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Objective Patients with HER2-positive metastatic breast cancer (MBC) benefit from trastuzumab-based therapy but eventually develop intrinsic or acquired resistance. Whether plasma HER2 gene copy number (GCN) could predict survival after trastuzumab treatment remained controversial. We evaluated the prognostic value of plasma HER2 GCN using low-coverage whole-genome sequencing (LC-WGS). Methods The plasma was collected from HER2-positive MBC patients whose pre-therapeutic samples were available before first-line trastuzumab-based treatment. Plasma DNA was extracted and assessed by LC-WGS for HER2 GCN. The optimal cut-off point for HER2 GCN to shorter survival was determined by receiver operating characteristic (ROC) curve analysis. Results A total of 49 patients were retrieved from 2013 to 2017, among whom 21 had multiple organ involvement (≥3 sites). Variations of HER2 GCN in pre-therapeutic plasma ranged from 1.89 to 23.86 (median = 2.59). ROC analysis identified the optimal cut-off point for HER2 GCN as 2.82 (P = 0.005), with 23 patients had high-level HER2 GCN and 26 in the low-level group. Both progression-free survival (PFS, P = 0.032) and overall survival (OS, P = 0.006) were adversely associated with high-level HER2 GCN. In multivariate analyses, high HER2 GCN was independently associated with shorter PFS [hazard ratio (HR) = 2.042, P = 0.037], while both high HER2 GCN (HR = 4.909, P = 0.004) and more metastatic organs (HR = 4.019, P = 0.011) were negative prognostic factors for OS. Conclusion In this population of patients with HER2-positive MBC, individuals with high HER2 GCNs in plasma had worse prognosis after trastuzumab-based therapy. Plasma HER2 GCN may be a prognostic marker in these patients.

Lianyuan Tao,1,2 Li Su,3 Chunhui Yuan,1 Zhaolai Ma,1 Lingfu Zhang,1 Shiping Bo,4 Yunyun Niu,4 Sijia Lu,4 Dianrong Xiu11Department of General Surgery, Peking University Third Hospital, Beijing 100191, People’s Republic of China; 2Department of Hepatobiliary Surgery, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, School of Clinical Medicine, Henan University, Zhengzhou, Henan 450003, People’s Republic of China; 3Medical and Health Analytical Center, Peking University Health Science Center, Beijing 100191, People’s Republic of China; 4Department of Clinical Research, Yikon Genomics Co. Ltd., Shanghai, People’s Republic of ChinaPurpose: The aim of this study was to evaluate the value of flow cytometry (FCM) detection of portal vein circulating tumor cells (CTCs) in predicting postoperative metastasis.Methods: Samples of portal venous blood and peripheral blood were collected from 39 patients during surgery, and CTCs were detected by FCM, with confirmation by laser confocal microscopy and single-cell sequencing.Results: Among all patients, a portal EpCAM+CD45- percentage ≥24.5×10−4 (P=0.06), peripheral EpCAM+CD45- count ≥97/5 mL (P=0.034), peripheral EpCAM+CD45- percentage ≥4.4×10−4 (P=0.042), and CA242≥3.5U/mL (P=0.027) were significant predictors of metastasis. Further analysis showed that the portal EpCAM+CD45- ratio ≥24.5×10−4 is a predictor of metastasis (P=0.025) in pancreatic cancer after curative resection.Conclusion: CTCs detected by FCM in portal venous blood are of significant value for the prediction of postoperative metastasis in pancreatic or periampullary tumors.Keywords: circulating tumor cells, flow cytometry, portal vein, pancreatic cancer, metastases &nbsp

Background HER2 status is significant to trastuzumab therapy; however, it is difficult to determine HER2 status accurately with few pieces of biopsies from advanced gastric cancer (AGC) due to highly heterogeneity and invasive behaviour, which will be investigated in this study. Methods Fifty-six patients with AGC were included in this study. Primary tumour tissues and matched plasmas before medication from 36 patients were retrospectively collected, and the other 20 patients with primary tumour tissues and paired plasmas were prospectively collected. HER2 expression and amplification in 56 tumour tissues were determined by immunohistochemistry (IHC) and dual in situ hybridisation (DISH), and HER2 copy number in 135 circulating tumour DNAs (ctDNAs) was judged by next-generation sequencing. Results For tumour tissues, HER2 amplification by DISH was most commonly found in patients with HER2 score 3+by IHC. For plasmas, HER2 amplification defined as HER2 copy number >2.22 was identified in 26 of 56 patients. There was a high concordance of HER2 amplification between ctDNA and tumour tissues, suggesting that ctDNA could function as an alternative to screen HER2-targeted population. Moreover, the changes of HER2 copy number in ctDNA could efficiently monitor trastuzumab efficacy, the power of which was superior to commonly used markers carcinoembryonic antigen (CEA) and CA199, suggesting its potential role in clinical practice. Conclusion ctDNA for HER2 analysis was strongly recommended to serve as a surrogate to screen trastuzumab-suitable population and monitor trastuzumab efficacy.

Objective Patients with HER2-positive metastatic breast cancer (MBC) benefit from trastuzumab-based therapy but eventually develop intrinsic or acquired resistance. Whether plasma HER2 gene copy number (GCN) could predict survival after trastuzumab treatment remained controversial. We evaluated the prognostic value of plasma HER2 GCN using low-coverage whole-genome sequencing (LC-WGS). Methods The plasma was collected from HER2-positive MBC patients whose pre-therapeutic samples were available before first-line trastuzumab-based treatment. Plasma DNA was extracted and assessed by LC-WGS for HER2 GCN. The optimal cut-off point for HER2 GCN to shorter survival was determined by receiver operating characteristic (ROC) curve analysis. Results A total of 49 patients were retrieved from 2013 to 2017, among whom 21 had multiple organ involvement (≥3 sites). Variations of HER2 GCN in pre-therapeutic plasma ranged from 1.89 to 23.86 (median = 2.59). ROC analysis identified the optimal cut-off point for HER2 GCN as 2.82 (P = 0.005), with 23 patients had high-level HER2 GCN and 26 in the low-level group. Both progression-free survival (PFS, P = 0.032) and overall survival (OS, P = 0.006) were adversely associated with high-level HER2 GCN. In multivariate analyses, high HER2 GCN was independently associated with shorter PFS [hazard ratio (HR) = 2.042, P = 0.037], while both high HER2 GCN (HR = 4.909, P = 0.004) and more metastatic organs (HR = 4.019, P = 0.011) were negative prognostic factors for OS. Conclusion In this population of patients with HER2-positive MBC, individuals with high HER2 GCNs in plasma had worse prognosis after trastuzumab-based therapy. Plasma HER2 GCN may be a prognostic marker in these patients.

Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

Reciprocal translocations (RecT) and Robertsonian translocations (RobT) are among the most common chromosomal abnormalities that cause infertility and birth defects. Preimplantation genetic testing for aneuploidy using comprehensive chromosome screening for in vitro fertilization enables embryo selection with balanced chromosomal ploidy; however, it is normally unable to determine whether an embryo is a translocation carrier. Here we report a method named “Mapping Allele with Resolved Carrier Status” (MaReCs), which enables chromosomal ploidy screening and resolution of the translocation carrier status of the same embryo. We performed MaReCs on 108 embryos, of which 96 were from 13 RecT carriers and 12 were from three RobT carriers. Thirteen of the sixteen patients had at least one diploid embryo. We have confirmed the accuracy of our carrier status determination in amniotic fluid karyotyping of seven cases as well as in the live birth we have thus far. Therefore, MaReCs accurately enables the selection of translocation-free embryos from patients carrying chromosomal translocations. We expect MaReCs will help reduce the propagation of RecT/RobT in the human population.

Single cell whole genome sequencing helps to decipher the genome heterogeneity within a cell population and facilitates the analysis of trace amounts of genetic material, such as is found in human embryos. The mitochondrial genome, although an important part of the genetic composition of eukaryotic cells, is often neglected in single cell genome analysis. A recently developed single cell whole genome amplification method was used, known as multiple annealing and looping based amplification cycles (MALBAC-NGS), for simultaneous analysis of chromosomal and mitochondrial genomes at the single cell level. The platform was validated by a series of technical and biological replicates and used for chromosomal and mitochondrial copy number analysis in 399 in-vitro fertilized embryos from 81 couples. A positive correlation of maternal age with increased mitochondria quantity (β = 0.176, P = 0.001) was observed after adjusting for the impact of cell type. Lower numbers of mitochondria were detected in successfully implanted embryos, although the difference was not significant. It is proposed that MALBAC-NGS could potentially be used for an advanced pre-implantation genetic screening procedure with both chromosomal constitution and mitochondrial copy number being evaluated.

Introduction Reciprocal translocations (RecT) and Robertsonian translocations (RobT) are among the most common chromosomal abnormalities that cause infertility and birth defects. Preimplantation genetic testing for aneuploidy using comprehensive chromosome screening for in vitro fertilization enables embryo selection with balanced chromosomal ploidy. However, it is normally unable to determine whether an embryo is a translocation carrier. We have reported a method named “Mapping Allele with Resolved Carrier Status” (MaReCs), which enables chromosomal ploidy screening and resolution of the translocation carrier status of the same embryo ( J. Xu et al. 2017 ). Here we introduce MaReCs and update the clinical study results. Material & Methods From September, 2017 to now, we have performed MaReCs on 69 patients, among them 55 were RecT carriers and 14 were RobT carriers. Result Totally 176 embryos from patients were identified ploidy-balanced, of which embryos from 41 patients were successfully identified as normal or balanced translocation. Other embryos’ translocation status could not be identified because of lacking reference embryos and these patients have accepted further identification in next cycles. And we have confirmed the accuracy of our carrier status determination in amniotic fluid karyotyping of seven cases as well as in the live birth we have thus far. Conclusions MaReCs accurately enables the selection of translocation-free embryos from patients carrying chromosomal translocations. It can work as an effective technology to help reduce the propagation of RecT/RobT in the human population.

With 4 figures and 3 tables Abstract Endogenous non-coding small RNAs usually consist of 20–24 nucleotides, are found ubiquitously throughout the genome and play important regulatory roles in most eucaryotes. In this study, a new kind of PCR-based marker approach using multi-mapped small RNA sequences, called inter small RNA polymorphism (iSNAP), was developed. iSNAP primers were designed based on multi-mapped small RNA and its two end conserved flanking sequences. To demonstrate the marker system, 10 rice and 30 tobacco iSNAP primers were developed and used to construct a rice genetic map and to fingerprint eight tobacco varieties. The results indicate that this technique can be effectively applied in the mapping of rice and fingerprinting of species (such as tobacco) with extremely low genetic diversity and thus provides a new option of molecular marker techniques.

Bamboo occupies an important phylogenetic node in the grass family and plays a significant role in the forest industry. We produced 1.2 Mb of tetraploid moso bamboo (Phyllostachys pubescens E. Mazel ex H. de Leh.) sequences from 13 bacterial artificial chromosome (BAC) clones, and these are the largest genomic sequences available so far from the subfamily Bambusoideae. The content of repetitive elements (36.2%) in bamboo is similar to that in rice. Both rice and sorghum exhibit high genomic synteny with bamboo, which suggests that rice and sorghum may be useful as models for decoding Bambusoideae genomes.

e13018Background: Only a subset of patients with HER2 positive (HER2+) metastatic breast cancer (MBC) responds to anti-HER2 therapy. This study aimed at detecting plasma HER2 gene copy number (GCN)...

Bacillus subtilis is an aerobic spore-forming Gram-positive bacterium that is a model organism and of great industrial significance as the source of diverse novel functional molecules. Here we present, to our knowledge, the first genome sequence of Bacillus subtilis strain gtP20b isolated from the marine environment. A subset of candidate genes and gene clusters were identified, which are potentially involved in production of diverse functional molecules, like novel ribosomal and nonribosomal antimicrobial peptides. The genome sequence described in this paper is due to its high strain specificity of great importance for basic as well as applied researches on marine organisms.

Background Endogenous non-coding small RNAs (21-24 nt) play an important role in post-transcriptional gene regulation in plants. Domestication selection is the most important evolutionary force in shaping crop genomes. The extent of polymorphism at small RNA loci in domesticated rice and whether small RNA loci are targets of domestication selection have not yet been determined. Results A polymorphism survey of 94 small RNA loci (88 MIRNAs, four TAS3 loci and two miRNA-like long hairpins) was conducted in domesticated rice, generating 2 Mb of sequence data. Many mutations (substitution or insertion/deletion) were observed at small RNA loci in domesticated rice, e.g. 12 mutation sites were observed in the mature miRNA sequences of 11 MIRNAs (12.5% of the investigated MIRNAs). Several small RNA loci showed significant signals for positive selection and/or potential domestication selection. Conclusions Sequence variation at miRNAs and other small RNAs is higher than expected in domesticated rice. Like protein-coding genes, non-coding small RNA loci could be targets of domestication selection and play an important role in rice domestication and improvement.