Expression Pattern and Prognostic Value of CTLA-4, CD86, and Tumor-Infiltrating Lymphocytes in Rectal Cancer after Neoadjuvant Chemo(radio)therapy.

The synergistic effect of combining immune checkpoint inhibitors (ICIs) with neoadjuvant chemo(radio)therapy (nCRT) in colorectal cancer is still limited. We aimed to understand the impact of nCRT on the tumor microenvironment and to explore favorable immune markers of this combination. Herein, we investigated the expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), CD86, CD4, and CD8 after nCRT and its association with clinicopathological characteristics. Immunostaining of immune-related molecules was performed in 255 surgically resected specimens from rectal cancer patients treated with nCRT. CD4 and CD8 expression on the tumor (tCD4/CD8), stroma (sCD4/CD8), and invasive front (iCD4/CD8) was evaluated. The expression levels of immune-related molecules were significantly lower in the nCRT-treated group, except for CTLA-4 and sCD8. However, patients with higher sCD8+ cell density and CTLA-4 expression had better progression-free survival (PFS) and distant metastasis-free survival (DMFS). In addition, higher CD86 expression was associated with poorer overall survival (OS). Higher CTLA-4 expression was associated with higher tCD8+ cell density, whereas CD86 expression was correlated with the cell density of t/sCD8. Prognostic analysis confirmed that the relationships between CTLA-4 and DMFS as well as CD86 and OS were significantly correlated in low rather than high CD8+ cell density. Further the combination of CD8+ cell density and CD86 expression was shown to be an independent prognostic factor of OS, whereas the combination of CTLA-4 was not for DMFS. Together, these results demonstrate significant correlations between CD86 expression and t/sCD8+ cell density in rectal cancer after nCRT and could potentially have clinical implications for combining ICIs and nCRT.

Conversion chemotherapy with capecitabine and oxaliplatin for colorectal cancer with potentially resectable liver metastases: A phase II, open-label, single-arm study.

AIM: The aim of this is study is to assess the efficacy and safety of conversion capecitabine plus oxaliplatin (XELOX) in Chinese patients with potentially resectable colorectal liver metastases (CLMs). PATIENTS AND METHODS: Thirty patients (median age 57.5 years) with potentially resectable CLMs were treated with XELOX in a single-arm, open-label, nonrandomized, multicenter clinical trial. RESULTS: The objective response rate in the 30 patients was 40% (95% confidence interval: 22.7%-59.4%), and the rate of conversion to resectable CLMs was 43.3%. Patients who underwent liver resection (n = 11) had a longer median progression-free survival and overall survival than those who did not. XELOX showed an acceptable safety profile. CONCLUSION: XELOX may effectively convert potentially resectable CLM into resectable CLM, providing survival benefits with a favorable safety profile. CLINICAL TRIALS.GOV IDENTIFIER: NCT 00997685.

Neuroendocrine-like cells -derived CXCL10 and CXCL11 induce the infiltration of tumor-associated macrophage leading to the poor prognosis of colorectal cancer.

Our previous study revealed that neuroendocrine differentiation in colorectal cancer is one of the important factors leading to worse prognosis. In this study, we apply immunohistochemical staining, Western-blot, RT-PCR and ELISA to investigate the underlying mechanism that how the neuroendocrine differentiation to affect the prognosis of colorectal cancer. The interaction of colorectal cancer cells, neuroendocrine-like cells and tumor-associated macrophages in colorectal cancer progress is also investigated. By analyzing 82 cases of colorectal cancer patients treated in our institution, we found that colorectal adenocarcinoma with neuroendocrine differentiation had increasing number of tumor-associated macrophages and worse prognosis. Further evaluation of cytology showed that neuroendocrine cells have the ability to recruit tumor-associated macrophages to infiltrate the tumor tissue, and the tumor-associated macrophages enhance the proliferation and invasion abilities of the colon cancer cells. Moreover, we confirmed that CXCL10 and CXCL11 are the key chemokines in neuroendocrine-like cells and they promote the chemotaxis activity of tumor-associated macrophages. The secretion of CXCL10 and CXCL11 by neuroendocrine-like cells can recruit tumor-associated macrophages to infiltrate in tumor tissues. The latter enhances the proliferation and invasion of colorectal cancer cell and lead to poor prognosis.

Bmi1 combines with oncogenic KRAS to induce malignant transformation of human pancreatic duct cells in vitro.

It is critical to understand the pathogenesis of preinvasive stages of pancreatic duct adenocarcinoma (PDAC) for developing novel potential diagnostic and therapeutic targets. The polycomb group family member B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi1) is overexpressed and involved in cancer progression in PDAC; however, its role in the multistep malignant transformation of human pancreatic duct cells has not been directly demonstrated. In this study, we stably expressed Bmi1 in a model of telomerase-immortalized human pancreatic duct-derived cells (HPNE) and showed that Bmi1 promoted HPNE cell proliferation, migration, and invasion but not malignant transformation. We then used mutant KRASG12D as a second oncogene to transform HPNE cells and showed that it further enhanced Bmi1-induced malignant potential. More importantly, coexpression of KRASG12D and Bmi1 caused anchorage-independent growth transformation in vitro but still failed to produce tumors in nude mice. Finally, we found that mutant KRASG12D induced HPNE-Bmi1 cells to undergo partial epithelial-mesenchymal transition (EMT) likely via upregulation of snail. Knockdown of KRASG12D significantly reduced the expression of snail and vimentin at both the messenger RNA (mRNA) and protein level and further impaired the anchorage-independent growth capability of invasive cells. In summary, our findings demonstrate that coexpression of Bmi1 and KRASG12D could lead to transformation of HPNE cells in vitro and suggest potential new targets for diagnosis and treatment of PDAC.

Prognostic significance of neuroendocrine differentiation in colorectal adenocarcinoma after radical operation: a meta-analysis.

BACKGROUND: The phenomenon of neuroendocrine differentiation has been observed in colorectal adenocarcinoma. However, the ability of neuroendocrine differentiation to predict the outcome of colorectal adenocarcinoma remains controversial. METHODS: We conducted an extensive search of research studies related to neuroendocrine differentiation using scientific databases, including the PubMed, Embase, OVID, BIOSIS Previews, and Cochrane Central Register of Controlled Trials (up to July, 2013), according to the established search terms. RevMan version 5.2 statistical program was used to analyze the data. An odds ratio (OR) with a 95% confidence interval (CI) was used for the dichotomous data. RESULTS: Eleven studies with a total of 1,587 patients were included. Patients with neuroendocrine differentiation who underwent a radical operation had a lower 5-year survival rate (pooled OR 0.60, 95% CI 0.37-0.97) compared with those without neuroendocrine differentiation, with evidence of moderate heterogeneity (I (2) = 37%, p = 0.10). A sensitivity analysis and meta-regression showed that the different classification criteria of neuroendocrine differentiation used in these studies were the main source of heterogeneity. When the strong positive rates of neuroendocrine differentiation indicators between the higher (stage III + IV) and the lower (stage I + II) clinical stages were compared, the pooled OR was 1.84 (703 patients; 95% CI 0.98-3.43) without evidence of heterogeneity (I (2) = 0 %, p = 0.89). However, comparisons between consecutive stages showed different ORs: stage II vs. I (203 patients; OR = 0.52, 95% CI 0.17-1.56), stage III vs. II (569 patients; OR = 2.27, 95% CI 1.03-4.98), and stage IV vs. III (375 patients; OR = 1.81, 95% CI 1.00-3.29). CONCLUSION: The patients with strong positive indicators of neuroendocrine differentiation had a lower 5-year survival rate. The ability to detect neuroendocrine indicators using conventional methods could improve the prognosis judgment of colorectal adenocarcinoma.

Clinicopathological features and prognosis of gastroenteropancreatic neuroendocrine tumors: analysis from a single-institution.

BACKGROUND: The gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN) is the most common type of neuroendocrine neoplasm. We summarized data in our centre to investigate the clinicopathological features, diagnostic methods, therapeutic approaches and prognosis for this neoplasm to increase knowledge of this disease in Asian populations. METHOD: A total of 122 patients treated at Sun Yet-san Memorial Hospital of Sun Yat-sen University between January 2000 and December 2011 were analyzed retrospectively. RESULTS: Pancreas was the most common site of involvement (65/122, 53.3%); this disease has no special symptoms; positive rates of chromogranin A (CgA) and synaptophysin (Syn) were 81.1% and 87.7%, respectively. The positive rate of Syn had statistical difference among the three grades, but not CgA. Some 68 patients had G1 tumors, 32 G2 tumors and 22 G3 tumors, and Chi-square test showed that higher grading was correlated with worse prognosis (χ2=32.825, P=0.0001). A total of 32 patients presented with distant metastasis, and 8 cases emerged during following up. Cox proportional hazards regression modeling showed that the tumor grade (P=0.01), lymphatic metastasis (P=0.025) and distant metastasis (P=0.031) were predictors of unfavorable prognosis. The overall 5-year survival rate was 39.6%, the 5-year survival rate of G1 was 55.7%, and the G2 and G3 were 34.2% and 0%, respectively. CONCLUSIONS: The incidence of gastroenteropancreatic neuroendocrine tumors has risen over the last 12 years. All grades of these diseases metastasize readily, and further research regarding the treatment of patients after radical surgery is needed to prolong disease-free survival.

Maturation induction of human peripheral blood mononuclear cell-derived dendritic cells.

The aim of the present study was to explore an optimal method for maturation induction of dendritic cells (DCs). Human monocyte-derived DCs were induced in the presence of GM-CSF and IL-4. On Day 6, the maturation of DCs was induced with CD40L, LPS, TNF-α and cocktail of cytokines (TNF-α, IL-6, IL-1ß and PGE2), respectively, for 24 h. Then, DCs were harvested and subjected to flow cytometry (FCM) for the detection of CD80, CD83, CD86 and HLA-DR. FITC-dextran endocytic activity was measured by FCM, IL-12 production by ELISA and T lymphocyte proliferation following DC stimulation by MTT assay. CD40L, LPS, TNF-α and a cocktail of cytokines induced DC maturation. Induction with the cocktail of cytokines was the most efficient, and the expression rate of CD83 was 66.91% (P<0.05). The FITC-dextran endocytic activity of mature DCs was significantly reduced, and IL-12 production was dramatically increased in mature DCs, particularly in those following induction using the cocktail of cytokines. The mature DCs had potent ability to stimulate the proliferation of lymphocytes. The cocktail of cytokines is a favorable strategy for the induction of DC maturation.

Expression of heat shock protein 27 in the esophageal tissue of rats with reflux esophagitis.

BACKGROUND: Little attention has been paid to the expression of heat shock protein 27 (HSP27) in patients with reflux esophagitis (RE), and few studies of the importance of HSP27 in esophagitis have been carried out in animal models. This study aimed to explore the expression of HSP27 in the esophageal tissue of rats with RE. METHODS: Eighty female Wistar rats were randomly divided into experimental groups A and B and control groups C and D (n = 20 in each group). To establish RE, rats in the two experimental groups received pylorus and forestomach ligations, while rats in the control group received gastrostomy and gastric perforation repair. The rats in groups A and C were sacrificed 7 days after surgery, and the rats in groups B and D were sacrificed 14 days after surgery. In groups A and B, 10 and 8 rats were diagnosed with RE by pathological examination, respectively (they were included in groups A' and B', respectively). The histopathological diagnosis of all the lower esophageal tissues in groups C and D was normal and 20 normal specimens were randomly selected for groups C' and D' with 10 specimens in each group. Macroscopic and microscopic esophagitis scores were assessed for the specimens in groups A' and B'. Lower esophageal tissues were collected from groups A', B', C', and D', and paraffin-embedded slices were made using part of the tissues. The expression of HSP27 in the tissues was detected using the two-step streptavidin-peroxidase immunohistochemical method. Some collected tissues were frozen, and expressions of HSP27 mRNA were detected using fluorescence quantitative polymerase chain reaction (FQ-PCR). RESULTS: Median macroscopic and microscopic esophagitis scores in groups A' (n = 10) and B' (n = 8) were 1.0 and 1.5, and 2.0 and 2.5, respectively. There were no significant differences in the macroscopic or microscopic esophagitis scores between the two groups (Z = -0.330, P = 0.741; Z = -0.142, P = 0.887, respectively). Immunohistochemical staining showed that HSP27 was expressed in all layers of the esophageal epithelia in RE and control rats. FQ-PCR showed that HSP27 mRNA levels in the lower esophageal tissue in RE group (groups A' and B') were higher than those in control group (groups C' and D') (Z = -0.249, P = 0.001). HSP27 mRNA expression in the lower esophageal tissue was significantly different in groups B' and D' (Z = -3.027, P = 0.002). And the levels of HSP27 mRNA expression in severe RE group (microscopic esophagitis score: 3) were higher than in mild RE group (microscopic esophagitis score: 1-2) and control group (Z = -3.396, P = 0.001; Z = -3.855, P < 0.001). CONCLUSIONS: HSP27 mRNA expression in the lower esophageal tissue of rats with RE is significantly higher than in the normal controls. Although reflux is a persistent stimulating factor, increased expression of HSP27 in the lower esophageal tissue of rats with RE requires aggravated esophageal injury.

Proteomic analysis identifies translationally controlled tumor protein as a mediator of phosphatase of regenerating liver-3-promoted proliferation, migration and invasion in human colon cancer cells.

BACKGROUND: Considerable evidence suggests that phosphatase of regenerating liver-3 (PRL-3) plays multiple roles in cancer metastasis; however, the molecular mechanisms remain largely unknown. The aim of this study was to identify proteins associated with PRL-3-promoted colon cancer metastasis, by comparative proteomic analysis. METHODS: Proteomes of human colon cancer LoVo cells transfected with PRL-3 gene (LoVo-PRL-3) or empty vector PAcGFP-C3 (LoVo-control) were compared using 2D gel electrophoresis. Proteins that varied significantly in concentration were selected and identified using mass spectrometry. Expression of translationally controlled tumor protein (TCTP) mRNA and protein in LoVo-PRL-3 and LoVo-control cells was detected by real-time PCR and Western blotting. Small interfering RNA (siRNA) targeting TCTP was used for silencing TCTP expression in LoVo-PRL-3 cells. Functional significance of TCTP in PRL-3-promoted colon cancer cell proliferation, migration and invasion was investigated by Cell Counting Kit-8 assay and transwell chamber. RESULTS: Seventeen proteins displaying significant and reproducible differences between LoVo-PRL-3 and LoVo-control cells were identified. Ten proteins were upregulated and seven were downregulated in LoVo-PRL-3 cells when compared with LoVo-control cells. Eight identified proteins are associated with distinct steps of tumor metastasis: ubiquitin-like protein ISG15, interleukin-18, TCTP, serpin B5, annexin A3, macrophage-capping protein, ATP-dependent RNA helicase DDX3X, and cathepsin D. Real-time PCR and Western blotting results showed that both TCTP mRNA and protein were significantly increased in LoVo-PRL-3 cells compared to LoVo-control cells. Transfection with TCTP siRNA significantly reduced the expression of both mRNA and protein levels of TCTP in LoVo-PRL-3 cells. Knockdown of TCTP by siRNA inhibited PRL-3-promoted proliferation, migration and invasion of LoVo-PRL-3 cells. CONCLUSION: Our results imply that TCTP might be a mediator of PRL-3-promoted proliferation, migration and invasion of human colon cancer cells.

[Changes of serum cytokine caused by acute paraquat poisoning].

OBJECTIVE: To observe the change of cytokine interleukin IL-1 beta, IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha) occurred in acute paraquat (PQ) poisoning rats and to investigate the mechanism of acute lung injury caused by paraquat (PQ) poisoning. METHODS: All 72 healthy adult Wistar rats were random assigned into normal control groups, paraquat high dose group (120 mg/kg), paraquat middle dose (60 mg/kg) group, paraquat low dose group (30 mg/kg). Three observing periods of time included 8, 24, 72 h and the standards of TNF-alpha, IL-1 beta, IL-6, IL-10 were determined. RESULTS: Every index of the PQ group was significantly higher than that in the NS group at the same period of time (P<0.05 or P<0.01). In the 72 h group, the high dose group was significantly higher than the middle and low dose group (P<0.05), and there was no significantly difference between the middle and low dose group (P>0.05). For the comparison of index in the same dose group, the group of 72 h was much higher than 8 h group and 24 h group (P<0.05), and there was no difference between the 8h group and 24 h group (P>0.05). CONCLUSION: The cytokine may play an important role in paraquat-induced acute lung tissue injury.

[Application of adenosine triphosphate tumor chemosensitive assay system to individual chemotherapy for hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: Present chemotherapy for hepatocellular carcinoma (HCC) is not effective. To improve the effect of chemotherapy, in vitro chemo-drug sensitive testing system, adenosine triphosphate tumor chemosensitive assay (ATP-TCA) system, was used to evaluate efficacies of chemotherapeutic drugs, and to guide clinical individual chemotherapy for HCC patients. METHODS: ATP-TCA system was applied to test efficacies of 5-fluorouracil (5-FU), mitomycin (MMC), cisplatin (DDP), oxaliplatin (OXA), epirubicin (EPI), gemcitabine (GEM), irinotecan (CPT-11), etoposide (VP-16), and paclitaxel (PTX) on 50 HCC samples. Twenty-three HCC patients received ATP-TCA-directed chemotherapy (ATP-TCA group)û 20 HCC patients received surgery and routine treatments (control group). Clinical outcomes of these patients were observed for 162 weeks. RESULTS: The assessable rate of ATP-TCA result is 90.8%. In the 50 samples, the sensitive (moderate to high degree) rates were 46% to PTX, 44% to CPT-11, 36% to GEM, 14% to MMC, 12% to EPI, 8% to DDP, 6% to VP-16, 6% to OXA, and 4% to 5-FU, respectively. In clinical trial, at the research end-point, no significant differences were found in partial remission (PR), complete remission (CR), stable disease (SD), and mortality between ATP-TCA group and control group (P > 0.05), but progression disease (PD) rate was significantly higher in control group than in ATP-TCA group (60.00% vs.13.04%, P=0.003); significant differences were found in overall response rate (ORR) (60.86% vs. 30.00%, P =0.043), overall survival (OS) (78.91 weeks vs. 27.21 weeks, P=0.006), and progress-free survival (PFS) (30.52 weeks vs. 4.78 weeks, P=0.005) between ATP-TCA group and control group. CONCLUSIONS: ATP-TCA system might be useful in evaluating the efficacy of chemotherapeutic drugs on HCC samples, and in planning individualized chemotherapy regimen for HCC patients. PTX, CPT-11 and GEM might be potential drugs for the treatment of HCC. ATP-TCA-guided chemotherapy might prolong survival time of HCC patients.

[Study of embryonic stem cells induced to express hepatic cell functions in vitro in a pathologic niche containing cholestatic serum].

OBJECTIVE: To study the role of a pathologic niche inducing mouse embryonic stem cells (ESC) to express hepatic cell functions in vitro. METHODS: Embryoid bodies were developed from 5 to 7 day hanging-drop culture of mouse ESC, and their dissociated cells were planted in three differential systems: nothing added; with 20 ng/ml hepatocyte growth factor (HGF); and 5% rat cholestatic serum plus 20 ng/ml HGF added. Their differentiation was observed with inverted microscopes daily, and their hepatic functions were analyzed against their synthesis of glycogen, triglycerides, albumin, and urea nitrogen, and by their staining of indocyanine green (ICG) and fluorescein diacetate (FDA). RESULTS: ESC spontaneous differentiation was hardly being controlled to form three germ layers. HGF prompted the ESC to develop further into visceral endoderm and mesoderm (myocardium), but both of them only expressed a low level of hepatocyte-specific metabolic functions. With cholestatic serum added into the HGF-induced system, differentiated cells grew into similar angular cells, and had a higher level synthesis of glycogen, triglycerides, albumin and urea nitrogen with positive ICG and FDA staining. CONCLUSIONS: Spontaneous or HGF-induced ESC differentiation has only limited hepatic functions expressed. A pathologic niche in vitro induces ESC to develop into hepatic lineages, with a higher level of hepatic metabolic functions.

Selection, proliferation and differentiation of bone marrow-derived liver stem cells with a culture system containing cholestatic serum in vitro.

AIM: To explore the feasibility of direct separation, selective proliferation and differentiation of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Whole bone marrow cells of rats cultured in routine medium were replaced with conditioning selection media containing 20 mL/L, 50 mL/L, 70 mL/L, and 100 mL/L cholestatic sera, respectively, after they attached to the plates. The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures. Then the selected BDLSC were induced to proliferate and differentiate with the addition of hepatocyte growth factor (HGF). The morphology and phenotypic markers of BDLSC were characterized using immunohistochemistry, RT-PCR and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. RESULTS: Bone marrow cells formed fibroblast-like but not hepatocyte-like colonies in the presence of 20 mL/L cholestatic serum. In 70 mL/L cholestatic serum, BDLSC colonies could be selected but could not maintain good growth status. In 100 mL/L cholestatic serum, all of the bone marrow cells were unable to survive. A 50 mL/L cholestatic serum was the optimal concentration for the selection of BDLSC at which BDLSC could survive while the other populations of the bone marrow cells could not. The selected BDLSC proliferated and differentiated after HGF was added. Hepatocyte-like colony-forming units (H-CFU) then were formed. H-CFU expressed markers of embryonic hepatocytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors (HNF-1alpha and HNF-3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selected medium containing cholestatic serum can select BDLSC from whole bone marrow cells. It will be a new way to provide a readily available alternate source of cells for clinical hepatocyte therapy.

[Expression of growth hormone receptor in hepatocellular carcinoma and its significance].

BACKGROUND & OBJECTIVE: Recombinant human growth hormone (rhGH) has been proved effective in clinic, such as promoting protein synthesis and decreasing the mortality. But there are still many arguments on whether it can be used in hepatocellular carcinoma (HCC) patients. rhGH cannot work except that it combines to its own receptor growth hormone receptor (GHR). The current study was designed to explore the expression of GHR in HCC tissues and to investigate the viability of rhGH for HCC treatment. METHODS: Radioreceptor assays were used to determine growth hormone receptor (GHR) in 40 HCC tissues; 6 normal liver tissues were used as control. Receptor binding capacity(RT) and affinity constant(Kd) of GHR were calculated by Scatchard's method; the relationship between RT of GHR in cancer and clinicopathologic factors were also analyzed. RESULTS: The growth hormone- specific singular binding site, namely GHR, was detected respectively in 35 HCC cases and control tissues. The RT and Kd of GHR were 18.5416+/-4.1686 fmol/mg protein and 0.6319+/-0.1978 nmol/L in tumor tissues, 39.5467+/-3.4770 fmol/mg protein and 0.6167+/-0.1007 nmol/L in control tissues, respectively. Compared with the normal liver tissue, RT of GHR in tumor was lower (P< 0.05) but Kd did not show any difference(P >0.05). The RT of the GHR was negatively relevant to the tumor size and disease stage, but was not associated with differentiation of tumor, ages of the patients or whether the patient suffered from cirrhosis at the same time. GHR was undetectable in 5 cases. CONCLUSION: This study showed that most of the HCC tissues express low levels of GHR. Before their functions are well understood, rhGH should be very carefully used in HCC patients.