PureCN: copy number calling and SNV classification using targeted short read sequencing

Background

Matched sequencing of both tumor and normal tissue is routinely used to classify variants of uncertain significance (VUS) into somatic vs. germline. However, assays used in molecular diagnostics focus on known somatic alterations in cancer genes and often only sequence tumors. Therefore, an algorithm that reliably classifies variants would be helpful for retrospective exploratory analyses. Contamination of tumor samples with normal cells results in differences in expected allelic fractions of germline and somatic variants, which can be exploited to accurately infer genotypes after adjusting for local copy number. However, existing algorithms for determining tumor purity, ploidy and copy number are not designed for unmatched short read sequencing data.

Results

We describe a methodology and corresponding open source software for estimating tumor purity, copy number, loss of heterozygosity (LOH), and contamination, and for classification of single nucleotide variants (SNVs) by somatic status and clonality. This R package, PureCN, is optimized for targeted short read sequencing data, integrates well with standard somatic variant detection pipelines, and has support for matched and unmatched tumor samples. Accuracy is demonstrated on simulated data and on real whole exome sequencing data.

Conclusions

Our algorithm provides accurate estimates of tumor purity and ploidy, even if matched normal samples are not available. This in turn allows accurate classification of SNVs. The software is provided as open source (Artistic License 2.0) R/Bioconductor package PureCN (http://bioconductor.org/packages/PureCN/).

     

Predicting Prostate Biopsy Outcomes: A Preliminary Investigation on Screening with Ultrahigh B-Value Diffusion-Weighted Imaging as an Innovative Diagnostic Biomarker

Background

Routine screening of prostate specific antigen (PSA) is no longer recommended because of a high rate of over-diagnosis of prostate cancer (PCa).

Objective

To evaluate the efficacy of diffusion-weighted magnetic resonance imaging (DW-MRI) for PCa detection, and to explore the clinical utility of ultrahigh b-value DW-MRI in predicting prostate biopsy outcomes.

Methodology

73 male patients were selected for the study. They underwent 3T MRI using T2WI conventional DW-MRI with b-value 1000 s/mm², and ultrahigh b-value DW-MRI with b-values of 2000 s/mm² and 3000 s/mm². Two radiologists evaluated individual prostate gland images on a 5-point rating scale using PI-RADS, for the purpose of region-specific comparisons among modalities. Sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratios (LR) were investigated for each MRI modality. The area under the receiver operating characteristic (ROC) curve (AUC) was also calculated.

Results

Results showed the improved diagnostic value of ultrahigh b-value DWI-MRI for detection of PCa when compared to other b values and conventional MRI protocols. Sensitivity values for 3000 s/mm² in both peripheral zone (PZ) and transition zone (TZ) were significantly higher than those observed with conventional DW-MRI—Specificity values for 3000 s/mm² in the TZ were significantly higher than other b-value images, whereas specificity values using 3000 s/mm² in the PZ were not significantly higher than 2000 s/mm² images. PPV and NPV between 3000 s/mm² and the other three modalities were significantly higher for both PZ and TZ images. The PLRs and NLRs of b-value 3000 s/mm² DW-MRI in the PZ and TZ were also recorded. ROC analysis showed greater AUCs for the b value 3000 s/mm² DWI than for the other three modalities.

Conclusions

DW-MRI with a b-value of 3000 s/mm² was found to be the most accurate and reliable MRI modality for PCa tumor detection and localization, particularly for TZ lesion discrimination. It may be stated that the b-value of 3000 s/mm² is a novel, improved diagnostic biomarker with greater predictive accuracy for PCa prior to biopsy.     

Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease

Background

High resolution melting (HRM) is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population.

Methods

A total of 315 blood samples from 147 CHD patients (male72, female 75) and 168 healthy controls (male 92, female 76) were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs) from unconditional logistic regression.

Results

All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%.

Conclusion

MTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping.     

Effect of Immunotherapy on Seizure Outcome in Patients with Autoimmune Encephalitis: A Prospective Observational Registry Study

Objective

To evaluate the seizure characteristics and outcome after immunotherapy in adult patients with autoimmune encephalitis (AE) and new-onset seizure.

Methods

Adult (age ≥18 years) patients with AE and new-onset seizure who underwent immunotherapy and were followed-up for at least 6 months were included. Seizure frequency was evaluated at 2–4 weeks and 6 months after the onset of the initial immunotherapy and was categorized as “seizure remission”, “> 50% seizure reduction”, or “no change” based on the degree of its decrease.

Results

Forty-one AE patients who presented with new-onset seizure were analysed. At 2–4 weeks after the initial immunotherapy, 51.2% of the patients were seizure free, and 24.4% had significant seizure reduction. At 6 months, seizure remission was observed in 73.2% of the patients, although four patients died during hospitalization. Rituximab was used as a second-line immunotherapy in 12 patients who continued to have seizures despite the initial immunotherapy, and additional seizure remission was achieved in 66.6% of them. In particular, those who exhibited partial response to the initial immunotherapy had a better seizure outcome after rituximab, with low adverse events.

Conclusion

AE frequently presented as seizure, but only 18.9% of the living patients suffered from seizure at 6 months after immunotherapy. Aggressive immunotherapy can improve seizure outcome in patients with AE.     

Cerebral Glioma Grading Using Bayesian Network with Features Extracted from Multiple Modalities of Magnetic Resonance Imaging

Many modalities of magnetic resonance imaging (MRI) have been confirmed to be of great diagnostic value in glioma grading. Contrast enhanced T1-weighted imaging allows the recognition of blood-brain barrier breakdown. Perfusion weighted imaging and MR spectroscopic imaging enable the quantitative measurement of perfusion parameters and metabolic alterations respectively. These modalities can potentially improve the grading process in glioma if combined properly. In this study, Bayesian Network, which is a powerful and flexible method for probabilistic analysis under uncertainty, is used to combine features extracted from contrast enhanced T1-weighted imaging, perfusion weighted imaging and MR spectroscopic imaging. The networks were constructed using K2 algorithm along with manual determination and distribution parameters learned using maximum likelihood estimation. The grading performance was evaluated in a leave-one-out analysis, achieving an overall grading accuracy of 92.86% and an area under the curve of 0.9577 in the receiver operating characteristic analysis given all available features observed in the total 56 patients. Results and discussions show that Bayesian Network is promising in combining features from multiple modalities of MRI for improved grading performance.     

Covered versus Uncovered Self-Expandable Metal Stents for Managing Malignant Distal Biliary Obstruction: A Meta-Analysis

Aim

To compare the efficacy of using covered self-expandable metal stents (CSEMSs) and uncovered self-expandable metal stents (UCSEMSs) to treat objective jaundice caused by an unresectable malignant tumor.

Methods

We performed a comprehensive electronic search from 1980 to May 2015. All randomized controlled trials comparing the use of CSEMSs and UCSEMSs to treat malignant distal biliary obstruction were included.

Results

The analysis included 1417 patients enrolled in 14 trials. We did not detect significant differences between the UCSEMS group and the CSEMS group in terms of cumulative stent patency (hazard ratio (HR) 0.93, 95% confidence interval (CI) 0.19–4.53; p = 0.93, I² = 0%), patient survival (HR 0.77, 95% CI 0.05–10.87; p = 0.85, I² = 0%), overall stent dysfunction (relative ratio (RR) 0.85, M-H, random, 95% CI 0.57–1.25; p = 0.83, I² = 63%), the overall complication rate (RR 1.26, M-H, fixed, 95% CI 0.94–1.68; p = 0.12, I² = 0%) or the change in serum bilirubin (weighted mean difference (WMD) -0.13, IV fixed, 95% CI 0.56–0.3; p = 0.55, I² = 0%). However, we did detect a significant difference in the main causes of stent dysfunction between the two groups. In particular, the CSEMS group exhibited a lower rate of tumor ingrowth (RR 0.25, M-H, random, 95% CI 0.12–0.52; p = 0.002, I² = 40%) but a higher rate of tumor overgrowth (RR 1.76, M-H, fixed, 95% CI 1.03–3.02; p = 0.04, I² = 0%). Patients with CSEMSs also exhibited a higher migration rate (RR 9.33, M-H, fixed, 95% CI 2.54–34.24; p = 0.008, I² = 0%) and a higher rate of sludge formation (RR 2.47, M-H, fixed, 95% CI 1.36–4.50; p = 0.003, I² = 0%).

Conclusions

Our meta-analysis indicates that there is no significant difference in primary stent patency and stent dysfunction between CSEMSs and UCSEMSs during the period from primary stent insertion to primary stent dysfunction or patient death. However, when taking further management for occluded stents into consideration, CSEMSs is a better choice for patients with malignant biliary obstruction due to their removability.     

尼罗罗非鱼染色体的C带、Ag带和减数分裂联会复合体的研究

以Sumner法和界面铺张——硝酸银技术,对尼罗罗非鱼(Tilapia nilotica)染色体C带、Ag染带及减数分裂前期精母细胞联会复合体(SC)进行了显微和亚显微结构观察。 尼罗罗非鱼的2n=44,核型可分为三个组:第一组为4对亚中着丝粒染色体;第二组为17对亚端着丝粒染色体;第三组为具1对端着丝粒的特大染色体。 结构异染色质主要分布于着丝粒附近,其中Nos.6、8、15亚中着丝粒染色体短臂全部深染。带有银染核仁组织者(Ag-NORs)染色体的数目为2—6条,NORs均位于6、8、15亚中着丝粒染色体短臂。 银染色可清楚地显示尼罗罗非鱼的联会复合体(SC)结构和减数分裂行为。SC组型与有丝分裂染色体的组型有较好的一致性。     

疏花水柏枝幼苗生物量与构件对模拟土壤地下水位变化的响应

通过模拟不同地下水位的方法,对疏花水柏枝（Myricaria laxiflora（Franch.）P.Y.Zhang et Y.J.Zhang）一年生幼苗在不同条件下地上与地下部生物量及构件的变化进行测定,分析幼苗生长对地下水位变化的响应。结果显示：随着地下水位的降低,疏花水柏枝幼苗的生长特征指标均呈先增加后减少的趋势,其中地上、地下部分生物量的最高值分别为0.0438、0.0100 g,最低值分别为0.0177、0.0026 g。幼苗地上部生物量在-10 cm处理水平最高;地下部生物量在-15 cm处理水平最高。幼苗直径、根表面积、株高、主根长度、根体积、一级枝数、二级枝数等指标也分别在-10 cm或-15 cm处理水平达到最高值。疏花水柏枝幼苗主要构件的生长与地下水位的变化存在显著相关性。主成分分析结果表明,幼苗的地下部分更容易受到土壤地下水位变化的影响,幼苗性状症候群随地下水位的变化而发生移动,说明该物种幼苗在不同地下水位时的生长投资策略具有较大差异。     

The effect of in vivo treatment with triiodothyronine on the in vitro synthesis of protein-poly(ADP)-ribose adducts by isolated cardiocyte nuclei and the separation of poly(ADP)-ribosylated proteins by phenol extraction and electrophoresis

In vivo treatment of rats with triiodothyronine (30 micrograms/100 g of body weight for 4 consecutive days) inhibited poly(ADP)-ribose polymerase activity of cardiocyte nuclei, but low enzymatic activity of nuclei of noncardiocyte origin remained unaffected. RNA synthesis in cardiocyte nuclei isolated from triiodothyronine-treated rats was augmented. A positive correlation was observed between the degree of inhibition of poly(ADP)-ribose polymerase and cardiac ventricular enlargement in triiodothyronine-treated animals. RNA synthesis in isolated cardiocyte nuclei was inhibited by in vitro poly(ADP)-ribosylation only when cardiocyte nuclei were obtained from triiodothyronine-treated animals. In vitro poly(ADP)-ribosylated proteins were isolated from cardiocyte nuclei by solvent partitioning between phenol and aqueous phases. About 90% of the protein-poly(ADP)-ribose adducts partitioned into the aqueous fraction, and the chain length of polymers in this phase was between medium (n = 4-9) and long (n greater than 32), whereas the phenol phase contained protein-oligomer and monomer adducts. Not only the chain length of oligomers but the nature of modified proteins appeared to participate in determining the partitioning of polymer-protein adducts, and different proteins were separated from the two phases by gel electrophoresis. More than 90% of protein-polymer adducts formed by cardiocyte nuclei were not extracted by 0.25 N HCl, indicating prevalence of nonhistone proteins as polymer acceptors. Gel electrophoresis and near quantitative recovery of adducts in a gel system that protected from degradation of adducts to free polymers confirmed the predominance of nonhistone proteins as main acceptors and demonstrated an artifact of autoradiography that seemed to indicate histone H1 as a significant acceptor. Treatment with triiodothyronine diminished poly(ADP)-ribosylation of certain groups of proteins more than others, implying some degree of selectivity of action of the hormone. Catabolism of the polymer in vitro was not affected by triiodothyronine treatment.     

Computed tomography in the diagnosis of malignant renal neoplasms and their dissemination]

CT, performed in 66 patients with suspected renal tumors, showed renal cell carcinoma in 36. Tumor spreading was correctly established in 80.6%. Accurate diagnosis was made in 64 of 66 cases. The authors regard CT as an effective method for the recognition of sizable processes and differential diagnosis of solid tumors. Among the visual methods of investigation, used to define a tumor stage, CT turned out to be the most effective one.