Gene alterations and epigenetic changes in intrahepatic cholangiocarcinoma.

INTRODUCTION: Intrahepatic cholangiocarcinoma (ICC) is a highly malignant neoplasm. The incidence of ICC has been increasing world-wide over the past several decades. Areas covered: The pathogenesis of ICC is a complex process involving the alteration of specific genes and epigenetic changes. This review summarizes the advances in research of gene alterations and epigenetic changes in ICC; it also aims to promote the findings of new biomarkers that can be used to diagnose ICC and to provide novel ideas for the combination of molecular targeted therapies. Expert commentary: Surgical resection is regarded as the most effective curative treatment, although in many cases, resectability and curability are unsatisfactory. Furthermore, many patients are diagnosed at advanced stages and have to resort to chemotherapy, which is rarely curative. Many of the molecular mechanisms of ICC are yet to be determined; once these determinations are made, then ICC may be able to be treated by precise targeted drugs.

Laparoscopic splenectomy for primary immune thrombocytopenia: Current status and challenges.

Primary immune thrombocytopenia (ITP) is an immune-mediated disorder affecting both adults and children, characterised by bleeding complications and low platelet counts. Corticosteroids are the first-line therapy for ITP, but only 20%-40% of cases achieve a stable response. Splenectomy is the main therapy for patients failing to respond to corticosteroids for decades, and about two-thirds of patients achieve a long-lasting response. Although some new drugs are developed to treat ITP as second-line therapies in recent years, splenectomy is still the better choice with less cost and more efficiency. Laparoscopic splenectomy (LS) for ITP proves to be a safe technique associated with lower morbidity and faster recovery and similar hematological response when compared to traditional open splenectomy. Based on the unified hematological outcome criteria by current international consensus, the response rate of splenectomy should be reassessed. So far, there are not widely accepted preoperative clinical indicators predicting favorable response to LS. Since the patients undergoing surgery take the risk of complications and poor hematological outcome, the great challenge facing the doctors is to identify a reliable biomarker for predicting long-term outcome of splenectomy which can help make the decision of operation.

Proteomics-based identification of haptoglobin as a favourable serum biomarker for predicting long-term response to splenectomy in patients with primary immune thrombocytopenia.

BACKGROUND: Splenectomy is the most effective treatment for patients with primary immune thrombocytopenia (ITP) who fail to respond to steroid therapy. Thus far, there is no effective means to predict the long-term haematological response of the procedure. The purpose of this study was to identify serum biomarkers as predictors of long-term response based on a proteomics approach. METHODS: The serum samples of ITP patients were collected before splenectomy and seven days after surgery. After depletion of the abundant serum proteins, pooled preoperative serum samples from four responders to splenectomy, four nonresponders and four healthy controls were subjected to two-dimensional gel electrophoresis (2-DE). Nine protein spots with at least a five-fold alteration in expression between responders and nonresponders were all identified as haptoglobin (Hp) by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometer (MS) analysis. The validation of serum Hp expression was performed using enzyme-linked immunosorbent assays (ELISA) in thirty-seven responders, thirteen nonresponders and twenty-one healthy controls. RESULTS: The preoperative serum levels of Hp in the nonresponders (925.9 ± 293.5 µg/ml) were significantly lower than those in the responders (1417.4 ± 315.0 µg/ml, p <0.001) and the healthy controls (1409.1 ± 354.2 µg/ml, p <0.001), while there was no significant difference between the latter two groups. The postoperative serum levels of Hp in responders and nonresponders were (1414.1 ± 225.0 µg/ml) and (952.9 ± 202.4 µg/ml), respectively. There were no significant differences between the serum Hp levels before and after surgery in both responders and nonresponders (p>0.05). The preoperative serum levels of Hp did not significantly correlate with preoperative platelet count of the same blood samples (r = 0.244, p = 0.087), while it positively correlated with postoperative peak platelet count (r = 0.622, p < 0.001). The optimal cutoff value of preoperative serum Hp levels (1173.80 µg/ml) derived from the receiver operating characteristic (ROC) curve led to 78.4% sensitivity and 84.6% specificity. CONCLUSIONS: These results suggest that serum Hp levels may serve as a favourable predictor for the long-term response to splenectomy in ITP and may help to understand the pathophysiological differences between responders and nonresponders.

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.

Laparoscopic splenectomy for immune thrombocytopenic purpura at a teaching institution.

BACKGROUND: High anatomic location, fragility, and generous blood supply of the spleen makes laparoscopic splenectomy (LS) difficult to master, and few patients need splenectomy for benign disorders. The aim of this research was to assess operative outcomes and hematological results of a large series of patients treated with LS for chronic immune thrombocytopenic purpura (ITP) and to determine which clinical variables predict favorable hematological outcome. METHODS: LS was successfully performed for 154 patients with chronic ITP from September 1999 to April 2009 at the First Affiliated Hospital of Sun Yat-sen University. Operative outcomes were assessed retrospectively. Long-term follow-up data were obtained from outpatient medical records and phone interviews. Clinical and laboratory variables (including gender, age, disease duration before surgery, previous response to steroids, preoperative platelet count, and postoperative peak platelet count) were evaluated by univariate analysis to identify potential predictors of hematological outcome. Multivariate Logistic regression model was used to determine independent predictors of hematological outcome. RESULTS: One patient died from subphrenic abscess and postoperative sepsis. The overall major morbidity rate was 8.4%. None of the patients required a second surgery for complications. Of the 127 patients available for a mean follow-up of 43.6 months (range 9 - 114 months), the overall initial response (i.e., at two months after LS) and long-term response to LS were achieved in 89.0% and 80.3%, respectively. Five patients (3.9%) developed pneumonia 3 - 35 months after LS. Univariate analysis showed a significant difference in mean age between responders (29.1 years) and nonresponders (38.8 years; P < 0.05). Patients who responded to steroid therapy had better hematological outcome than those who did not respond (P < 0.05). Compared to nonresponders, responders to LS had a significantly higher postoperative peak platelet count (404 × 10(9)/L versus 213 × 10(9)/L, P < 0.001). Multivariate Logistic regression analysis identified postoperative peak platelet count as the only independent predictor of favorable response to LS (P < 0.001). CONCLUSIONS: LS is a safe and effective treatment for chronic ITP. Postoperative peak platelet count may serve as a major predictor of long-term response.

[Multicenter cross-sectional study of anemia in patients with gastric and colorectal cancer before and after the operation].

OBJECTIVES: To determine the prevalence of cancer-related anemia and iron deficiency anemia (IDA) in patients with gastric and colorectal cancer in North of China. METHODS: A cross-sectional study of 262 inpatients diagnosed with gastric or colorectal cancer admitted to eight general hospitals in Beijing from August 2009 to December 2009 was performed. The blood samples were took on the day after admission and the seventh day after operation for the tests of hemoglobin, serum iron and ferritin. The morbidity of cancer-related anemia and IDA before and after the surgery was also compared respectively. RESULTS: The preoperative morbidity of cancer-related anemia was 36.6% in 131 patients with gastric cancer, and the morbidity of IDA was 52.1%. The mean age of the anemic patients was higher than that in cases without anemia [(62 ± 11) yrs vs. (57 ± 12) yrs, P < 0.05]; the postoperative morbidity of IDA increased to 72.6% (P < 0.05). In the 131 cases with colorectal cancer, the preoperative incidence of cancer-related anemia and IDA was 37.4% and 61.2%, respectively. About 45% of the cases with anemia had a tumor in the right colon. Postoperative incidence of IDA was significantly higher than that before the surgery (76.7%, P < 0.05). Only 10.3% of the anemic patients were treated with chalybeate therapy before surgical procedures, and the proportion was 22.7% after the operation. More than 50% of anemic patient received blood transfusion. CONCLUSIONS: Cancer-related anemia is a common clinical manifestation in patients with gastrointestinal cancer, and anemia occurs more frequently in elder and patients with right colon tumor. The treatment to cancer-related anemia is insufficient and a systematic therapy is needed to be established.

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.

[Experimental study of human colon cell line Lovo transfected with soluble vascular endothelial growth factor receptor 1 gene].

OBJECTIVE: To investigate the effect of transfection with human soluble vascular endothelial growth factor receptor-1(sFlt-1) gene on cell growth and vascular endothelial growth factor (VEGF) concentration of the culture supernatant in human colon cancer cell line Lovo. METHODS: The recombinant plasmid pcDNA3-sFlt-1 containing sFlt-1 gene was transfected into Lovo cells by Lipofectamine 2000, which was identified by RT-PCR and ELISA. The effect of sFlt-1 protein on cell growth and VEGF expression in Lovo cells were investigated by MTT and ELISA. RESULTS: The recombinant plasmid pcDNA3-sFlt-1 was successfully transfected into Lovo cells. The sFlt-1 expression was identified by RT-PCR and ELISA, which inhibited the growth of Lovo cells and reduced the VEGF concentration in the culture supernatant compared with control. The inhibitory rates of proliferation of Lovo cells via MTT assay after 2,14,21 and 28 days were(23.92+/-9.16)%, (13.98+/-10.21)%,(22.54+/-11.92)% and (33.43+/-9.34)% respectively. Compared with the control groups, the differences were significant (P<0.05, P<0.01). CONCLUSION: Transfection with sFlt-1 gene into Lovo cells results in the expression of sFlt-1 protein, which possesses high biological activity and inhibits the growth of cancer cells.

Laparoscopic splenectomy: the latest technical evaluation.

AIM: To introduce our latest innovation on technical manipulation of laparoscopic splenectomy. METHODS: Under general anesthesia and carbon dioxide (CO(2)) pneumoperitoneum, 86 cases of laparoscopic splenectomy (LS) were performed. The patients were placed in three different operative positions: 7 cases in the lithotomic position, 31 cases in the right recumbent position and 48 cases in the right lateral position. An ultrasonic scissors was used to dissect the pancreaticosplenic ligament, the splenocolicum ligament, lienorenal ligament and the lienophrenic ligament, respectively. Lastly, the gastrosplenic ligament and short gastric vessels were dissected. The splenic artery and vein were resected at splenic hilum with Endo-GIA. The impact of different operative positions, spleen size and other events during the operation were studied. RESULTS: The laparoscopic splenectomy was successfully performed on all 86 patients from August 1997 to August 2002. No operative complications, such as peritoneal cavity infection, massive bleeding after operation and adjacent organs injured were observed. There was no death related to the operation. The study showed that different operative positions could significantly influence the manipulation of LS. The right lateral position had more advantages than the lithotomic position and the right recumbent position in LS. CONCLUSION: Most cases of LS could be accomplished successfully when patients are placed in the right lateral position. The right lateral position has more advantages than the conventional supine approach by providing a more direct view of the splenic hilum as well as other important anatomies. Regardless of operation positions, the major axis of spleen exceeding 15 cm by B-ultrasound in vitro will surely increase the difficulties of LS and therefore prolong the duration of operation. LS is a safe and feasible modality for splenectomy.

[Influence of combination of low dosage 131I-labeled anti-carcinoembryonic antigen antibody C50 and 5-fluorouracil on tumor growth of colorectal cancer xenografts in nude mice].

BACKGROUND & OBJECTIVE: The aim of this study was to investigate the influence of low dosage (131)I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibody C50 ((131)I-C50) on tumor growth and the therapeutic efficacy of combination of low dosage (131)I-C50 with chemotherapy using 5-fluorouracil (5-FU) on human colorectal cancer xenografts in nude mice. METHODS: Human colorectal cancer xenografts with positive CEA expression were established in nude mice with LoVo cell line. 5-FU, 2,775 kBq (131)I-C50, and 5-FU combined with (131)I-C50 were given to nude mice through tail vein to treat xenografts at 9(th) day after implantation of tumor cells. Two of the mice of each group were sacrificed randomly at 7(th) day after the treatment; and cellular ultrastructure of tumor tissues was examined under electron microscope. Pathological changes of tumor tissues were examined under light microscope. Tumor volume, tumor doubling time, and inhibition rate of each group were calculated. Tumor volumes of all groups at 30(th) day after implantation were compared with each other. RESULTS: There was significant difference of tumor volumes at 30(th) day after implantation between each other among control group, chemotherapy group, radioimmunotherapy (RAIT) group, and RAIT+chemotherapy group (P< 0.001). Tumor doubling time of these groups was prolonged and tumor inhibition rates increased successively. CONCLUSION: Low dosage (131)I-C50 can inhibit tumor growth of human colorectal cancer xenografts in nude mice. Efficacy of tumor growth inhibition can be enhanced by combination of low dosage (131)I-C50 with chemotherapy.

Changes in the level of serum liver enzymes after laparoscopic surgery.

AIM: The purpose of this study was to investigate the effect of laparoscopic surgery on liver function in humans and the possible mechanisms behind such effect. METHODS: Blood samples from 286 patients who underwent laparoscopic cholecystectomy (LC) and 40 patients who underwent open cholecystectomy (OC) were tested for liver function by measuring the level of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) before and after the operations. The same tests were also applied to 18 laparoscopic colorectal cancer resection (LCR) patients and 23 open colorectal cancer resection (OCR) patients to determine whether CO(2) pneumoperitoneum could alter the serum liver enzymes. RESULTS: The level of serum ALT and AST increased significantly during the first 48 hours post operations in both LC and LCR patients. However, no significant change of the serum liver enzymes was detected in both OC and OCR patients. As a result, there was statistically significant difference in change of both ALT and AST levels between LC and OC patients and LCR and OCR patients, respectively. By the 7(th) day post operation, the level of both enzymes returned to normal values in LC, OC and OCR patients except LCR patients whose enzymes remained at a higher level. CONCLUSION: Transient elevation of hepatic transaminases occurred after laparoscopic surgery. The major causative factor seemed to be the CO(2) pneumoperitoneum. In most of the laparoscopic surgery patients, the transient elevation of serum liver enzymes showed no apparent clinical implications. However, if preoperative liver function was very poor, laparoscopic surgery may not be the best choice for the treatment of patients with certain abdominal diseases.