Effects of Pre-analytical Variables on Cell-free DNA Extraction for Liquid Biopsy

BACKGROUND: Extraction of cell-free DNA (cfDNA) is a key step for determining the quality of cfDNA-related molecular diagnostics. We evaluated the effect of sample containers and sample storage conditions on cfDNA extraction. METHODS: The cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit from five healthy controls and five lung cancer patients was evaluated according to the type of sample container and storage conditions: K2-EDTA container, <1, 6, 24, and 48 hr storage at 4℃ after immediate plasma separation; and Cell-Free DNA BCT container, <1, 3, 7, and 14 days stored at room temperature. Mutation analysis of EGFR exons 18–21 was performed. To assess the effect of a delay in centrifugation, EDTA whole blood samples from five healthy individuals were stored at 4℃ for 6, 12, and 24 hr before plasma separation. RESULTS: There was no significant difference in the amount and nucleic acid size of cfDNA in both controls and patients with cancer when EDTA plasma was stored at 4℃ up to 48 hr. The amount and size of cfDNA in the BCT container were not different up to 7 days; however, the 14-day sample showed an increase in cfDNA concentration due to genomic DNA contamination. EGFR mutations were detected on EDTA containers up to 48 hr and with BCT containers up to 14 days. When EDTA whole blood was stored at 4℃ and plasma separation was delayed, the cfDNA concentration increased from 24 hr. CONCLUSIONS: The cfDNA extraction was affected by the sample containers and storage conditions.

Nitric Oxide-Induced Apoptosis of Human Dental Pulp Cells Is Mediated by the Mitochondria-Dependent Pathway

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway.

CT-based quantitative evaluation of radiation-induced lung fibrosis: a study of interobserver and intraobserver variations

PURPOSE: The degree of radiation-induced lung fibrosis (RILF) can be measured quantitatively by fibrosis volume (VF) on chest computed tomography (CT) scan. The purpose of this study was to investigate the interobserver and intraobserver variability in CT-based measurement of VF. MATERIALS AND METHODS: We selected 10 non-small cell lung cancer patients developed with RILF after postoperative radiation therapy (PORT) and delineated VF on the follow-up chest CT scanned at more than 6 months after radiotherapy. Three radiation oncologists independently delineated VF to investigate the interobserver variability. Three times of delineation of VF was performed by two radiation oncologists for the analysis of intraobserver variability. We analysed the concordance index (CI) and inter/intraclass correlation coefficient (ICC). RESULTS: The median CI was 0.61 (range, 0.44 to 0.68) for interobserver variability and the median CIs for intraobserver variability were 0.69 (range, 0.65 to 0.79) and 0.61(range, 0.55 to 0.65) by two observers. The ICC for interobserver variability was 0.974 (p < 0.001) and ICCs for intraobserver variability were 0.996 (p < 0.001) and 0.991 (p < 0.001), respectively. CONCLUSION: CT-based measurement of VF with patients who received PORT was a highly consistent and reproducible quantitative method between and within observers.

Parotid gland sparing effect by computed tomography-based modified lower field margin in whole brain radiotherapy

PURPOSE: Parotid gland can be considered as a risk organ in whole brain radiotherapy (WBRT). The purpose of this study is to evaluate the parotid gland sparing effect of computed tomography (CT)-based WBRT compared to 2-dimensional plan with conventional field margin. MATERIALS AND METHODS: From January 2008 to April 2011, 53 patients underwent WBRT using CT-based simulation. Bilateral two-field arrangement was used and the prescribed dose was 30 Gy in 10 fractions. We compared the parotid dose between 2 radiotherapy plans using different lower field margins: conventional field to the lower level of the atlas (CF) and modified field fitted to the brain tissue (MF). RESULTS: Averages of mean parotid dose of the 2 protocols with CF and MF were 17.4 Gy and 8.7 Gy, respectively (p or =20 Gy were observed in 15 (28.3%) for CF and in 0 (0.0%) for MF. The whole brain percentage volumes receiving >98% of prescribed dose were 99.7% for CF and 99.5% for MF. CONCLUSION: Compared to WBRT with CF, CT-based lower field margin modification is a simple and effective technique for sparing the parotid gland, while providing similar dose coverage of the whole brain.

Neurogenic Bladder in Lyme Disease / 대한배뇨장애요실금학회지

Lyme disease is a multi-systemic, tick-borne infectious disease caused by a spirochete, Borrelia burgdorferi. Various urologic symptoms are associated with Lyme disease, which can be primary or late manifestations of the disease. Although voiding dysfunction is a rarely reported symptom in patients with Lyme disease, it is one of the most disabling complications of Lyme disease. Korea is not an endemic area of Lyme disease, thus, fewer cases have been reported. Herein, we report a case of a 32-year-old man with rapidly progressive bilateral ptosis, dysphagia, spastic paraparesis, and voiding difficulty in whom Lyme disease was diagnosed through serologic tests for antibodies and Western blot testing. A urodynamic study demonstrated detrusor areflexia and bulbocavernosus reflex tests showed delayed latency, indicating demyelination at S2-S4 levels. He received a 4-week course of intravenous ceftriaxone (2 g/day). The patient has recovered from the bilateral ptosis and spastic paraparesis but still suffers from neurogenic bladder.

Factors Influencing Readmission of Home Ventilator-Assisted Children

PURPOSE: This study was conducted to analyze factors affecting readmission of children with home ventilator care. METHODS: To collect patient data, a retrospective chart review was done of medical records of children admitted between June 1, 2007 and May 31, 2010 at one children's hospital located in Seoul. During that period 30 children were discharged with a home ventilator. RESULTS: Twenty-one of these children had a total of 63 readmissions during the study period, averaging 2.1 readmissions per child with a mean duration of hospitalization of 7.4 days. Children with nasogastric tubes were more frequently readmitted (t=7.232, p=.012) and duration of hospitalization was significantly longer (t=4.761, p=.038). Children who had cardio-pulmonary comorbidity were more frequently readmitted and had longer hospitalization than children without comorbidity (t=5.444, p=.027). When home ventilator assisted children were admitted via emergency room, they were hospitalized longer (t=14.686, p<.001). Cardio-pulmonary morbidity and readmission via ER explained 38.1% of variation for readmission. Feeding method explained 15.0% of variation in length of hospitalization. CONCLUSION: The results suggest that health care providers must give individualized education on home ventilator care to parents with children who are at risk for readmission due to cardio-pulmonary comorbidities, nasogastric tube, or readmission via ER.

Analysis of an Association between Single Nucleotide Polymorphisms of Vascular Endothelial Growth Factor Gene and Risk of Cerebral White Matter Lesion

BACKGROUND: Endothelial dysfunction is suggested to be one of the pathogenesis of cerebral white matter lesion (cWML). Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and integrity of vascular endothelial cell, and altered expression of VEGF gene induces vascular diseases including cerebrovascular diseases. The objective of this study is to investigate whether single nucleotide polymorphism (SNP) of VEGF gene confers an increased risk of cWML. METHODS: Total 337 study subjects without history of stroke were included. The presence and severity of cWML were measured on fluid-attenuated inversion recovery image. Genotypes of VEGF -2578G>A, -1154G>A, -634G>C and +936C>T were analyzed. RESULTS: Among 337 study subjects, cWML was found in 208 patients (62%), and fifty-eight cases (17%) of them had overt cWML. In univariate analysis, age, female sex and plasma total homocysteine level (tHcyt) were higher in the mild and overt cWML group than no cWML group (p<0.05). The percentage of previous history of hypertension and the value of systolic blood pressure were higher in overt cWML group than no cWML group. In univariate and logistic regression analysis, none of four tested VEGF SNPs was significantly different between control group, mild and overt cWML groups. There was no difference between plasma tHcyt levels and each VEGF SNPs in control group and cWML groups. CONCLUSIONS: In this study, old age, female sex, hypertension and plasma tHcyt were associated with cWML. However, we failed to find an association between cWML and VEGF gene polymorphism, which may indicate that genetic polymorphism of VEGF does not play a direct role in the pathogenesis of cWML.

Knowledge, Self-Efficacy, and Perceived Barriers on the Low-Iodine Diet among Thyroid Cancer Patients Preparing for Radioactive Iodine Therapy

The purposes of the study were to assess knowledge, self-efficacy, and perceived barriers on a low-iodine diet among thyroid cancer patients and to identify strategies for nutrition education. A self-administered questionnaire was developed based on a review of literature and pilot-tested. A total of 121 female thyroid cancer patients participated in a survey and 117 responses were used for data analysis. An average knowledge score of the thyroid cancer patients was 4.5 point (available score: 0-10 point). Majority of the respondents knew that seaweeds such as lavers, brown seaweeds, and sea tangles contain large amount of iodine. However they mistook the low iodine diet as a low salt diet and were not aware of foods and seasonings that are allowed on the low iodine diet. While self-efficacy related to consuming various fruits and vegetables, to choosing potatoes and sweet potatoes for snacks, and restricting consumption of eggs, milk and milk products, and processed foods was rated highly, self-efficacy for preparing foods without using sea salts was rated low. The self-efficacy score increased as their interest on the dietary life and perceived cooking skills were greater. Most perceived barriers toward practicing the low iodine diet were related to preparation of the low iodine menus. As their interest in the dietary life and cooking and perceived cooking skills were greater, the patients perceived barriers on practicing the low iodine diet less. While the patients showed higher self-efficacy and lower barrier perception on selecting foods low in iodine and restricting food high in iodine, they showed lower self-efficacy and higher barrier perceptions on preparing low iodine meals. Clinical dietitians should recognize the gap between what the patients should know and what they really know and identify strategies on how to improve self-efficacy and reduce perceived barriers on the low iodine diet. Recent literature and the findings of the study reveal that incorporating cooking classes into nutrition education for thyroid patients is effective to enhance self-efficacy and to reduce perceived barriers on the low iodine diet.

Association between Single Nucleotide Polymorphisms of alpha2A-, alpha2B-, and alpha2C-Adrenergic Receptor Genes and Risk of Cerebral White Matter Lesion / 대한뇌졸중학회지

BACKGROUND: An alpha2-adrenergic receptor (alpha2-AR, ADRA2) mediates induction of hypotension and inhibition of lipolysis and insulin secretion. We evaluated whether single nucleotide polymorphisms (SNPs) of alpha2A (ADRA2A), alpha2B (ADRA2B), and alpha2C (ADRA2C) adrenergic receptors are associated with cerebral white matter lesion (cWML). METHODS: Total 336 study subjects who had no stroke were enrolled in this study. The Indices of cWML include total WML (TWML), periventricular WML (PVWML), and subcortical WML (SCWML) on brain fluid-attenuated inversion recovery (FLAIR) image. Common genetic variants of ADRA2A (1780G>A), ADRA2B (Ins/Del301-303), and ADRA2C (Ins/Del322-325) were examined. RESULTS: Among 336 study subjects, cWML was found in 66 patients (20%). In multivariate analysis, there were no significant effects of all tested ADRA2 polymorphisms on TWML. Significant association of ADRA2A 1780 AA genotype was found in PVWML (OR: 3.368, 95% CIs: 1.280-8.865, adjusted p-value after false discovery rate (FDR) correction=0.014) but not SCWML. CONCLUSION: Although SNPs of three ADRA2 subtypes failed to reach a significance in overall risk for cWML, the ADRA2A 1780G>A polymorphism may be associated with development of PVWML.

Difference in the Set-up Margin between 2D Conventional and 3D CT Based Planning in Patients with Early Breast Cancer / 대한방사선종양학회지

PURPOSE: Simulation using computed tomography (CT) is now widely available for radiation treatment planning for breast cancer. It is an important tool to help define the tumor target and normal tissue based on anatomical features of an individual patient. In Korea, most patients have small sized breasts and the purpose of this study was to review the margin of treatment field between conventional two-dimensional (2D) planning and CT based three-dimensional (3D) planning in patients with small breasts. MATERIALS AND METHODS: Twenty-five consecutive patients with early breast cancer undergoing breast conservation therapy were selected. All patients underwent 3D CT based planning with a conventional breast tangential field design. In 2D planning, the treatment field margins were determined by palpation of the breast parenchyma (In general, the superior: base of the clavicle, medial: midline, lateral: mid - axillary line, and inferior margin: 2 m below the inframammary fold). In 3D planning, the clinical target volume (CTV) ought to comprise all glandular breast tissue, and the PTV was obtained by adding a 3D margin of 1 cm around the CTV except in the skin direction. The difference in the treatment field margin and equivalent field size between 2D and 3D planning were evaluated. The association between radiation field margins and factors such as body mass index, menopause status, and bra size was determined. Lung volume and heart volume were examined on the basis of the prescribed breast radiation dose and 3D dose distribution. RESULTS: The margins of the treatment field were smaller in the 3D planning except for two patients. The superior margin was especially variable (average, 2.5 cm; range, -2.5 to 4.5 cm; SD, 1.85). The margin of these targets did not vary equally across BMI class, menopause status, or bra size. The average irradiated lung volume was significantly lower for 3D planning. The average irradiated heart volume did not decrease significantly. CONCLUSION: The use of 3D CT based planning reduced the radiation field in early breast cancer patients with small breasts in relation to conventional planning. Though a coherent definition of the breast is needed, CT-based planning generated the better plan in terms of reducing the irradiation volume of normal tissue. Moreover it was possible that 3D CT based planning showed better CTV coverage including postoperative change.

Limbic Encephalitis Caused by Human Herpesvirus-6 Infection in Allogenic Hematopoietic Stem Cell Transplantation Patient

No abstract available.

Effects of Arc Number or Rotation Range upon Dose Distribution at RapidArc Planning for Liver Cancer / 의학물리

In this paper, we evaluated the performance of 3D CRT, IMRT and three kind of RA plannings to investigate the clinical effect of RA with liver cancer case. The patient undergoing liver cancer of small volume and somewhat constant motion were selected. We performed 3D CRT, IMRT and RA plannings such as 2RA, limited triple arcs (3RA) and 3MRA with Eclipse version 8.6.15. The same dose volume objectives were defined for only CTV, PTV and body except heart, liver and partial body in IMRT and RA plannings. The steepness of dose gradient around tumor was determined by the Normal Tissue Objective function with the same parameters in place of respective definitions of dose volume objectives for the normal organs. The approach between the defined dose constraints and the practical DVH of CTV, PTV and Body was the best in 3MRA and the worst in IMRT. The DVHs were almost the same among RAs. Plans were evaluated using Conformity Index (CI), Homogeneity Index (HI) and Quality of coverage (QoC) by RTOG after prescription with dose level surrounding 98% of PTV in the respective plans. As a result, 3MRA planning showed the better favorable indices than that of the others and achieved the lowest MUs. In this study, RA planning is a technique that is possible to obtain the faster and better dose distribution than 3D CRT or IMRT techniques. Our result suggest that 3MRA planning is able to reduce the MUs further, keeping a similar or better targer dose homogeneity, conformity and sparing normal tissue than 2RA or 3RA.

First Clinical Experience about RapidArc Treatment with Prostate Cancer in Ajou University Hospital / 의학물리

In this study, the patient with localized prostate cancer who had previously been treated at Ajou University Hospital was randomly selected since March, 2009. we performed IMRT and 2RA plans and the same dose objectives were used for CTVs, PTVs, rectum, bladder, and femoral head of the respective plans. Arc optimizations and dose calculations were performed using Eclipse versions 8.6. In this paper, we evaluated the performance of IMRT and RA plans to investigate the clinical effect of RA for prostate cancer case. In our comparison of treatment techniques, RA was found to be superior to IMRT being better dose conformity of target volume. As for the rectum and bladder, RA was better than IMRT at decreasing the volume irradiated. RA has the ability to avoid critical organs selectively through applied same dose constraints while maximally treating the target dose. Therefore, this result suggests that there should be less rectal toxicity with RA compared with IMRT, with no compromise in tumor margin. These findings, which show more favorable rectal, bladder, and femoral head DVHs with RA, imply that should not result in excess risk of toxicity when this technique is used. Many experiences with RA have shown not only dosimetric advantage, but also improved clinical toxicity when comparing with IMRT. The main drawbacks of RA are the more complex and time-consuming treatment planning process and the need for more exact physics quality assurance (QA).

Orbital Myositis of the Lateral Rectus Muscle Presenting With Adduction Impairment

No abstract available.

Ape1/Ref-1 Stimulates GDNF/GFR alpha1-mediated Downstream Signaling and Neuroblastoma Proliferation

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor alpha1 (GFR alpha1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRalpha1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFR alpha1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRalpha1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFR alpha1-specific RNA experiments demonstrated that the downregulation of GFR alpha1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLC gamma-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFR alpha signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.

Deletion of Y-Chromosome Specific Genes in Testicular Germ Cell Tumor / 대한비뇨기과학회지

PURPOSE: We hypothesized that the deletion of Y-chromosome specific genes is associated with testicular tumors. To test this hypothesis, we analyzed the expressions of five Y-chromosome specific genes in testicular tumor samples. MATERIALS AND METHODS: Thirty-five human testicular tumor specimens were processed for the micro-dissection of pure epithelial cells. The DNA was extracted, and polymerase chain reactions performed using five different specific primers (ZFY, DYS203, SMCYM45, GDB187507 and RH38676). These primers were designed based on sequences available in the public genome data bank. RESULTS: Deletion was observed in 88.6% of the testicular tumor specimens with at least one Y-chromosome specific gene. The losses of DYS203, ZFY, SMCYM45, RH38676 and GDB187507 were shown in 51.4, 42.9, 40, 28.6 and 20% of cases, respectively. There was a different pattern of loss of the Y-chromosome specific genes according to the histologic types of germ cell tumor. The losses of the DYS203 and GDB187507 genes were seen more frequently in the advanced stages. CONCLUSIONS: There was a significant loss of the Y-chromosome specific genes in testicular germ cell tumors. The loss of the DYS203 gene was observed in about half the cases, and was more frequent in the higher stages of testicular tumor. The loss of Y-chromosome specific genes is associated with testicular tumors, suggesting their role in the pathogenesis of this disease.

Pattern of Exon Deletions of Dystrophin Gene in Korean Patients with Duchenne Muscular Dystrophy

OBJECTIVE: To investigate the pattern of exon deletions in Korean patients with Duchenne muscular dystrophy (DMD), and to find the correlation of the exon-deletion with clinical symptoms or laboratory findings. METHOD: Genomic DNA of the nine children with DMD were analyzed by the sets of multiplex PCR and one singlet PCR in total of fifteen primers of the dystrophin gene. The primers were made from the promotor, and the exons 3, 4, 6, 8, 12, 13, 43, 44, 47, 48, 50, 51, 52 and 60 of the dystrophin gene, respectively. RESULTS: Eight out of nine patients revealed exon deletions. The exon 3 was most commonly deleted (6 patients), and exon 48, 50 and 60 were second most common (2 patients). The exons 4, 6, 13, 44, 47 and 52 were not deleted in all patients. CONCLUSION: We found that the exons 3, 48, 50 and 60 are frequently deleted in Korean patients with DMD. The pattern of deletion was not correlate with clinical symptoms or laboratory findings.

Clinicopathologic Characteristics of Replication Error-Positive Gastric Adenocarcinoma in Korean

The purpose of this study is to obtain the clinicopathological characteristics of replication error-positive (RER ) gastric adenocarcinoma in Korean, and to identify the significance of RER in adenoma stage of gastric carcinogenesis. Microsatellite instability was examined at D2S71, D2S119, D3S1067, D6S87, D11S905, DM, AR, VWF, HPRT, and BAT-26 loci. Frameshift mutation of BAX gene was analyzed in RER tumors. Normal and tumor DNA of 76 cases of gastric carcinoma and 25 cases of adenoma were examined. RER was found in 8 of 76 cases (10.5%), and it was more frequently found in adenocarcinoma of female (17.7%) than those of male (4.8%). The frequency of RER was not different between the histologic types, age of the patient, anatomical location of the carcinoma, and the stage. The RER found in adenoma suggests that RER contributes to the malignant transformation early in the adenoma stage of the gastric carcinogenesis. None of the RER tumors revealed frameshift mutation of the BAX gene.

Commissionning of Dynamic Wedge Field Using Conventional Dosimetric Tools / 대한치료방사선과학회지

PURPOSE: To collect beam data for dynamic wedge fields using conventional measurement tools without the multi-detector system, such as the linear diode detectors or ionization chambers. MATERIALS AND METHODS: The accelerator CL 2100 C/D has two photon energies of 6MV and 15MV with dynamic wedge angles of 15o, 30o, 45o and 60o. Wedge transmission factors, percentage depth doses(PDD's) and dose profiles were measured. The measurements for wedge transmission factors are performed for field sizes ranging from 4x4cm2 to 20x20cm2 in 1-2cm steps. Various rectangular field sizes are also measured for each photon energy of 6MV and 15MV, with the combination of each dynamic wedge angle of 15o, 30o, 45o and 60o. These factors are compared to the calculated wedge factors using STT(Segmented Treatment Table) value. PDD's are measured with the film and the chamber in water phantom for fixed square field. Converting parameters for film data to chamber data could be obtained from this procedure. The PDD's for dynamic wedged fields could be obtained from film dosimetry by using the converting parameters without using ionization chamber. Dose profiles are obtained from interpolation and STT weighted superposition of data through selected asymmetric static field measurement using ionization chamber. RESULTS: The measured values of wedge transmission factors show good agreement to the calculated values. The wedge factors of rectangular fields for constant Y-field were equal to those of square fields. The differences between open fields' PDDs and those from dynamic fields are insignificant. Dose profiles from superposition method showed acceptable range of accuracy(maximum 2% error) when we compare to those from film dosimetry. CONCLUSION: The results from this superposition method showed that commissionning of dynamic wedge could be done with conventional dosimetric tools such as point detector system and film dosimetry winthin maximum 2% error range of accuracy.