hoechst 33342, from the NSCLC cell lines A549 and H460. RESULTS: Compared with non-SP cells, mRNA of vascular endothelial growth factor (VEGF)-A, VEGF-B, angiopoietin (ang)-1, ang-2, fibroblast growth factor-2 (FGF-2), cyclooxygenase-2 (Cox-2) and interleukin-8 (IL-8) were over-expressed in SP cells accompanied by over-expression of ABCG2 and MDR1 mRNA. The supernatant of cultured SP cells could significantly induce migration of human umbilical vein endothelial cells, while recombinant human endostatin (Endostar 25(®)) could inhibit the migration. CONCLUSIONS: This study revealed that the NSCLC SP cells might represent CSCs and possess pro-angiogenic properties, and antiangiogenesis represent a potential therapy.

Clinical safety of induced CTL infusion through recombinant adeno-associated virus-transfected dendritic cell vaccination in Chinese cancer patients.

AIM: The aim of the study is to explore the safety of cytotoxic T lymphocytes (CTLs) infusion by transfected dendritic cells (DCs) with recombinant adeno-associated virus vector (rAAV) carrying CEA cDNA among advanced cancer patients. PATIENTS AND METHODS: A total of 27 cancer patients with tumor tissue expression positivity and/or sera-elevated level of CEA were subsequently divided into cohort A and B resulted from the ex vivo expansion number of CTLs generated from co-culture of specific transfected DCs with autologous T lymphocytes. Based on the variations of infused number of specific CTL derived from different yields of individualized patients who had experienced various anti-cancer treatments, we compared the patients of low number of CTL cells (2-8 × 10(8) infused, cohort A, 6 cases) with those of higher number (above 8 × 10(8) infused, cohort B, 21 cases) to testify the possible adverse reactions caused by amount of infused CTLs. This study resembled a phase I study aiming for setting up clinical trial of adoptive cellular therapy that conceptually comes from conventional cytotoxic drugs. RESULTS: The results showed that one case from the each cohort had experienced moderate fever, and four cases with fatigue were seen in cohort B. The symptoms were transient without serious adverse events. For the consideration of clinical response 2 partial remission (8.0 %, 2/25), 1 minor remission, and 9 stable disease (40 %, 10/25) were observed in 25 patients eligible for evaluation. Sera levels of CEA assay were lowered in six patients. During a median follow-up of 8.1 months, we could not observe severe or chronic adverse reactions related to rAAV-DC infusions. Meanwhile, the variation of number of CTLs infused in this setting did not alter the status of peripheral lymphocyte population. CONCLUSIONS: These preliminary data suggest that the rAAV-DC immunotherapy is well-tolerated and showed no severe adverse reactions in cancer patients.

HER2-specific T lymphocytes kill both trastuzumab-resistant and trastuzumab-sensitive breast cell lines in vitro.

OBJECTIVE: Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer, the resistance to anti-HER2 therapy is a growing clinical dilemma. We aim to determine whether HER2-specific T cells generated from dendritic cells (DCs) modified with HER2 gene could effectively kill the HER2-positive breast cancer cells, especially the trastuzumab-resistant cells. METHODS: The peripheral blood mononuclear cells (PBMCs) from healthy donors, whose HLA haplotypes were compatible with the tumor cell lines, were transfected with reconstructive human adeno-association virus (rhAAV/HER2) to obtain the specific killing activities of T cells, and were evaluated by lactate dehydrogenase (LDH) releasing assay. RESULTS: Trastuzumab produced a significant inhibiting effect on SK-BR-3, the IC50 was 100ng/ml. MDA-MB-453 was resistant to trastuzumab even at a concentration of 10,000 ng/ml in vitro. HER2-specific T lymphocytes killed effectively SK-BR-3 [(69.86±13.41)%] and MDA-MB-453 [(78.36±10.68)%] at 40:1 (effector:target ratio, E:T), but had no significant cytotoxicity against HER2-negative breast cancer cell lines MDA-MB-231 or MCF-7 (less than 10%). CONCLUSION: The study showed that HER2-specific T lymphocytes generated from DCs modified by rhAAV/HER2 could kill HER2-positive breast cancer cell lines in a HER2-dependent manner, and result in significantly high inhibition rates on the intrinsic trastuzumab-resistant cell line MDA-MB-453 and the tastuzumab-sensitive cell line SK-BR-3. These results imply that this immunotherapy might be a potential treatment to HER2-positive breast cancer.

Activation of sonic hedgehog signaling pathway is an independent potential prognosis predictor in human hepatocellular carcinoma patients.

OBJECTIVE: The activation of hedgehog (HH) pathway is implicated in the development of human malignancies including hepatocellular carcinoma (HCC). However, the clinical impact of HH activation in HCC patients is still unclear. This study was conducted to confirm whether the expression of HH pathway components was associated with HCC progression and clinical outcome. METHODS: This study was a sample-expanded and prolonged follow up of one of our previous studies. It included 46 HCC patients who underwent surgical treatment from 2002 to 2005. The expression of sonic HH (SHH), patched-1 (PTCH1), smoothened (SMOH) and glioma-associated oncogene-1 (GLI1) genes in tumor and adjacent normal tissues extracted from the patients were examined by reverse transcription-polymerase chain reaction (RT-PCR) to explore the relationship between these genes and the clinical prognosis of HCC. RESULTS: The expression levels of SHH, PTCH1, SMOH and GLI1 in HCC tissues were 60.87%, 50.00%, 32.61% and 54.35%, respectively. The expression levels of SHH-related molecules were relatively intense in cancer tissue, but insignificantly correlated with any clinicopathological factors of tumor. Transcriptional factor GLI1 was the only molecule associated with poor prognosis among the HCC patients. The expression of GLI1 gene in tumor tissues was significantly related with disease-free survival (DFS) (P=0.042) and overall survival (OS) (P=0.030). The simultaneous expression of GLI1 in tumor and adjacent normal liver tissues correlated with DFS (P<0.029) and OS (P<0.025). CONCLUSIONS: HH signaling activation is an important event in the development of human HCC. The expression of GLI1 in SHH pathway is possibly involved in HCC progression, which may be a useful prognostic indicator of HCC.

[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.

DNA methyltransferase inhibitor CDA-2 synergizes with high-dose thiotepa and paclitaxel in killing breast cancer stem cells.

It has been suggested that breast cancer stem cells (CSCs), which characterized by CD44(+)CD24(-/low), may result in treatment failure in patients with breast cancer. It is possible therefore that that inhibiting such subpopulation might subsequently improve clinical outcome. In the present study, we found that the CD44(+)/CD24(-/low) CSCs, isolated from both human breast cell line MCF-7 and MDA-MB-231, were more resistant to thiotepa, paclitaxel and anthracycline, when compared with the non-breast cancer stem cell subset from the same cell lines, whereas the chemosensitivities were remarkably reversed by higher concentration of thiotepa and paclitaxel except for adriamycin. The percentage of CSCs was significantly decreased with an addition of DNA methyltransferase inhibitor CDA-2 and the expression of Smo, Shh, and Gli-1 of Hedgehog signaling pathway in CSCs was decreased. Of important findings, combination of thiotepa or paclitaxel with CDA-2 could significantly inhibit the proliferation of CSCs regardless of their dosages. These results unveiled that the selection of cytotoxic agents and increasing their dosage might be of great importance in the respect of eliminating CSCs. DNA methyltransferase inhibitor CDA-2 exhibited a synergistic effect with cytotoxic drugs, which might provide a conceptually new therapeutic strategy.

MUC1-positive circulating tumor cells and MUC1 protein predict chemotherapeutic efficacy in the treatment of metastatic breast cancer.

Chemotherapy plays an important role in the treatment of metastatic breast cancer. It is important to monitor chemotherapeutic efficacy, to find a simple and efficient tool to guide treatment, and to predict the efficacy of treatment in a timely and accurate manner. This study aimed to detect mucin-1 (MUC1)-positive circulating tumor cells and MUC1 protein in the peripheral blood of patients with metastatic breast cancer and to investigate their relationship to chemotherapeutic efficacy. MUC1 mRNA was detected in the peripheral blood of 34 patients with newly diagnosed metastatic breast cancer by reverse transcription-polymerase chain reaction. The positive rates of MUC1 mRNA were 88.2% before chemotherapy and 70.6% after chemotherapy, without a significant difference (P=0.564); MUC1 mRNA expression before chemotherapy had no correlation with treatment effectiveness (P=0.281). The response rate of MUC1 mRNA-negative patients after first-cycle chemotherapy was significantly higher (P=0.009) and the progression-free survival (PFS) was clearly longer than those of MUC1 mRNA-positive patients (P=0.095). MUC1 protein in peripheral blood plasma was detected by an ELISA competitive inhibition assay. The patients with decreased MUC1 protein after chemotherapy had a significantly longer PFS than those with elevated MUC1 protein (P=0.044). These results indicate that the outcomes of MUC1 mRNA-negative patients after chemotherapy are better than those of MUC1 mRNA-positive patients. In addition, patients with decreased expression of MUC1 protein have a better PFS.

Cancer/testis antigens and clinical risk factors for liver metastasis of colorectal cancer: a predictive panel.

PURPOSE: Accumulating evidence suggests that cancer/testis antigens may serve as indicators of tumor malignant phenotype. The purpose of this study is to evaluate cancer/testis antigen genes in predicting metastasis of colorectal cancer to the liver. METHODS: The expression levels of 25 cancer/testis antigen genes were determined by reverse-transcription polymerase chain reaction in 288 colorectal cancer tissue samples from the primary tumor or liver metastasis. Pearson chi2 and multiple logistic regression analyses were performed to assess the association between risk factors and probability of liver metastasis of colorectal cancer. RESULTS: No significant difference was detected between the primary tumor and liver metastasis in expression pattern of cancer/testis antigen genes in colorectal cancer tissue samples. However, 3 cancer/testis antigen genes (PAGE4, SCP-1, and SPANX) and 3 clinicopathologic parameters (lymph node involvement, vessel cancer embolus, and tumor invasion depth) correlated significantly with liver metastasis of colorectal cancer (P < .05). A logistic regression model was constructed for prediction of liver metastasis based on a panel consisting of PAGE4, lymph node involvement, and presence or absence of vessel cancer embolus. The predicted risk of liver metastasis based on the panel was consistent with the actual risk observed. The probability of developing liver metastasis as estimated by the panel was 86.9% when all 3 factors were positive, representing an up to 20% improvement in the prediction level compared with the classic methods of lymph node involvement and vessel cancer embolus. CONCLUSIONS: A new predictive panel including PAGE4 expression may help predict liver metastasis of colorectal cancer.

[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.