A Novel Assay Coupling Dephosphorylation and Blue/White Colony Screening for the G > A Hotspot Mutation at Codon 13 of KRAS Gene.

Development of sensitive assay for detection of hotspot mutations of cancer driving gene is crucial for circulating tumor DNA analysis. This study tested the possibilities of applying restriction enzyme digestion and dephosphorylation coupled with blue/white screening technology for analyzing a hotspot point mutation in codon 13 of KRAS gene. The present study has documented that the combination of PCR with restriction digestion, dephosphorylation, blue/white screening and Sanger's sequencing can identify rare mutations with sensitivities at 0.003%. This novel assay with high sensitivity may have application in the diagnosis of early cancer targeting ctDNAs.

Rs56288038 (C/G) in 3'UTR of IRF-1 Regulated by MiR-502-5p Promotes Gastric Cancer Development.

BACKGROUND/AIMS: Interferon regulatory factor 1 (IRF-1) has been shown to function as a transcriptional activator or repressor of a variety of target genes. However, its upstream, non-coding RNA-related regulatory capacity remains unknown. In this study, we focus on the miRNA-associated single nucleotide polymorphisms (SNPs) in the 3'untranslated region (UTR) of IRF-1 to further investigate the functional relationship and potential diagnostic value of the SNPs and miRNAs among Chinese gastric cancer (GC) patients. METHODS: We performed a case-control study with 819 GC patients and 756 cancer-free controls. Genotyping by realtime PCR assay, cell transfection, and the dual luciferase reporter assay were used in our study, and the 5-year overall survival rate and relapse-free survival rate in different groups were investigated. RESULTS: We found that patients suffering from Helicobacter pylori (Hp) infection were the susceptible population compared to controls. SNP rs56288038 (C/G) in IRF-1 3'UTR was involved in the occurrence of GC by acting as a tumor promoter factor. SNP rs56288038 (C/G) could be up-regulated by miR-502-5p, which caused a down-regulation of IRF-1 in cell lines and decreased apoptosis induced by IFN-γ. Carrying the G genotype was related to significantly low expression of IRF-1 and Hp infection, poor differentiation, big tumor size, invasion depth, as well as the high probability of metastasis, and moreover, the C/G SNP was associated with shorter survival of GC patients with five years of follow-up study. CONCLUSIONS: our findings have shown that the SNP rs56288038 (C/G) in IRF-1 3'UTR acted as a promotion factor in GC development through enhancing the regulatory role of miR-502-5p in IRF-1 expression.

Prediction of cancer progression in a group of 73 gastric cancer patients by circulating cell-free DNA.

BACKGROUND: Circulating cell-free DNA (ccf-DNA) in plasma may contain both specific and non-specific of tumor markers. The concentration and integrity of ccf-DNA may be clinical useful for detecting and predicting cancer progression. METHODS: Plasma samples from 40 healthy controls and 73 patients with gastric cancers (two stage 0, 17 stage I, 11 stage II, 33 stage III, and 10 stage IV according to American Joint Committee on Cancer stage) were assessed respectively. qPCR targeting the Alu repeats was performed using two different sets of primers amplifying the long and short segments. DNA integrity was calculated as a ratio of the long to the short fragments of Alu repeats. RESULTS: Plasma DNA concentration was significantly higher in patients with stage III and IV gastric cancers than in healthy controls (p = 0.028 and 0.029 respectively). The receiver operating characteristic (ROC) curve for discriminating patients with stage III and IV gastric cancers from healthy controls had an area under the curve (AUC) of 0.744 (95% CI, 0.64 to 0.85). Circulating cell-free DNA concentration increased within 21 days following surgery and dropped by 3 months after surgery. CONCLUSIONS: Concentration of ccf-DNA is a promising molecular marker for assessing gastric cancer progression. TRIAL REGISTRATION: Current Controlled Trials ChiCTR-DDT-12002848 , 8 October 2012.

JARID1B modulates lung cancer cell proliferation and invasion by regulating p53 expression.

Although three therapeutic modalities (surgical resection, chemotherapy, and radiotherapy) have been established, long-term survival for lung cancer patients is still generally poor. Until now, the mechanisms of lung cancer genesis remain elusive. The JARID1B is a histone demethylase that has been proposed as oncogene in several types of human cancer, but its clinical significance and functional role in human non-small cell lung cancer (NSCLC) remain unclear. In present study, we found that JARID1B was overexpressed in lung cancer cell lines and lung cancer tissues but not in normal lung tissues. The proliferation and invasive potential of lung cancer cells was significantly increased by ectopic expression of JARID1B. Contrarily, RNA interference targeting JARID1B in lung cancer cells significantly decreased the proliferation and invasive potential of cells. Moreover, we also found that the expression of p53 was modulated by JARID1B. Overexpressed JARID1B cell exhibited greatly decreased p53 expression, whereas silencing of JARID1B expression dramatically increased p53 expression at both the messenger RNA (mRNA) and protein levels. Inhibition of p53 by small interfering RNA (siRNA) reversed the shJARID1B-induced suppression of proliferation and invasion. Our results collectively suggested that JARID1B expressed in lung cancer played a role in lung cancer cells proliferation and invasion, which may be partly associated with the p53 expression.

A Nanoscale Mutation-Sensitive On/Off Switch Based Assays for the Detection of Hepatitis B Virus Lamivudine-Resistant Mutations.

Long-term usage of lamivudine in the treatment of chronic hepatitis B virus (HBV) infection induces the emergence of drug resistance. Sensitive and specific methods aimed at detecting the mutants are clinically useful and required. The purpose of this study was to develop methods for detecting the mutations of YMDD, rtL180M, and rtV173L by nanoscale mutation-sensitive switch consisting of high fidelity polymerase and phosphorothioate-modified allele specific primers. Four assays for these hotspot mutations have been developed with the sensitivity of 100 copies and specificity of at least three log scales for matched templates over mismatched templates. In the condition of multiplex PCR, the sensitivities of these assays are approximately 1000 copies and specificities with two log scales in discrimination of mutant alleles over wild type sequences. These newly developed assays are rapid, accurate, and cost-efficient in detection of lamivudine-related HBV mutants.

Pharmacokinetic evaluation of novel oral fluorouracil antitumor drug S-1 in Chinese cancer patients.

AIM: S-1 is an oral anticancer fluoropyrimidine formulation consisting of tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate. The aim of this study was to evaluate the pharmacokinetics and bioequivalence of a newly developed generic formulation of S-1 in Chinese cancer patients in comparison with the branded reference formulation of S-1. METHODS: A single-dose, randomized-sequence, open-label, two-way self-crossover study was conducted in 30 Chinese cancer patients. The subjects alternatively received the two formulations (40 mg/m(2), po) with a 7-d interval. Plasma concentrations of FT, CDHP, Oxo, and 5-Fu were determined using LC-MS/MS. Pharmacokinetic parameters, including Cmax, Tmax, t1/2, AUC0-t, and AUC0-∞ were determined using non-compartmental models with DAS2.0 software. Bioequivalence of the two formulations were to be evaluated according to 90% CIs for the log-transformed ratios of AUC and Cmax of S-1. Adverse events were evaluated through monitoring the symptom, physical and laboratory examinations, ECGs and subject interviews. RESULTS: The mean values of Cmax, AUC0-t, and AUC0-∞ of FT, 5-Fu, CDHP, and Oxo for the two formulations had no significant differences. The 90% CIs for natural log-transformed ratios of Cmax, AUC0-t, and AUC0-∞ were within the predetermined bioequivalence acceptance limits. A total of 11 mild adverse events, including fatigue, nausea and vomiting, anorexia, diarrhea and myelosuppression, were observed, and no serious and special adverse events were found. CONCLUSION: The newly developed generic formulation and reference formulation of S-1 have similar pharmacokinetics with one dose (40 mg/m(2)) in Chinese cancer patients. Both the formulations of S-1 are well tolerated.

Novel Method for Detection of PIK3CA Mutations in Circulating Tumor DNA of Patients with Colorectal Cancer.

The PIK3CA mutation is considered a potential target for treatment of colorectal cancer. We evaluated a PIK3CA mutation assay on plasma cell-free DNA (cfDNA) using a newly developed PCR with restriction digestion integrated and followed by Sanger's sequencing. We analyzed PIK3CA mutation in plasma with our newly developed assays and in matching tumor tissues by routine methods. We detected the PIK3CA gene mutation status by both methods in samples from 40 colorectal cancer patients. Three H1047R mutations of PIK3CA gene were detected in the cfDNA of the 40 patients by restriction digestion PCR. Neither E545K nor H1047R mutations were detected in the cfDNA by routine PCR/sequencing. The PIK3CA H1047R and E545K mutations in cfDNA can be sensitively detected with our newly developed assays. The colorectal cancer has been used as a clinical example in testing our new assays, which indicates that the new assays may have wider applications in detecting mutations in precision oncology. Trial registration: Current Controlled Trials ChiCTR-DDT-12002848, 8 October 2012.

Identification of the mitochondrial protein ADCK2 as a therapeutic oncotarget of NSCLC.

The aarF domain containing kinase 2 (ADCK2) is a mitochondria-locating protein, important for fatty acid metabolism and coenzyme Q biosynthesis. The bioinformatics results show that elevated ADCK2 transcripts in NSCLC correlate with poor overall survival and poor anti-PD-1/PD-L1 therapy response. ADCK2 is overexpressed in local human NSCLC tissues and various primary and established NSCLC cells. In NSCLC cells, ADCK2 shRNA or CRISPR/Cas9 knockout remarkably suppressed cell viability, proliferation, cell cycle progression, cell mobility, and provoked cell apoptosis. Moreover, ADCK2 depletion disrupted mitochondrial functions in NSCLC cells, causing cytochrome C release, mitochondrial depolarization, DNA damage and ATP reduction. Contrarily, ectopic ADCK2 overexpression promoted NSCLC cell growth. Further studies revealed that ADCK2 depletion inactivated Akt-mTOR signaling in primary NSCLC cells. NSCLC xenograft growth in nude mice was significantly hindered after ADCK2 silencing or knockout. ADCK2 depletion, apoptosis induction and oxidative injury as well as ATP reduction and Akt-mTOR inactivation were detected in ADCK2-silenced or ADCK2-knockout NSCLC xenograft tissues. Together overexpressed ADCK2 is important for the growth of NSCLC cells, representing an important therapeutic molecular oncotarget.

Cancer stage-dependent alterations in cell-free DNA in patients with colorectal cancer.

PURPOSE: Cell-free DNA (cfDNA) in plasma is a useful resource for liquid biopsy. The concentration and integrity of cfDNA may be clinical informative for detecting and predicting cancer progression. METHODS: Plasma from 40 healthy controls and 90 colorectal cancer patients was assessed. qPCR targeting the arithmetic-logic unit (Alu) repeats were performed using two different sets of primers amplifying the long and short segments. DNA integrity was calculated by the ratio of the long to the short fragments of amplified Alu repeats. RESULTS: cfDNA concentration was significantly higher in the patients than that in healthy controls. Patients with stage III colorectal cancer showed no significant difference in their cfDNA levels as compared with the healthy controls. In colorectal cancer, cfDNA level of stage IV patients was higher than that of stage 0-III (p=0.049). The DNA integrity was significantly lower in patients with stage I and II cancer than that in normal controls (p=0.007, 0.029 respectively). The receiver operating characteristic (ROC) curve for discriminating patients with colorectal cancer from normal controls had an area under the curve of 0.672 (95%CI, 0.572 to 0.772) and cfDNA concentration increased within 21 days following surgery and dropped by 3 months after surgery. CONCLUSION: Concentration of cfDNA is a promising molecular marker for assessing colorectal cancer progression. Both the cfDNA concentration and its integrity are highly variable. Some cancer stage dependent changes were observed, which warrants further investigation with more patients included.

Successful Rescue of the Victim Exposed to a Super High Dose of Iridium-192 during the Nanjing Radiological Accident in 2014.

Here we report on the interventions taken to treat a patient exposed to high-dose radiation and provide a protocol for treating such patients in the future. The patient, Mr. Wang, was a 58-year-old male janitor who was accidentally exposed to a 192Ir source with an activity of 966.4 GBq or 26.1 Ci. The dose estimated to the lower right limb was 4,100 Gy, whereas the whole-body effective dose was 1.51 Gy. The diagnosis was made according to the results of the patient dose estimation and clinical manifestations. Systemic treatment included stimulating bone marrow hematopoietic cells, enhancing immunity, anti-infection and vitamin supplements. The treatment of radiation-induced skin lesions consisted of several debridements, two skin-flap transplantations and application of mesenchymal stem cells (MSCs). Skin-flap transplantations and MSCs play important roles in the recovery of skin wound. A combination of antibiotics and antimycotic was useful in reducing inflammation. The application of vacuum sealing drainage was effective in removing necrotic tissue and bacteria, ameliorating ischemia and hypoxia of wound tissue, providing a fresh wound bed for wound healing and improving skin or flap graft survival rates. The victim survived the accident without amputation, and function of his highly exposed right leg was partially recovered. These results demonstrate the importance of collaboration among members of a multidisciplinary team in the treatment of this patient.

Primerless Amplification for Circulating Tumor DNA Assays.

A primerless amplification suitable for enrichment of particular genotype cfDNA which is a one-dimensional material has been developed. This primerless amplification coordinated by two thermostable enzymes of endonuclease and proofreading polymerase, functions as a genotype switch in analyzing cfDNA. The endonuclease digests the wild-typed fragments into mega-primer and discriminately destroys the wild-type DNA alleles. The DNA polymerase proofreads the megaprimer and then extends the mega-primer using the mutant DNA as the template. The prototypes of this technology were applied to two hotspot mutations of APC and EGFR with confirmed by DNA sequencing analysis. Genotype switch was then employed to clinical cfDNA assay targeting PIK3CA. Data from the clinical application suggest its potential in early cancer diagnosis.

Clinical value of ALU concentration and integrity index for the early diagnosis of ovarian cancer: A retrospective cohort trial.

CA125 amounts have a large overlap in ovarian cancer and benign diseases. We conducted a retrospective cohort trial to assess the clinical value of circulating cell-free DNA concentration and integrity index for the diagnosis of ovarian cancer. A total of 150 patients were recruited. Plasma samples of 24 ovarian cancer patients, 12 benign ovarian cysts, and 12 healthy controls were assessed. By amplifying short ALU-115 repeat and long ALU-219 fragments, circulating cell-free DNA concentrations and integrity index were measured. Plasma ALU-219 fragment levels and integrity index were significantly higher in the ovarian cancer group compared with the benign disease and healthy control groups (p = 0.023 and p = 0.004, respectively). These findings indicated that plasma ALU-219 levels and integrity may have a clinical value in the early diagnosis of ovarian cancer.

Multiple Organ Lesions in a Case of Contamination With Multiple Radionuclides After 38 Years.

The patient was contaminated with multiple radionuclides 38 years ago due to an accident. To investigate the effects of radionuclide contamination on humans, he has been followed up by examinations for many years. Long-term effects gradually emerge in these years. Lung cancer was diagnosed by medical examinations. Besides, chronic gastritis with intestinal metaplasia was indicated by gastroscopic biopsies, while colorectal polyps found by colonoscopy. All 13 colorectal polyps were removed, and radical surgery for lung cancer was performed. Fortunately, pathological examinations indicated that it was early lung cancer. The ground glass nodule (GGN) in left lung identified during the follow-up will be resected when needed. It is speculated that multiple manifestations of the patient may be related to radiation, and different lesions in the organs may be related to systemic adaptive response. However, longer follow-up is needed due to a lack of effective and direct evidence. This work is expected to provide experiences for similar patients' treatment and follow-up.

Circulating DNA and its methylation level in inflammatory bowel disease and related colon cancer.

Both of chronic inflammation and abnormal immune in inflammatory bowel disease can induce colon cancer. Previous research showed that cell apoptosis and necrosis become the main source of circulating DNA in the peripheral blood during tumorigenesis that reduced along with methylation degree. However, its role in the process of colitis transforming to colon cancer is not clarified. Drinking 3% DSS was used to establish colitis model, while 3% dextran sodium sulfate (DSS) combined with azo oxidation methane (AOM) intraperitoneal injection was applied to establish colitis related colon cancer model. Circulating DNA and its methylation level in peripheral blood were tested. Morphology observation, HE staining, and p53 and ß-catenin expression detection confirmed that drinking 3% DSS and 3% DSS combined with AOM intraperitoneal injection can successfully establish colitis and colitis associated colorectal cancer models. Circulating DNA level in colitis and colon cancer mice increased by gradient compared with control, while significant difference was observed between each other. Circulating DNA methylation level decreased obviously in colitis and colon cancer, and significant difference was observed between each other. Abnormal protein expression, circulating DNA and its methylation level in ulcerative colitis associated colorectal tissues change in gradient, suggesting that circulating DNA and its methylation level can be treated as new markers for colitis cancer transformation that has certain significance to explore the mechanism of human ulcerative colitis canceration.

Effect of environmental factors on chemoresistance of HepG2 cells by regulating hypoxia-inducible factor-1α.

BACKGROUND: Accumulating evidence demonstrates that the microenvironment of the host has an important effect on the chemoresistance of tumors. We also found that the formation of intrinsic multidrug resistance is related to environmental factors that are common with tumor growth of hepatocellular carcinoma. The aim of this study was to explore the molecular mechanisms by which multidrug resistance of hepatocellular carcinoma is induced by the microenvironment. In particular, the regulation of nuclear transcription factor (hypoxia-inducible factor-1α, HIF-1α) activation in the process of multidrug resistance formation was investigated. METHODS: HepG2 cells were exposed to different microenvironmental conditions respectively, such as hypoxia, stimulation of glucose deprivation and transfection of plasmid PcDNA3/HBx. In the HepG2 cells, the expression of the related MDR proteins, HIF-1α protein expression and localization, activity of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) were detected. Specific inhibitor U0126 was used to block ERK/MAPK signal pathway, the alteration of HIF-1α and the related MDR proteins were investigated. Multivariate analysis of variance (MANOVA) repeated measures and one-way analysis of variance (ANOVA) followed by Tukey test or t-test were used to determine differences over time and effects of the treatments. RESULTS: The above three microenvironment factors increase the expression of the related MDR proteins (including P-gp, LRP, and MRP1) and induce MDR of HepG2 cells. HIF-1α was induced at the protein and mRNA levels and the nuclear translocation was also increased. The activity of ERK/MAPK was also increased in HepG2 cells. But when ERK/MAPK pathway was inhibited, the mRNA and protein expression of MDR1, MRP1, and LRP was to some extent decreased. Inhibition of ERK/MAPK significantly reduced activated HIF-1α protein and the nuclear translocation of HIF-1α, whereas HIF-1α mRNA levels were not affected. CONCLUSIONS: The microenvironmental factors could induce MDR of HepG2 cells by the activity of HIF-1α. The activity of HIF-1α is regulated by the ERK/MAPK pathway at the phosphorylation level. As an important nuclear transcription factor, HIF-1α controls the transcription of MDR-related genes and the synthesis of their corresponding proteins by ERK/MAPK signal pathway in HepG2 cells.