Promising biomarkers for predicting the outcomes of patients with KRAS wild-type metastatic colorectal cancer treated with anti-epidermal growth factor receptor monoclonal antibodies: a systematic review with meta-analysis.

KRAS mutations have been established as a major predictive biomarker for resistance to the treatment of metastatic colorectal cancer (mCRC) with anti-epidermal growth factor receptor monoclonal antibodies (anti-EGFR MoAbs). However, many patients with KRAS wild-type tumors still do not respond to the treatment. We conducted a systematic review with meta-analysis to assess whether BRAF mutations, PIK3CA mutations and PTEN loss can predict the outcomes of patients with KRAS wild-type mCRC treated with anti-EGFR MoAbs. Studies that explored the association of one or more of the three biomarkers with progression-free survival (PFS), overall survival (OS) and/or objective response rate (ORR) were identified through August 2012. Summary hazard ratios (HRs) and rate differences (RDs) and corresponding 95% confidence intervals (CIs) were calculated by using the random-effects model. BRAF mutations, PIK3CA exon 20 mutations and PTEN loss were all associated with shorter PFS (HR = 2.59, 95% CI 1.67-4.03; HR = 2.52, 95% CI 1.33-4.78 and HR = 1.75, 95% CI 1.19-2.56, respectively), shorter OS (HR = 2.74, 95% CI 1.79-4.19; HR = 3.29, 95% CI 1.60-6.75 and HR = 1.85, 95% CI 1.30-2.64, respectively) and lower ORR (RD = -36%, 95% CI -44 to -28%; RD = -38%, 95% CI -51 to -24% and RD = -41%, 95% CI -68 to -14%, respectively). PIK3CA exon 9 mutations were associated with none of the outcomes. Studies with relevant data consistently demonstrated a stronger predictive power of combined multiple biomarkers as compared to one alteration alone. These results suggest that BRAF mutations, PIK3CA exon 20 mutations and PTEN loss are predictive of worseoutcomes in KRAS wild-type mCRC treated with anti-EGFR MoAbs [corrected]. However, the quality of included studies varied, and some of the meta-analyses were limited by significant between-study heterogeneity. In the future, well-designed large randomized controlled trials conducted in KRAS wild-type mCRC patients with subgroup analysis according to BRAF, PIK3CA exon 20 and PTEN status are essential to fully assess the clinical relevance of these biomarkers.

KRAS p.G13D mutation and codon 12 mutations are not created equal in predicting clinical outcomes of cetuximab in metastatic colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: The authors conducted a systematic review and meta-analysis to examine whether patients who had metastatic colorectal cancer (mCRC) with the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) p.G13D mutation (an amino acid substitution at position 13 in KRAS from a glycine to an aspartic acid) and received cetuximab treatment had better clinical outcomes than patients who had mCRC tumors with KRAS codon 12 mutations. METHODS: Relevant studies were identified by a search of MEDLINE, EMBASE, the Chinese Biomedical Database, and Wan Fang Digital Journals from inception to October 2011. The primary clinical outcomes included the objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). The pooled relative risk (RR) or hazard ratio (HR) was estimated by using fixed-effects or random-effects models according to heterogeneity between studies. RESULTS: Ten studies were considered eligible that included 1487 patients with mCRC. Patients who had tumors with the KRAS p.G13D mutation had a significantly higher ORR (10 studies; RR, 1.642; 95% confidence interval [CI], 1.131-2.384), longer PFS (1 study; HR, 0.54; 95% CI, 0.36-0.81), and longer OS (1 study; HR, 0.52; 95% CI, 0.33-0.80) than patients who had tumors with KRAS codon 12 mutations. Compared with patients who had KRAS wild-type tumors, patients with the p.G13D mutation had a significantly lower ORR (9 studies; RR, 0.540; 95% CI, 0.381-0.765) and nonsignificantly shorter PFS (1 study; HR, 0.99; 95% CI, 0.68-1.45) and OS (1 study; HR, 1.01; 95% CI, 0.66-1.54). CONCLUSIONS: Patients who had mCRC with the KRAS p.G13D mutation appeared to benefit more from cetuximab than patients who had tumors with KRAS codon 12 mutations. However, because of the limited sample sizes in the current meta-analysis, these results should be interpreted with caution.

Mechanisms of tumor-associated macrophages affecting the progression of hepatocellular carcinoma.

Tumor-associated macrophages (TAMs) are essential components of the immune cell stroma of hepatocellular carcinoma. TAMs originate from monocytic myeloid-derived suppressor cells, peripheral blood monocytes, and kupffer cells. The recruitment of monocytes to the HCC tumor microenvironment is facilitated by various factors, leading to their differentiation into TAMs with unique phenotypes. TAMs can directly activate or inhibit the nuclear factor-κB, interleukin-6/signal transducer and signal transducer and activator of transcription 3, Wnt/ß-catenin, transforming growth factor-ß1/bone morphogenetic protein, and extracellular signal-regulated kinase 1/2 signaling pathways in tumor cells and interact with other immune cells via producing cytokines and extracellular vesicles, thus affecting carcinoma cell proliferation, invasive and migratory, angiogenesis, liver fibrosis progression, and other processes to participate in different stages of tumor progression. In recent years, TAMs have received much attention as a prospective treatment target for HCC. This review describes the origin and characteristics of TAMs and their mechanism of action in the occurrence and development of HCC to offer a theoretical foundation for further clinical research of TAMs.

The Roles of circRNAs in Liver Cancer Immunity.

Circular RNAs (circRNAs) are stable covalently closed non-coding RNAs (ncRNAs). Many studies indicate that circRNAs are involved in the pathological and physiological processes of liver cancer. However, the functions of circRNAs in liver cancer immunity are less known. In this review, we summarized the functions of circRNAs in liver cancer, including proliferative, metastasis and apoptosis, liver cancer stemness, cell cycle, immune evasion, glycolysis, angiogenesis, drug resistance/sensitizer, and senescence. Immune escape is considered to be one of the hallmarks of cancer development, and circRNA participates in the immune escape of liver cancer cells by regulating natural killer (NK) cell function. CircRNAs may provide new ideas for immunotherapy in liver cancer.

The prognostic value of phosphatase and tensin homolog negativity in breast cancer: A systematic review and meta-analysis of 32 studies with 4393 patients.

The prognostic value of phosphatase and tensin homolog (PTEN) negativity in breast cancer has been evaluated by many studies but remains controversial. We conducted a meta-analysis to assess the association of PTEN negativity with overall survival and disease-free survival. Thirty-two studies with 4393 patients were identified. PTEN negativity was significantly associated with unfavorable overall survival in breast cancer (hazard ratio=1.89, 95% confidence interval 1.58-2.26), with low heterogeneity among the studies (I(2)=25%, P=0.160) and no evidence for publication bias. Meta-analysis of multivariate hazard ratios and sensitivity analyses did not materially change the results. The data on disease-free survival was heterogeneous (I(2)=61.9%, P<0.001), with a summary hazard ratio of 1.57 (95% confidence interval 1.31-1.89). The exact source of heterogeneity remains unclear. We thus concluded that PTEN negativity was significantly associated with unfavorable prognosis in terms of overall survival in breast cancer.

The prognostic value of phosphorylated Akt in breast cancer: a systematic review.

The prognostic value of phosphorylated Akt (pAkt) overexpression in breast cancer has been investigated by many studies with inconsistent results. This systematic review was conducted to evaluate the association of pAkt overexpression with breast cancer prognosis in terms of overall survival and disease-free survival. Three electronic databases (PubMed, EMBASE and Chinese Biomedical Literature Database) were comprehensively searched. Hazard ratios (HRs) with 95% confidence intervals (CIs) from different studies were combined using the random-effects model. In total, 33 studies with 9,836 patients were included for final analysis. The summary HR for overall survival and disease-free survival was 1.52 (95% CI: 1.29-1.78) and 1.28 (95% CI: 1.13-1.45), respectively, indicating higher risk of death and disease recurrence associated with pAkt overexpression. The results were robust in sensitivity analyses by omitting one study each time and by using the fixed-effects model instead. Subgroup and meta-regression analyses did not show that the prognostic effect of pAkt overexpression would change materially with such factors as population, status of hormone receptors, hormonal or trastuzumab treatment given, analyzing method (univariate versus multivariate) and methodological quality of the original studies. In conclusion, the available evidence suggests that pAkt overexpression is an adverse prognostic factor for breast cancer.

[Conditions for large-scale production of genetically engineered E.coli bearing humanized anti- HBsAg Fab].

OBJECTIVE: To define the conditions for large-scale production of genetically engineered E.coli bearing humanized anti-HBsAg Fab. METHOD: Characteristic growth and expression of the engineered E.coli were observed during fermentation in the shaking flask to define the optimal culture conditions to achieve the highest production levels. On the basis of the observation results, the E.coli was cultured in a fermentor using the fed-batch method to determine the optimal production techniques. RESULTS: Observation of the bacterium in the shaking flask showed initiation of the induction procedure in the mid-log growth phase at 25 degrees Celsius; with 0.2% arabinose resulted in the highest production of anti-HBsAg Fab. The D(600) value of the culture reached 55.2, equivalent to 110 g/L wet weight of the bacterium, using the DO-stat fed-batch method. The resultant Fab showed well-preserved biological activity. CONCLUSION: Reliable techniques for rapid and massive production of the Fab have been developed.

Co-expression of RAGE and HMGB1 is associated with cancer progression and poor patient outcome of prostate cancer.

Receptor for advanced glycation end products (RAGE), along with its ligand high mobility group box 1 (HMGB1), is believed to play an important role in prostate cancer. The aim of this retrospective study was to investigate the expression of RAGE and HMGB1 and their clinical impact on prostate cancer progression and prognosis. The expression of RAGE and HMGB1 was assessed by immunohistochemistry in cancer lesions from 85 confirmed prostate cancer cases. We determined the potential association between the expression level of these two proteins and the clinicopathological features and overall patient survival. RAGE and HMGB1 were expressed in 78.8% (67/85) and 68.2% (58/85) cases of prostate cancer, respectively, and in the majority (54/85) of cases, these two proteins were co-expressed. There was a strong correlation between RAGE and HMGB1 expressions (P<0.001). The expression of RAGE, HMGB1 and their co-expression were all associated with advanced tumor clinical stage (P<0.05 for all). RAGE expression was also associated with the prostate specific antigen (PSA) level (P=0.014). However, neither the individual expression of those genes nor their co-expression was significantly related with age or Gleason score. The co-expression of RAGE and HMGB1 was associated with poor overall survival in patients with stage III and IV prostate cancer (P=0.047). These results suggest that the expression of RAGE and HMGB1 is associated with the progression and poor prognosis of prostate cancer. RAGE and HMGB1 could be new prognostic biomarkers for prostate cancer as well as molecular target for novel forms of therapies.

Gemcitabine plus erlotinib for advanced pancreatic cancer: a systematic review with meta-analysis.

BACKGROUND: This study aims to comprehensively summarize the currently available evidences on the efficacy and safety of gemcitabine plus erlotinib for treating advanced pancreatic cancer. METHODOLOGY/PRINCIPAL FINDINGS: PubMed, EMBASE, The Cochrane Library and abstracts of recent major conferences were systematically searched to identify relevant publications. Studies that were conducted in advanced pancreatic cancer patients treated with gemcitabine plus erlotinib (with or without comparison with gemcitabine alone) and reporting objective response rate, disease control rate, progression-free survival, time-to-progression, overall survival, 1-year survival rate and/or adverse events were included. Data on objective response rate, disease control rate, 1-year survival rate and adverse events rate, respectively, were combined mainly by using Meta-Analyst software with a random-effects model. Data on progression-free survival, time-to-progression and overall survival were summarized descriptively. Sixteen studies containing 1,308 advanced pancreatic cancer patients treated with gemcitabine plus erlotinib were included. The reported median progression-free survival (or time-to-progression), median overall survival, 1-year survival rates, objective response rates and disease control rates were 2-9.6 months, 5-12.5 months, 20%-51%, 0%-28.6% and 25.0%-83.3%, respectively. The weighted 1-year survival rate, objective response rate and disease control rate based on studies reporting robust results were 27.9%, 9.1% and 57.0%, respectively. According to the studies with relevant data, the incidences of total and severe adverse events were 96.3% and 62.9%, respectively. The most frequently reported adverse events were leucopenia, rash, diarrhea, vomitting, neutropenia, thrombocytopenia, anaemia, stomatitis, drug-induced liver injury, fatigue and fever. Compared with gemcitabine alone, the progression-free survival and overall survival with gemcitabine plus erlotinib were significantly longer, but there were also more deaths and interstitial lung disease-like syndrome related to this treatment. CONCLUSIONS/SIGNIFICANCE: Gemcitabine plus erlotinib represent a new option for the treatment of advanced pancreatic cancer, with mild but clinically meaningful additive efficacy compared with gemcitabine alone. Its safety profile is generally acceptable, although careful management is needed for some specific adverse events.

[Establishment of BGC-823/pBaBb-puro-MACC1 gastric cancer cell line stably expressing MACC1 and its tumor-related gene expression profiles].

OBJECTIVE: To establish a gastric cancer cell line with stable expression of metastasis-associated in colon cancer 1 (MACC1) and detect the changes in tumor-related gene expression profiles for investigating the possible regulation mechanisms between MACC1 and the differentially expressed genes. METHODS: The full-length MACC1 cDNA was amplified from human embryonic kidney 293FT cells and cloned into the pBaBb-puro vector. The recombinant pBaBb-puro-MACC1 expression vector, after identification with restriction enzyme digestion, was transfected into 293FT cells, and the expression of fluorescent reporter gene was observed. pBaBb-puro-MACC1 vector was transfected into human gastric cancer BGC-823 cell line to establish BGC-823/pBaBb-puro-MACC1 cell line stably expressing MACC1. Quantitative RT-PCR and Western blotting were used to detect MACC1 expression in both BGC-823/pBaBb-puro-MACC1 and control BGC-823 cells. High-throughout cDNA microarray was used to screen the effects of MACC1 on the gene expression profiles of gastric cancer cells. RESULTS: The recombinant pBaBb-puro-MACC1 plasmid was successfully constructed and verified by PCR and sequencing. BGC-823/pBaBb-puro-MACC1 cells showed significantly increased MACC1 mRNA expression as compared with the control cells. The results of cDNA microarray identified 33 up-regulated and 24 down-regulated genes in the cells after MACC1 transfection involved were in various cellular functions. CONCLUSION: The established BGC-823/pBaBb-puro-MACC1 gastric cancer cell line show some important molecular changes caused by MACC1.

EGFR gene copy number as a predictive biomarker for the treatment of metastatic colorectal cancer with anti-EGFR monoclonal antibodies: a meta-analysis.

BACKGROUND: Epidermal growth factor receptor gene copy number (EGFR GCN) has been heavily investigated as a potential predictive biomarker for the treatment of metastatic colorectal cancer (mCRC) with anti-EGFR monoclonal antibodies (MAbs). The objective of this study was to systematically review current evidences on this issue. METHODS: PubMed, EMBASE, The Cochrane Library, Chinese Biomedical Literature Database, Wanfang Data, and the conference abstracts of American Society of Clinical Oncology and European Society of Medical Oncology were comprehensively searched. Studies that reported the objective response rate (ORR), progression-free survival, and/or overall survival of mCRC patients treated with anti-EGFR MAbs, stratified by EGFR GCN status, were included. The effect measures for binary outcome (response) and time-to-event outcomes (progression-free survival and overall survival) were risk difference and hazard ratio, respectively. Statistical heterogeneity among the studies was assessed by the Cochran's Q-test and the I2 statistic. If appropriate, a quantitative synthesis of data from different studies would be conducted with a random-effects model. RESULTS: Nineteen eligible studies were identified. The criteria for increased EGFR GCN (GCN+) were highly inconsistent across different studies. The prevalence of GCN + ranged from 6.9% to 88.9%, and the difference in ORR between patients with GCN + and those with non-increased EGFR GCN (GCN-) varied from -28% to 84%. Because of the significant heterogeneity, no quantitative synthesis of data was performed. There was a general trend towards higher ORR in patients with GCN+. The difference in ORRs between patients with GCN + and those with GCN- was even greater in KRAS wild-type patients, while in KRAS mutated patients the difference often did not exist. Almost all patients with EGFR amplification responded to the treatment. However, the prevalence of EGFR amplification was generally low. Incomplete data on progression-free survival and overall survival seemingly supported the findings on ORR. CONCLUSIONS: Although increased EGFR GCN is generally associated with a better outcome of anti-EGFR MAbs treatment, especially among patients with wild-type KRAS, the clinical utility of this biomarker for selecting recipients of anti-EGFR MAbs would be severely limited by the heterogeneous scoring system and the poor reproducibility of EGFR GCN enumeration due to technical reasons.

[Effect of anti-EGFR monoclonal antibody on chemosensitivity of human colon cancer cells and the mechanism].

OBJECTIVE: To investigate the effect of anti-EGFR monoclonal antibody on the chemosensitivity of human colon cancer cells and explore the possible molecular mechanism. METHODS: The inhibitory effect of irinotecan (CPT-11), oxaliplatin (L-OHP) and fluorouracil (5-Fu), used alone or in combination with anti-EGFR monoclonal antibody, on the proliferation of LoVo cells in vitro was assessed by MTT assay. The expressions of PI3K and Akt protein in the treated cells were examined by Western blotting, and their mRNA expressions were detected by RT-PCR. RESULTS: Both h-R3 and C-225 treatments significantly increased the chemosensitivity of LoVo cells to irinotecan and oxaliplatin. 5-Fu and h-R3 coadministered showed a synergistic effect on the cells, but 5-Fu and C-225 had an antagonistic action. Treatment with C-225 or h-R3 resulted in lowered expressions of PI3K and Akt in LoVo cells. CONCLUSION: Anti-EGFR monoclonal antibody can increase the chemosensitivity of human colon cancer cells to most chemotherapeutic drugs, and such effect might be attributed to the blocking of PI3K/Akt signaling pathway by these antibodies.

[Reversion of drug-resistant hepatocellular carcinoma cell line BEL-7402/FU by sorafenib].

AIM: To investigate the reversion of multidrug resistance of drug-resistant hepatocellular carcinoma (HCC) cell line BEL-7402/FU by sorafenib and the possible mechanisms. METHODS: Flow cytometry was applied to detect the expression of rhodamine 123 (Rho123), immunohistochemistry was applied to observe the expression of P-glycoprotein (P-gp) on the cell membrane, the fluorescence quantitative PCR assay was applied to determine the changes of the multidrug-resistant gene mdr1 mRNA level and the Western blot assay was applied to test the changes of the expression level of the mdr1 gene product P-gp. RESULTS: After 4 micromol/L treatment by sorafenib, Rho123 accumulation was increased by BEL-7402/FU and exocytosis was slowed down, which evidently reduced the expression of cell surface P-gp; the mdr1 mRNA level was reduced by 35.4% compared to its level prior to drug administration; the expression level of the mdr1 gene product P-gp was significantly suppressed, 14.3% fewer than that of BEL-7402/FU cells. CONCLUSION: Sorafenib can inhibit the expression of the mdr1 gene product P-gp and increase the intracellular concentration of chemotherapeutic drugs, so as to reverse the multidrug resistance of HCC cells.

[Sorafenib reverses multidrug resistance of hepatoma cells in vitro].

OBJECTIVE: To explore the role of sorafenib in reversing multidrug resistance (MDR) in hepatoma BEL-7402/FU cells and its possible mechanisms. METHODS: MTT colorimetric assay was used to obtain the dose-response curve of sorafenib in BEL-7402/FU cells, and flow cytometry performed to assess the effect of sorafenib on Rho123 concentration in the cells. The optimal dose of sorafenib for cell treatment was determined according to the results of MTT assay and flow cytometry. MTT assay was employed to evaluate the effect of sorfenib on the cytotoxicity of the antitumor drugs, flow cytometry performed to determine the expression of cell membrane transport protein (P-gp), and RT-PCR used to detect mdr1 gene expression in the cells treated with sorafenib at the optimal dose. RESULTS: Sorafenib at the concentration of 4 micromol/L, efficiently reversed the MDR of the cells with minimal side effects. At the concentration of 4 micromol/L, sorafenib partially reversed the drug resistance of BEL-7402/FU cells to ADM, 5-FU, GEM and DDP, with reversal indexes of 2.98, 7.16, 1.99 and 10.08, respectively. Treatment of the cells with 4 micromol/L, sorafenib also partially down-regulated P-gp expression in BEL-7402/FU cells, and caused a reduction of mdr1 gene expression by 27.3% in comparison with the control cells. CONCLUSION: Sorafenib can reverse MDR in human hepatoma cells probably in association with down-regulation of mdr1 gene expression and increased accumulation of the chemotherapeutic agents in the cells.

[Effects of bevacizumab and cisplatin on human lung adenocarcinoma A549/DDP xenografts in nude mice].

OBJECTIVE: To explore the effects of bevacizumab with or without cisplatin (DDP) on the growth of lung adenocarcinoma A549/DDP cell xenografts in mice. METHODS: Human lung cancer A549/DDP cells was subcutaneously transplanted in to 25 nude mice, which were randomly divided into control group (group A), bevacizumab group (group B), DDP group (group C), combined treatment group (group D) and half-dose combined treatment group (group E). After corresponding treatments for 4 consecutive weeks, the tumor inhibition rate was evaluated, tumor microvessel density (MVD) measured with immunohistochemistry, and the mRNA expression of apoptosis-associated gene (bcl-2) and multidrug resistance genes (LRP and GST-pi) assessed by RT-PCR. RESULTS: The tumor growth inhibition rates in groups B, D, and E with bevacizumab treatment were 20.96%, 51.67% and 50.95%, respectively, and the two combined treatment groups showed better effects. MVD in these 3 groups were 18.6-/+1.14, 13.6-/+1.14, and 14.4-/+0.55, respectively, and no significant difference was found in MVD between DDP group and the control group. Compared with the control group, the 3 bevacizumab-treated groups showed decreased expression of bcl-2 genes in A549/DDP tumors at a comparable amplitude, and LRP and GST-pi mRNA expression showed no significant differences between the 5 groups. CONCLUSION: Bevacizumab has synergetic inhibitory effect with conventional chemotherapy against lung adenocarcinoma A549/DDP cell xenografts in mice by inhibiting angiogenesis of the tumor, and may enhance the sensitivity of A549/DDP cells to DDP by inducing cell apoptosis.

[Avastin combined with cisplan inhibits malignant ascites production in nude mice bearing transplanted ovary carcinoma with high VEGF expression].

OBJECTIVE: To establish a nude mouse model of malignant ascites with human ovarian carcinoma cell line OVCAR3 which highly expresses VEGF and evaluate the therapeutic of Avastin combined with cisplan. METHODS: Forty-eight nude mice with malignant ascites resulting from intraperitoneal transplantation of human ovarian carcinoma cell line OVCAR3 were treated with intraperitoneal injection of Avastin, cisplan, their combination, and PBS, respectively, to observe the effect on ascites development, VEGF content in the ascites, peritoneal permeability, development of new vessels and number of tumor cells in the ascites. RESULTS: Avastin obviously inhibited ascites accumulation and peritoneal capillary permeability, reduced VEGF protein level and microvascular density in the tumor tissues and the number of red cells and tumor cells in the malignant ascites, and prolonged the survival of the mice. The combination of Avastin and cisplan further enhanced the therapeutic efficacy of Avastin. CONCLUSION: The bio-chemotherapeutic strategy with Avastin combined with cisplan can be a promising method for treatment of malignant ascites.

[Adsorption condition optimization for anti-HBsAg Fab fragment separation and purification from E. coli using Streamline SP].

OBJECTIVE: To optimize the adsorption condition of cation-exchange chromatographic media Streamline SP for separation and purification of anti-HBsAg Fab fragment from E. coli. METHODS: The adsorption of the target protein for separation and purification by the cation-exchange chromatographic media Streamline SP was tested using test tube method in balanced buffer solution with different pH values and ion concentrations. The adsorption effect was then verified by cation-exchange chromatography using 1-ml Streamline SP prepacked column and 28-ml Streamline SP self-assembly column. RESULTS: According to the experiment results of test tube method, the loading buffer with pH of 4.4 and ionic concentration of 100 to 600 mmol/L could achieve optimal target protein adsorption effect by cation-exchange chromatographic media Streamline SP, as verified by cation-exchange chromatography with 1-ml SP prepacked column and 28-ml Streamline SP self-assembly column. CONCLUSION: The optimal condition of cation-exchange chromatography selected by test tube method can be applied for separation and purification of anti-HBsAg Fab fragment from E. coli.