Construction and Validation of an m6A RNA Methylation Regulators-Based Prognostic Signature for Esophageal Cancer.

PURPOSE: N6-methyladenosine (m6A) is reported to play a critical role in cancer through various mechanisms. We aimed to construct and validate an m6A RNA methylation regulators-based prognostic signature for Esophageal cancer (ESCA). MATERIALS AND METHODS: The RNA sequencing transcriptome data of 13 m6A RNA methylation regulators as well as clinical data were obtained from The Cancer Genome Atlas (TCGA) ESCA database. The differential expression of the regulators between ESCA tissues and normal tissues was assessed. Consensus clustering was conducted to explore the different ESCA clusters based on the expression of these regulators. LASSO Cox regression analysis was used to generate a prognostic signature based on m6A RNA methylation regulators expression. RESULTS: Eight regulators (KIAA1429, HNRNPC, RBM15, METTL3, WTAP, YTHDF1, YTHDC1, and YTHDF2) were found to be significantly upregulated in ESCA tissues. Significant differences of survival rate and clinicopathological features were found between the two clusters. A prognostic signature, which consists of HNRNPC and ALKBH5, was constructed based on the TCGA ESCA cohort, which can serve as an independent prognostic predictor. The results of bioinformatics analysis were further successfully validated in the clinical ESCA cohort by qRT-PCR and immunohistochemistry staining. CONCLUSION: Our study constructed and validated an m6A RNA methylation regulators-based prognostic signature. This might provide important information for developing diagnostic and therapeutic strategies.

The quality of total mesorectal excision specimen: A review of its macroscopic assessment and prognostic significance.

As a surgical procedure which could significantly lower the recurrence rate of cancers, total mesorectal excision (TME) has been the gold standard for middle and lower rectal cancer treatment. However, previous studies have shown that the procedure did not achieve the ideal theoretical local recurrence rates of rectal cancers. Some researchers pointed out it was very likely that not all so-called TME treatments completely removed the mesorectum, implying that some of these TME surgical treatments failed to meet oncological quality standards. Therefore, a suitable assessment tool for the surgical quality of TME is necessary. The notion of "macroscopic assessment of mesorectal excision (MAME)" was put forward by some researchers as a better assessment tool for the surgical quality of TME and has been confirmed by a series of studies. Besides providing rapid and accurate surgical quality feedbacks for surgeons, MAME also effectively assesses the prognosis of patients with rectal cancer. However, as a new assessment tool used for TME surgical quality, MAME has an only limited influence on the current guidelines and is yet to be widely applied in most countries. The aims of this review are to provide a detailed introduction to MAME for clinical practice and to summarize the current prognostic significance of MAME.

Pulmonary metastasis in rectal cancer: a retrospective study of clinicopathological characteristics of 404 patients in Chinese cohort.

OBJECTIVES: This study aim to investigate the incidence, timing and risk factors of metachronous pulmonary recurrence after curative resection in patients with rectal cancer. DESIGN: A retrospective cohort study. SETTING: This study was conducted at a tertiary referral cancer hospital. PARTICIPANTS: A total of 404 patients with rectal cancer who underwent curative resection from 2007 to 2012 at Beijing Hospital were enrolled in this study. INTERVENTIONS: The pattern of recurrence was observed and evaluated. PRIMARY AND SECONDARY OUTCOME MEASURES: The incidence and timing of recurrences by site were calculated, and the risk factors of pulmonary recurrence were analysed. RESULTS: The 5-year disease-free survival for the entire cohort was 77.0%. The most common site of recurrence was the lungs, with an incidence of 11.4%, followed by liver. Median interval from rectal surgery to diagnosis of pulmonary recurrence was much longer than that of hepatic recurrence (20 months vs 10 months, P=0.022). Tumour location, pathological tumor-node-metastasis (TNM) stage and positive circumferential resection margin were identified as independent risk factors for pulmonary recurrence. A predictive model based on the number of risk factors identified on multivariate analysis was developed, 5-year pulmonary recurrence-free survival for patients with 0, 1, 2 and 3 risk factors was 100%, 90.4%, 77.3% and 70.0%, respectively (P<0.001). CONCLUSIONS: This study emphasised that the lung was the most common site of metachronous metastasis in patients with rectal cancer who underwent curative surgery. For patients with unfavourable risk profiles, a more intensive surveillance programme that could lead to the early detection of recurrence is strongly needed.

High levels of serum platelet-derived growth factor-AA and human epidermal growth factor receptor-2 are predictors of colorectal cancer liver metastasis.

AIM: To develop predictive markers in blood for colorectal cancer liver metastasis. METHODS: Twenty colorectal cancer patients were selected and divided into two groups. Group A consisted of 10 patients whose pathological TNM stage was IIIC (T3-4N2M0), while another 10 patients with synchronous liver metastasis (TNM stage IV) were recruited for group B. During the surgical procedure, a 10-mL drainage vein (DV) blood sample was obtained from the DV of the tumor-bearing segment prior to the ligation of the DV. At the same time, a 10-mL peripheral vein (PV) blood sample was collected via peripheral venipuncture. The serum levels of 24 molecules that are potentially involved in the mechanism of liver metastasis in both DV blood and PV blood were analyzed by using high-throughput enzyme-linked immunosorbent assay technology. RESULTS: Univariate analysis revealed that platelet-derived growth factor AA (PDGFAA) in DV blood (dPDGFAA) (P = 0.001), PDGFAA in PV blood (pPDGFAA) (P = 0.007), and human epidermal growth factor receptor-2 in PV blood (pHER2) (P = 0.001), pMMP7 (P = 0.028), pRANTES (P = 0.013), and pEGF (P = 0.007) were significantly correlated with synchronous liver metastasis. Multivariate analysis identified dPDGFAA (HR = 1.001, P = 0.033) and pHER2 (HR = 1.003, P = 0.019) as independent predictive factors for synchronous liver metastasis. Besides, high peripheral HER2 level may also be a risk factor for metachronous liver metastasis, although the difference did not reach statistical significance (P = 0.06). Significant correlations were found between paired DV and PV blood levels for PDGFAA (r = 0.794, P < 0.001), but not for HER2 (r = 0.189, P = 0.424). CONCLUSION: PDGFAA in tumor drainage and HER2 in PV blood may be useful predictive factors for synchronous liver metastasis of colorectal cancer.

[Inhibitory Effect of High Concentration Insulin on K562 Cell Proliferation and Its Mechanism].

OBJECTIVE: To investigate the inhibitory effect of high concentration insulin on K562 cell proliferation and its underlying mechanism. METHODS: K562 cells were treated by different concentration of insulin and/or anti-IGF-1R antibody (IGF-1R-Ab), MTT assay and flow cytometry were used to detect the K562 cells proliferation and apoptosis, respectivety; Western blot was used to measure the expression and phosphorylation level of IGE-IR, Akt, Erk1/2 in K562 cells under the different concentration of insulin. RESULTS: MTT assay showed that less than 40 mU/ml insulin could promote K562 cell proliferation, while high concentration (> 40 mU/ml) insulin has been shown to inhibit K562 cell proliferation; Flow cytometry showed that 40 mU/ml insulin suppressed K562 cell apoptosis (P < 0.05), while 200 mU/ml insulin could significantly induce K562 cell apoptosis (P < 0.01); 0.01 to 1.0 µg/ml IGF-1R-Ab has significantly enhanced the inhibitory and inducing effects of high concentration (> 40 mU/ml) of insulin on K562 cell proliferation and apoptosis respectively (r = 0.962, P < 0.001); Western blot showed that after K562 cells were treated with different concentrations of insulin ERK, and the p-ERK expression did not change significantly, after K562 cells were treated with 200 mU/ml insulin, the expression of IGF-1R and AKT also not were changed obviously, while the phosphorylation level of IGF-1R and AKT increased. CONCLUSION: High concentration (>40 mU/ml) of insulin inhibits K562 cell proliferation and induces its apoptosis, and its mechanism may be related with the binding IGF-1R by insulin, competitively inhibiting the binding of IGF-1 and IGF-1R, the blocking the transduction of PI3K/AKT signal pathway.

Risk Factors for Nonclosure of a Temporary Defunctioning Ileostomy Following Anterior Resection of Rectal Cancer.

BACKGROUND: In patients with colorectal cancer, a defunctioning ileostomy is commonly constructed to reduce anastomotic complications. However, many patients do not undergo a subsequent procedure to have their temporary stoma closed. OBJECTIVE: This study investigated the incidence of nonclosure of ileostomies and identified factors associated with nonclosure. DESIGN: This study is a retrospective analysis of prospectively collected data. SETTING: This study was conducted at a tertiary referral cancer hospital. PATIENTS: A total of 296 patients who received anterior resection with a defunctioning ileostomy with protective intention from 2006 to 2013 were included. MAIN OUTCOME MEASURES: The primary outcomes measured were the incidence of nonclosure of ileostomy and associated risk factors. RESULTS: Patients were followed for a median time of 29 months (range, 21-100 months). At the end of the study, 51 (17.2%) patients were left with a permanent ileostomy. The median time interval from the creation of a defunctioning ileostomy to closure was 192 days (range, 14-865 days). Multivariate analyses using a logistic regression model showed that metastatic diseases (OR, 0.179, p < 0.001), Charlson Comorbidity Index score >1 (OR, 0.268; p < 0.01), and complications from the index surgery (OR, 0.391; p = 0.013) were significant independent risk factors for failing to close a defunctioning ileostomy. LIMITATIONS: Although our study has a large patient cohort, it is limited by its retrospective nature. It is difficult to fully evaluate stoma complications after hospital discharge, and the prevalence may be underestimated. CONCLUSION: One in 6 temporary ileostomies constructed during an elective anterior resection for rectal cancer was not closed. Patients should be told before the index surgery that there is a risk of nonclosure and possible complications associated with permanent ileostomy.

Subcutaneous vacuum drains reduce surgical site infection after primary closure of defunctioning ileostomy.

PURPOSE: Surgical site infection (SSI) is the most common complication after primary closure of defunctioning ileostomy. We use a subcutaneous vacuum drain (SVD) in our institution to prevent infection. This study aimed to analyze the risk factors of SSI and to assess the utility of an SVD for preventing SSI in patients undergoing primary closure of ileostomy. METHODS: Patients undergoing ileostomy closure in the Department of Colorectal Surgery, Peking University Cancer Hospital, from September 2006 to March 2013, were included in this study. The clinical features of these patients with or without a subcutaneous drain were reviewed, and the complication rate of SSI was analyzed. The primary endpoints were the incidence and risk factors of SSI, and the secondary endpoints were the rate of overall complications and their management. RESULTS: A total of 245 consecutive patients were enrolled in the study. The overall incidence of SSI was 8.6%. Eighty-five (34.7%) patients received placement of an SVD. The use of SVDs was associated with a significantly lower incidence of SSI compared with primary closure (PC) without an SVD (1.2 vs. 12.5%, p = 0.001). Multivariate analyses showed that the presence of an SVD (odds ratio (OR) 0.063, p = 0.012), total operation time >90 min (OR 4.862, p = 0.002), and postoperative complications (OR 10.576, p < 0.001) were independent risk factors of SSI. CONCLUSIONS: This study shows that an SVD is effective for reducing SSI in patients undergoing PC of ileostomy. Further randomized trials are required to confirm our findings and to compare SVDs with purse-string sutures.

Tumor regression grades: potential outcome predictor of locally advanced rectal adenocarcinoma after preoperative radiotherapy.

AIM: To analyze tumor regression grade (TRG) for prognosis of locally advanced rectal adenocarcinoma (LARA) treated with preoperative radiotherapy. METHODS: One hundred and ninety patients with clinical stage II/III LARA were studied. All patients underwent radical surgery (between 2004 and 2010) after 30-Gy/10-fraction preoperative radiotherapy (pre-RT). All 190 patients received a short course of pre-RT and were reassessed for disease recurrence and survival; the slides of surgical specimens were reviewed and classified according to Mandard TRG. We compared patients with good response (Mandard TRG1 or TRG2) vs patients with bad/poor response (Mandard TRG3-5). Outcomes evaluated were 5-year overall survival (OS), 5-year disease-free survival (DFS), and local, distant and mixed recurrence. Fisher's exact test or χ(2) test, log-rank test and proportional hazards regression analysis were used to calculate the probability that Mandard TRG was associated with patient outcomes. RESULTS: One hundred and sixty-six of 190 patients (87.4%) were identified as Mandard bad responders (TRG3-5). High Mandard grade was correlated with tumor height (41.7% < 6 cm vs 58.3% ≥ 6 cm, P = 0.050), ypT stage (75% ypT0-2 vs 25% ypT3-4, P = 0.000), and ypN stage (75% ypN0 vs 25% ypN1, P = 0.031). In univariate survival analysis, Mandard grade bad responders had significantly worse OS and DFS than good responders (TRG1/2) (OS, 83.1% vs 96.4%, P = 0.000; DFS, 72.3% vs 92.0%, P = 0.002). In multivariate survival analysis, Mandard bad responders had significantly worse DFS than Mandard good responders (DFS 3.8 years (95%CI: 1.2-12.2 years, P = 0.026). CONCLUSION: Mandard grade good responders had a favorable prognosis. TRG may be a potential predictor for DFS in LARA after pre-RT.

Phosphatidylinositol 3-kinase CB association with preoperative radiotherapy response in rectal adenocarcinoma.

AIM: To examine the correlation of phosphatidylinositol 3-kinase (PIK3) CB expression with preoperative radiotherapy response in patients with stage II/III rectal adenocarcinoma. METHODS: PIK3CB immunoexpression was retrospectively assessed in pretreatment biopsies from 208 patients with clinical stage II/III rectal adenocarcinoma, who underwent radical surgery after 30-Gy/10-fraction preoperative radiotherapy. The relation between PIK3CB expression and tumor regression grade, clinicopathological characteristics, and survival time was statistically analyzed. Western blotting and in vitro clonogenic formation assay were used to detect PIK3CB expression in four colorectal cancer cell lines (HCT116, HT29, LoVo, and LS174T) treated with 6-Gy ionizing radiation. Pharmacological assays were used to evaluate the therapeutic relevance of TGX-221 (a PIK3CB-specific inhibitor) in the four colorectal cancer cell lines. RESULTS: Immunohistochemical staining indicated that PIK3CB was more abundant in rectal adenocarcinoma tissues with poor response to preoperative radiotherapy. High expression of PIK3CB was closely correlated with tumor height (P < 0.05), ypT stage (P < 0.05), and high-degree tumor regression grade (P < 0.001). High expression of PIK3CB was a potential prognostic factor for local recurrence-free survival (P < 0.05) and metastasis-free survival (P < 0.05). High expression of PIK3CB was also associated with poor therapeutic response and adverse outcomes in rectal adenocarcinoma patients treated with 30-Gy/10-fraction preoperative radiotherapy. In vitro, PIK3CB expression was upregulated in all four colorectal cancer cell lines concurrently treated with 6-Gy ionizing radiation, and the PIK3CB-specific inhibitor TGX-221 effectively inhibited the clonogenic formation of these four colorectal cancer cell lines. CONCLUSION: PIK3CB is critically involved in response to preoperative radiotherapy and may serve as a novel target for therapeutic intervention.

Monocyte-induced NK cell inactivation: role of reactive oxygen and nitrogen metabolites.

Here in a co-cultivation system of natural killer (NK) cells and K562 cells, monocytes (MO) and/or interleukin (IL)-2/phytohemagglutinin (PHA) were administered. After MO were administered, reactive oxygen metabolites (ROM)/reactive nitrogen metabolites (RNM) productions increased, while tumor necrosis factor (TNF)-ß/interferon (IFN)-γ levels and NK cell cytotoxicity (NCC) decreased, the changes of which after administering tiopronin (TIP) or glutamylcysteinylglycine (GSH) were opposite. In conclusions, the activated MO could inhibit the NK cell activity to kill K562 cell by secreting ROM and RNM. And TIP and GSH could scavenge both ROM and RNM to reverse the inhibitory effect of MO.

[Effects of reactive nitrogen metabolites on NK cell-mediated killing of K562 cells].

OBJECTIVE: To explore the effects of the exogenous and endogenous reactive nitrogen metabolites (RNM) as NK cell inhibitors on NK cell-mediated killing of K562 cells and the influence of Tiopronin (TIP), glutamylcysteinylglycine (GSH) and histamine dihydrochloride (DHT) as RNM scavengers on reversing the suppressing effect of RNM. METHODS: The exogenous ONOO(-) was administered in the NK+K562 culture system, then the RNM scavengers were added in the NK+K562+ONOO(-) culture system, respectively. The concentrations of RNM, TNF-beta and IFN-gamma, K562 cell inhibition rate (KIR) and the percentage of living NK cells were examined. IL-2+PHA were used as monocyte (MO) activators in the culture system of MO+NK+K562. Then TIP, GSH and DHT were administered and the parameters of NK cell activity were analyzed. RESULTS: After exogenous ONOO(-) was administered in NK+K562 culture system, the percentage of living NK cells was decreased from (93.17 +/- 2.57)% to (71.87 +/- 1.02)% (P < 0.01) and KIR was decreased from (67.47 +/- 2.64)% to (43.44 +/- 2.87)% (P < 0.01). When TIP, GSH and DHT were administered into the systems, the percentage of living NK cells was increased to (91.13 +/- 3.67)% (P < 0.05), (88.03 +/- 1.46)% (P < 0.05), (73.60 +/- 2.76)% (P > 0.05), respectively; KIR was increased to (61.58 +/- 1.89)% (P < 0.05), (60.68 +/- 2.07)% (P < 0.05) and (45.26 +/- 3.31)% (P > 0.05), respectively. When IL-2/PHA were administered in the NK+K562+MO culture system, RNM products was increased from (82.10 +/- 6.60) micromom/L to (193.65 +/- 5.95) micromom/L(P < 0.01);KIR was decreased from (90.64 +/- 3.06)% to (61.29 +/- 2.22)% (P < 0.01). When the TIP, GSH and DHT were administered in the systems, RNM products were decreased to (91.32 +/- 6.81) micromom/L (P < 0.05), (84.66 +/- 5.99) micromom/L (P < 0.05) and (188.92 +/- 5.00) micromom/L (P > 0.05), respectively; KIR was increased to (84.31 +/- 4.56)%(P < 0.05), (81.65 +/- 3.09)% (P < 0.05) and (72.20 +/- 4.10)% (P < 0.05), respectively. CONCLUSION: NK Cell-mediated killing of K562 cells can be suppressed by exogenous and endogenous RNM administration. Both of TIP and GSH can protect NK cells by scavenging RNM and enhance the antineoplasmic activity of NK cells.

[Identification of a novel mutation of IDS gene from a Chinese pedigree with MPS II].

The purpose of this study was to understand the molecular genetic mechanism of mucopolysaccharidosis type II (MPS II) and to provide a prerequisite for future prenatal gene diagnosis. A preliminary diagnosis was made by qualitative detection of Urinary Glycosaminoglycans of the suspected MPS II proband. Then, mutation detection was performed on the proband and his family members with PCR and direct sequencing of PCR products. After the novel mutation of c.876 del 2 in IDS gene was detected, sequence analysis was performed on exon 6 of IDS gene of the 135 cases, which consisted of 120 randomly selected normal controls, and other 15 patients with MPS I, IV, and VI other than MPS II. Besides, the patho-genicity of the novel mutation was analyzed with the following 2 methods: conservative analysis of the sequence of muta-tion spots of different species and the direct test of the IDS enzyme activity of the patient and his relative family members. The result of uroscopy of the proband was strong positive (GAGs +++). There was a novel deletion mutation of c.876-877 del TC in the coding region of exon 6 of IDS gene, which was a hemizygous mutation. However, the mutation of his mother and sister was a heterozygous mutation. Detection of the exon 6 of IDS gene showed that the mutation was not found among normal controls and other patients with MPS I, IV, and VI other than MPS II. Homology comparison of amino acid sequences from different species showed that the phenylalanine (F) glutamine (Q) of the mutation site of c.876-877 del TC located in p.292-293 was highly conserved. The activity of IDS enzyme of the proband was only 2.3 nmol/4 h/mL, which was much lower than normal; but the activity of IDS enzyme of his father, mother and sister was 641.9 nmol/4 h/mL, 95.8 nmol/4h/mL and 103.2 nmol/4h/mL, respectively. These results illustrated that the deletion and frame-shift mutation of c.876-877 del TC detected was a novel pathologic mutation, which was the underlying cause of MPS II of this patient.