Personalised circulating tumour DNA assay with large-scale mutation coverage for sensitive minimal residual disease detection in colorectal cancer.

BACKGROUND: Postoperative minimal residual disease (MRD) detection using circulating-tumour DNA (ctDNA) requires a highly sensitive analysis platform. We have developed a tumour-informed, hybrid-capture ctDNA sequencing MRD assay. METHODS: Personalised target-capture panels for ctDNA detection were designed using individual variants identified in tumour whole-exome sequencing of each patient. MRD status was determined using ultra-high-depth sequencing data of plasma cell-free DNA. The MRD positivity and its association with clinical outcome were analysed in Stage II or III colorectal cancer (CRC). RESULTS: In 98 CRC patients, personalised panels for ctDNA sequencing were built from tumour data, including a median of 185 variants per patient. In silico simulation showed that increasing the number of target variants increases MRD detection sensitivity in low fractions (<0.01%). At postoperative 3-week, 21.4% of patients were positive for MRD by ctDNA. Postoperative positive MRD was strongly associated with poor disease-free survival (DFS) (adjusted hazard ratio 8.40, 95% confidence interval 3.49-20.2). Patients with a negative conversion of MRD after adjuvant therapy showed significantly better DFS (P < 0.001). CONCLUSION: Tumour-informed, hybrid-capture-based ctDNA assay monitoring a large number of patient-specific mutations is a sensitive strategy for MRD detection to predict recurrence in CRC.

Efficacy of Olaparib in Treatment-Refractory, Metastatic Breast Cancer with Uncommon Somatic BRCA Mutations Detected in Circulating Tumor DNA.

Poly(ADP-ribose) polymerase inhibitors have been shown dramatic responses in patients with BRCAness. However, clinical studies have been limited to breast cancer patients with germline mutations. Here, we describe a patient with metastatic breast cancer who had a rare BRCA1 somatic mutation (BRCA1 c.4336G>T (p.E1446*)) detected by cell-free DNA analysis after failing standard therapies. This tier III variant of unknown significance was predicted to be a pathogenic variant in our assessment, leading us to consider off-label treatment with olaparib. The patient responded well to olaparib for several months, with a decrease in allele frequency of this BRCA1 somatic mutation in cell-free DNA. Olaparib resistance subsequently developed with an increase in the allele frequency and new BRCA1 reversion mutations. To our knowledge, this is the first report confirming BRCA1 c.4336G>T (p.E1446*) as a mutation sensitive to olaparib in breast cancer and describing the dynamic changes in the associated mutations using liquid biopsy.

Differentially hypomethylated cell-free DNA and coronary collateral circulation.

BACKGROUND: The factors affecting cardioprotective collateral circulation are still incompletely understood. Recently, characteristics, such as CpG methylation of cell-free DNA (cfDNA), have been reported as markers with clinical utility. The aim of this study was to evaluate whether cfDNA methylation patterns are associated with the grade of coronary collateral circulation (CCC). RESULT: In this case-control study, clinical and angiographic data were obtained from 143 patients (mean age, 58 years, male 71%) with chronic total coronary occlusion. Enzymatic methyl-sequencing (EM-seq) libraries were prepared using the cfDNA extracted from the plasma. Data were processed to obtain the average methylation fraction (AMF) tables of genomic regions from which blacklisted regions were removed. Unsupervised analysis of the obtained AMF values showed that some of the changes in methylation were due to CCC. Through random forest preparation process, 256 differentially methylated region (DMR) candidates showing strong association with CCC were selected. A random forest classifier was then constructed, and the area under the curve of the receiver operating characteristic curve indicated an appropriate predictive function for CCC. Finally, 20 DMRs were identified to have significantly different AMF values between the good and poor CCC groups. Particularly, the good CCC group exhibited hypomethylated DMRs. Pathway analysis revealed five pathways, including TGF-beta signaling, to be associated with good CCC. CONCLUSION: These data have demonstrated that differential hypomethylation was identified in dozens of cfDNA regions in patients with good CCC. Our results support the clinical utility of noninvasively obtained epigenetic signatures for predicting collateral circulation in patients with vascular diseases.

Introduction to Single-Cell DNA Methylation Profiling Methods.

DNA methylation is an epigenetic mechanism that is related to mammalian cellular differentiation, gene expression regulation, and disease. In several studies, DNA methylation has been identified as an effective marker to identify differences between cells. In this review, we introduce single-cell DNA-methylation profiling methods, including experimental strategies and approaches to computational data analysis. Furthermore, the blind spots of the basic analysis and recent alternatives are briefly described. In addition, we introduce well-known applications and discuss future development.

Chimeric crRNAs with 19 DNA residues in the guide region show the retained DNA cleavage activity of Cas9 with potential to improve the specificity.

We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.

Whole-genome sequence analysis of Zika virus, amplified from urine of traveler from the Philippines.

Zika virus (ZIKV) (genus Flavivirus, family Flaviviridae) is an emerging pathogen associated with microcephaly and Guillain-Barré syndrome. The rapid spread of ZIKV disease in over 60 countries and the large numbers of travel-associated cases have caused worldwide concern. Thus, intensified surveillance of cases among immigrants and tourists from ZIKV-endemic areas is important for disease control and prevention. In this study, using Next Generation Sequencing, we reported the first whole-genome sequence of ZIKV strain AFMC-U, amplified from the urine of a traveler returning to Korea from the Philippines. Phylogenetic analysis showed geographic-specific clustering. Our results underscore the importance of examining urine in the diagnosis of ZIKV infection.

Identification of cystatin B as a potential serum marker in hepatocellular carcinoma.

PURPOSE: The poor survival rate of hepatocellular carcinoma (HCC) is in part due to the inability to diagnose patients at an early stage. Therefore, the aim of this study was to search for candidate serum marker for HCC and to test their ability to distinguish a HCC from benign liver disease. EXPERIMENTAL DESIGN: Genome-wide analysis by a microarray in 40 HCC patients was done between HCC and paired nontumor liver tissues. Expression of cystatin B (CSTB) was examined by mRNA expression analysis and immunohistochemistry. The serum CSTB levels were measured using a sandwich ELISA method in four groups, including normal healthy subjects (group 1, n = 52) and patients with noncirrhotic chronic hepatitis (group 2, n = 53), cirrhosis (group 3, n = 43), and HCC (group 4, n = 62). RESULTS: Microarray and statistical analyses identified 248 genes that were expressed differently between HCC and nontumor liver tissues. One of them, CSTB, was expressed preferentially in the HCCs compared with the nontumor tissues, 36 of 45 specimens (80%) by Northern blot and semiquantitative reverse transcription-PCR analyses. The serum CSTB level was much higher in HCC patients than in those with nonmalignant chronic liver disease (groups 2 and 3; P < 0.0001). The receiver operating characteristic curve indicated 5.34 ng/mL to be the optimal value for CSTB, and the sensitivity and specificity for this CSTB value were 85.5% (95% confidence interval, 74.2-93.1%) and 53.1% (95% confidence interval, 42.7-63.4%), respectively, in distinguishing between patients with HCC and those with nonmalignant chronic liver disease. CONCLUSION: CSTB is specifically overexpressed in most HCCs and is also elevated in the serum of a large proportion of HCC patients. CSTB or the combination of CSTB and alpha-fetoprotein may be a useful marker for diagnosing patients with HCC with a high sensitivity.

Association of BAF53 with mitotic chromosomes.

The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis.