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BRCA1/2 gene mutation testing, based on next-generation sequencing (NGS), has been gradually applied in the clinic to serve as preventive early screening for predisposed individuals or to provide treatment options for patients with hereditary breast or ovarian cancers. Here, we evaluated the accuracy of NGS-based mutation detection in BRCA1/2 and the consistency in variant interpretation among clinical laboratories to find the possible reasons underlying inaccurate results and discrepant variant interpretation. Laboratories were asked to use their routine procedures to detect six mimetic DNA samples with different BRCA1/2 germline variants. The results of variant detection were required to be submitted via a web-based evaluation system and were automatically scored, according to predefined criteria. The variant interpretation report, including the detailed clinical evidence, was summarized and analyzed for reasons underlying inconsistent results. Overall, only 55.2% (16/29) of laboratories, whose detection score was higher than 90 points, was found to be an acceptable detection capability level. 82.9% (29/35) of the errors were genotype errors. The variant classification results were generally consistent, and 77.8% (7/9) of the variants were given the consistent classification answer. Only two single nucleotide variants (SNVs) had a discrepant classification opinion across laboratories. The BRCA1/2 variant detection performance should be further improved, especially in reporting the correct genome coordinates. Inconsistent variant classification may be a result of the different clinical pieces of evidence collected by the laboratories. However, discordant clinical evidence also appeared within the same classification results. Therefore, our study provided clear clinical evidence assessment strategies for BRCA1/2 variants, which was aimed at obtaining a consistent variant classification strategy for providing accurate clinical reports to the clinicians.

Background Astrovirus (AstV), Sapovirus (SaV) and Poliovirus (PV) are important pathogens that cause infections in children under five years of age. It is a very important task to systematically monitor and evaluate the diagnostic performance of these viruses in clinical laboratories. Methods In our study, we performed a multicenter evaluation study among 21 laboratories across China using simulated stool samples spiked with self-designed AstV, SaV and PV pseudoviral particles. Results The testing capability of 80.0% (16/20, AstV), 52.6% (10/19, SaV), and 25.0% (2/8, PV) of the participating laboratories were found to be “competent” in reporting correct results for all samples. The main type of errors were false negatives. None of the laboratories identified the subtypes of AstV and SaV, and six laboratories specifically identified the subtypes of PV. Lacking of well-trained personnel and adequate funding were the main challenges. From the questionnaire results, 55.6% laboratories (10/18) believe that training personnel could improve the laboratory testing performance. Conclusions The laboratories showed a competent diagnostic performance for AstV, but inferior diagnostic performances for SaV and PV. Sensitivity of detection and the ability for virus typing should be improved clinically. Professional and standardized personnel training is urgently needed to further improve laboratory performance.

The results of EML4-ALK testing are critical to manage ALK tyrosine kinase receptor inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China in 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating three different variants and wild-type samples were collected between 2015 and 2019. Performance was analyzed by evaluating the detection methods, kits, and pre-analytic practices used to further display the landscape of changing conditions of the reliability of EML4-ALK testing. During the 5 years, 3224 results reported from 988 laboratories were evaluated, with an overall error rate of 5.36%. Along with an increasing number of participating laboratories, the error rate within each of the different methods showed a significantly downward trend over the years. No obvious differences in the error rates were found regarding the testing methods or kit manufacturers. Moreover, the individual performance of the laboratories improved when they participated in more PT scheme rounds. The data demonstrated that the performance of individual Chinese laboratories for EML4-ALK testing continuously improved over time by participating PT schemes, regardless of their method. However, care must be taken in standardized operations and validations.

Background Mismatch repair deficiency (dMMR) status induced by MLH1 protein deficiency plays a pivotal role in therapeutic decision‐making for cancer patients. Appropriate quality control (QC) materials are necessary for monitoring the accuracy of MLH1 protein deficiency assays used in clinical laboratories. Methods CRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein‐deficient cell lines. The positive cell lines were screened and validated by Sanger sequencing, Western blot (WB), and next‐generation sequencing (NGS) and were then used to prepare formalin‐fixed, paraffin‐embedded (FFPE) samples through xenografting. These FFPE samples were tested by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for suitability as novel QC materials for MLH1 protein deficiency testing. Results We successfully cultured 358 monoclonal cells, with a survival rate of 37.3% (358/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and named as GM12878Cas9_6 and GM12878Cas9_10. The NGS results further confirmed that the MLH1 gene mutation in these two cell lines would cause the formation of stop codons and terminate the expression of the MLH1 protein. The H&E staining and IHC results also verified the deficiency of the MLH1 protein, and FFPE samples from xenografts proved their similarity and consistency with clinical samples. Conclusions We successfully established MLH1 protein‐deficient cell lines. Followed by xenografting, we developed novel FFPE QC materials with homogenous, sustainable, and typical histological structures advantages that are suitable for the standardization of clinical IHC methods., CRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein‐deficient cell lines. Cell lines with MLH1 gene mutation and MLH1 protein deficiency were identified by Sanger sequencing and Western blot (WB). Followed by xenografting, we developed novel formalin‐fixed, paraffin‐embedded (FFPE) samples quality control (QC) materials with homogenous, sustainable, and typical histological structure advantages that are suitable for the standardization of clinical immunohistochemistry (IHC) methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to public health. Viral nucleic acid testing is the diagnostic gold standard and can play an important role in the prevention and control of this infection. In this study, bacteriophage MS2 virus-like particles encapsulating specific RNA sequences of SARS-CoV-2 and other coronaviruses were prepared by genetic engineering. The assessment panel, consisting of four positive samples with concentrations of 2.8, 3.5, 4.2, and 4.9 log10 copies/mL and five negative samples with other human coronaviruses, was prepared and distributed to evaluate the accuracy of routine viral RNA detection. Results of 931 panels from 844 laboratories were collected. The overall percentage agreement, positive percentage agreement (PPA), and negative percentage agreement, defined as the percentage of agreement between the correct results and total results submitted for all, positive, and negative samples were 96.8% (8109/8379), 93.9% (3497/3724), and 99.1% (4612/4655), respectively. For samples with concentrations of 4.9 and 4.2 log10 copies/mL, the PPAs were >95%. However, for 3.5 and 2.8 log10 copies/mL, the PPAs were 94.6% (881/931) and 84.9% (790/931), respectively. For all negative samples, the negative percentage agreement values were >95%. Thus, most laboratories can reliably detect SARS-CoV-2. However, further improvement and optimization are required to ensure the accuracy of detection in panel members with lower concentrations of viral RNA.

Exome sequencing has become an effective diagnostic method for Mendelian disorders. But the quality of services differs widely across laboratories in China, particularly in variant classification, even with the adoption of the ACMG guidelines. As an effort of quality control and improvement for better clinical utilization of exome sequencing, we assessed the exome data analysis and clinical reporting among Chinese laboratories. Five raw datasets of real clinical samples with associated phenotypes were sent to 53 laboratories. The participants independently performed secondary analysis, variant classification, and reporting. The first round of results was used for identifying problems associated with these aspects. Subsequently, we implemented several corrective actions and a training program was designed based on the identified issues. A second round of five datasets were sent to the same participants. We compared the performances in variant interpretation and reporting. A total of 85.7% (42/49) of participants correctly identified all the variants related with phenotype. Many lines of evidence using the ACMG guidelines were incorrectly utilized, which resulted in a large inter-laboratory discrepancy. After training, the evidence usage problems significantly improved, leading to a more consistent outcome. Participants improved their exome data analysis and clinical reporting capability. Targeted training and a deeper understanding of the ACMG guidelines helped to improve the clinical exome sequencing service in terms of consistency and accuracy in variant classification in China.

Microsatellite instability (MSI) became the spotlight after the US FDA' s approval of MSI as an indication of immunotherapy for cancer patients. Immunohistochemical detection of loss of MMR proteins and PCR amplification of specific microsatellite repeats are widely used in clinical practice. Next-generation sequencing is a promising tool for identifying MSI patients. Circulating tumour DNA provides a convenient alternative when tumour tissue is unavailable. MSI detection is an effective tool to screen for Lynch syndrome. Early-stage CRC patients with MSI generally have a better prognosis and a reduced response to chemotherapy; instead, they are more likely to respond to immunotherapy. In this review, we aimed to assess the clinical utility of MSI as a biomarker in CRC. We will provide an overview of the available methods for evaluation of the analytical validity of MSI detection and elaborate the evidence on the clinical validity of MSI in the management of CRC patients.

Immunotherapy has revolutionized the treatment of cancer due to its remarkable efficacy and extensive survival benefit in multiple tumor types. However, predictive biomarkers are required to identify patients who are likely to respond to immunotherapy. Recently, tumor mutational burden (TMB) has been shown to be associated with clinical outcomes in diverse cancers, such as melanoma, non-small-cell lung cancer and colorectal cancer. Several studies have demonstrated that high TMB can effectively predict the objective response rate and progression-free survival, but the ability of TMB to predict overall survival is limited. Thus, the clinical utility of TMB as a predictive and prognostic biomarker in immunotherapy is currently controversial. Importantly, multiple factors can affect the accurate assessment of TMB and further interfere with its prediction of clinical outcomes. These factors include preanalytical factors such as sample status, analytical factors such as differences in platforms and methods for determining TMB and variability of cutoff values, and postanalytical factors such as inconsistent interpretation and reporting of results. In addition, the optimal definition and quantification of TMB are unclear and require harmonization and standardization for reliable clinical application. This review elaborates on the factors affecting TMB status in primary tumors, summarizes the clinical utility of TMB as a biomarker in immunotherapy, and evaluates the impact of each analysis stage on the accurate estimation of TMB, especially its quantification, aiming to facilitate TMB assessment in routine clinical settings.

Targeted molecular therapies have markedly improved the therapeutic management of lung cancer, while the discovery of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has revolutionized the treatment of non-small cell lung cancer (NSCLC). However, the clinical benefit of targeted therapies is limited by the eventual emergence of resistance. Identifying and monitoring the underlying mechanism of EGFR-TKI resistance could lead to more precise therapy and advances in treatment. Presently, tissue biopsy remains the gold standard for genotyping but it is limited by sampling bias, lack of available tissue, and potential complications. Analysis of circulating tumour DNA (ctDNA) may overcome the current limitations of tissue biopsies and provide a comprehensive landscape of the resistance mechanisms in a minimally invasive manner. Well-developed, analytically valid detection technologies are prerequisites for integrating ctDNA detection into clinical cancer management. Here, we provide an overview of available methodologies for ctDNA detection and we also discuss the potential clinical applications of ctDNA to monitor the resistance mechanisms.

Genome-edited human cell lines are important resources for producing quality control materials for clinical molecular genetic testing. Generating cell lines with defined mutations through homology-directed repair-based methods are inefficient and can lead to unwanted insertions and deletions in the target loci. Nonhomologous end joining in the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system was harnessed to generate genome-engineered cell lines harboring target mutations. Donor plasmids containing target sites for the single guide RNA (sgRNA) and homologous DNA fragments harboring important cancer gene mutations were cotransfected with the Cas9/sgRNA vector into wild-type human cells. The introduced mutations were validated in-house and in 44 laboratories using various techniques, including next-generation sequencing. Exogenous sequences containing the target mutations were efficiently integrated into the ALK receptor tyrosine kinase (ALK) locus in HEK293T and A549 cells. Successful introduction of artificial mutations was confirmed via both Sanger sequencing and the amplification refractory mutation system. Results of external pilot testing revealed that the DNA samples derived from genome-edited cell lines were widely applicable across multiple platforms and laboratories. This study demonstrates that CRISPR/Cas9-induced nonhomologous end joining is a valuable and novel method for generating artificial mutants for use in quality control applications in clinical molecular genetics.

Background Reports serve as a bridge between laboratories and clinicians, help synthesize an overwhelming amount of raw data into evidence-based medicine, and play a significant role in designing clinical treatments. In an effort to guarantee high-quality epidermal growth factor receptor (EGFR) gene mutation testing and reporting performance, the National Center for Clinical Laboratories launched a proficiency testing (PT) scheme reflecting clinical practices in China since 2014. This study focuses on the quality assessment of gene mutation reports. Materials and methods Fifty-three laboratories that submitted reports in both 2014 and 2016 EGFR gene mutation PT schemes were selected for report analysis and comparison according to predefined evaluation criteria. Results The average score for reports from 2014 was 14 out of 30 points. The overall scores for reports from 2016 improved substantially, yielding an average score of 20 out of 30 points. Among the evaluation criteria, general items were well documented in the reports. However, items specific to molecular diagnosis were far from satisfactory, and some items were even missing. Conclusion The quality assessment of clinical written reports from 2014 and 2016 demonstrates that substantial improvements have been made in overall reporting performance. However, not all statements pertaining to important elements met expectations. To continue education, repeated PT schemes need to be executed in a timely fashion to expose and address existing shortcomings in clinical reports. There remains ample room for improvement towards generating concise, comprehensive, and readable reports. Implications for practice This article compares the quality of clinical gene mutation reports submitted in 2014 to those submitted in 2016 epidermal growth factor receptor proficiency testing schemes, exposes the existing shortcomings, and discusses ways to communicate results more effectively in the future. The findings demonstrate that notable progress was observed in the overall reporting performance. However, key points specific to molecular diagnosis were far from expectation, and some items were even missing. Standardization needs to be emphasized to improve the report format and content. This article provides a reference that laboratories can use to write concise, comprehensive, and readily accessible clinical reports.

Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.

// Rui Zhang 1, 3, * , Jiansheng Ding 1, 2, 3, * , Yanxi Han 1, 3 , Lang Yi 1, 2, 3 , Jiehong Xie 1, 3 , Xin Yang 1, 2, 3 , Gaowei Fan 1, 2, 3 , Guojing Wang 1, 2, 3 , Mingju Hao 1, 2, 3 , Dong Zhang 1, 2, 3 , Kuo Zhang 1, 3 , Guigao Lin 1, 3 , Jinming Li 1, 2, 3 1 National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People’s Republic of China 2 Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China 3 Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People’s Republic of China * These authors have contributed equally to this work Correspondence to: Jinming Li, email: jmli@nccl.org.cn Keywords: next-generation sequencing, reliable assurance, cancer-related genes, somatic mutations, external quality assessment Received: April 16, 2016 Accepted: July 27, 2016 Published: August 16, 2016 ABSTRACT To evaluate the proficiencies of laboratories utilizing next-generation sequencing (NGS) to detect somatic mutations in cancer-related genes, an external quality assessment (EQA) was implemented by the National Center for Clinical Laboratories of China in 2015. We prepared a panel of samples that comprised eight samples made by mixing synthetic mutated DNA fragments with normal human genomic DNA and one reference sample containing only genomic DNA. We validated our sample panel, and then distributed it to laboratories across China. We received complete results from 64 laboratories. The performances of 51.6 % (33/64) respondent labs were acceptable and 26.6 % (17/64) of the labs returned perfect results. In total, 449 mistakes were reported, including 201 false-negatives (201/449, 44.8 %) and 222 false-positives (222/449, 49.4 %) and 26 slightly discordant results (26/449, 5.8 %). We believe these unsatisfactory results and varied performances are mainly due to the enrichment methods used, the diverse sequencing chemistries of the different NGS platforms, and other errors within the sequencing process. The results indicate that our sample panel is suitable for use in EQA studies, and that further laboratory training in targeted NGS testing is urgently required. To address this, we propose a targeted NGS workflow with details on quality assurance procedures according to the current guidelines.

// Le Chang 1, 2 , Guojing Wang 1, 2 , Tingting Jia 3 , Lei Zhang 1, 4 , Yulong Li 1, 2 , Yanxi Han 1 , Kuo Zhang 1, 2 , Guigao Lin 1 , Rui Zhang 1 , Jinming Li 1, 2 , Lunan Wang 1, 2 1 National Center for Clinical Laboratories, Beijing Hospital, Beijing, People's Republic of China 2 Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, People's Republic of China 3 Department of Clinical Laboratory, Beijing Chaoyang Hospital, Capital Medical University, Beijing, People's Republic of China 4 Peking University Fifth School of Clinical Medicine, Beijing, People's Republic of China Correspondence to: Jinming Li, e-mail: jmli@nccl.org.cn Lunan Wang, e-mail: lunan99@163.com Keywords: hepatocellular carcinoma, maternally expressed gene 3, MS2 virus-like particles, epidermal growth factor receptor, GE11 Received: January 21, 2016 Accepted: March 02, 2016 Published: March 16, 2016 ABSTRACT Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide. However, the treatment of patients with HCC is particularly challenging. Long non-coding RNA maternally expressed gene 3 ( MEG3 ) has been identified as a potential suppressor of several types of tumors, but the delivery of long RNA remains problematic, limiting its applications. In the present study, we designed a novel delivery system based on MS2 virus-like particles (VLPs) crosslinked with GE11 polypeptide. This vector was found to be fast, effective and safe for the targeted delivery of lncRNA MEG3 RNA to the epidermal growth factor receptor (EGFR)-positive HCC cell lines without the activation of EGFR downstream pathways, and significantly attenuated both in vitro and in vivo tumor cell growth. Our study also revealed that the targeted delivery was mainly dependent on clathrin-mediated endocytosis and MEG3 RNA suppresses tumor growth mainly via increasing the expression of p53 and its downstream gene GDF15 , but decreasing the expression of MDM2. Thus, this vector is promising as a novel delivery system and may facilitate a new approach to lncRNA based cancer therapy.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive, inherited neuromuscular disorders, caused by pathogenic variants in the dystrophin gene that encodes the dystrophin protein. A number of mutations have been identified in the past years, producing dystrophin diversity and resulting in mild to severe phenotypes in patients. Mutations in the dystrophin gene can be characterized by laboratory testing to confirm a clinical diagnosis of DMD/BMD. Traditional genetic diagnostic strategy for DMD/BMD involves the initial detection of large mutations, followed by the detection of smaller mutations, where two or more analytical methods are employed. With the development of next generation sequencing (NGS) technology, comprehensive mutational screening for all variant types can be performed on a single platform in patients and carriers, although further optimization and validation are required. Furthermore, the discovery of cell-free fetal DNA (cffDNA) in maternal plasma provides basis for noninvasive prenatal diagnosis of DMD/BMD. Here, we discuss the correlation between genotype and phenotype, the current methods of molecular genetic testing and genetic diagnostic strategy for probands and female carriers of DMD/BMD, the diagnostic ability of a comprehensive targeted NGS strategy and the possibility of it replacing conventional methods.

In this era of precision medicine, molecular biology is becoming increasingly significant for the diagnosis and therapeutic management of non-small cell lung cancer. The specimen as the primary element of the whole testing flow is particularly important for maintaining the accuracy of gene alteration testing. Presently, the main sample types applied in routine diagnosis are tissue and cytology biopsies. Liquid biopsies are considered as the most promising alternatives when tissue and cytology samples are not available. Each sample type possesses its own strengths and weaknesses, pertaining to the disparity of sampling, preparation and preservation procedures, the heterogeneity of inter- or intratumors, the tumor cellularity (percentage and number of tumor cells) of specimens, etc., and none of them can individually be a “one size to fit all”. Therefore, in this review, we summarized the strengths and weaknesses of different sample types that are widely used in clinical practice, offered solutions to reduce the negative impact of the samples and proposed an optimized strategy for choice of samples during the entire diagnostic course. We hope to provide valuable information to laboratories for choosing optimal clinical specimens to achieve comprehensive functional genomic landscapes and formulate individually tailored treatment plans for NSCLC patients that are in advanced stages.

Pharmacogenomics has significantly added to our understanding of drug responses in clinical pharmacology, changing the paradigm of treatment decisions. Interrogations of both inherited and somatic variations for therapeutic purposes are increasingly being adopted in clinics, where quality control (QC) materials are required. However, for many pharmacogenomic tests, the acquisition of well-characterized QC materials is often difficult or impossible. In this review, several sources of appropriate QC materials for therapy-associated genetic testing are discussed. Among them, the novel methods for producing renewable controls that resemble patient samples are highlighted. Owing to technological complexity, more efforts are needed to develop proper controls for next-generation sequencing-based assay.

Background Quantitative immunoassays to detect IgG antibodies are the most commonly used tests for diagnosing toxoplasmosis. We investigated the current state of standardization of quantitative immunoassays used to measure anti-Toxoplasma IgG levels. Methods Four fully automated immunoassays (Architect i4000ISR, Immulite 2000 Xpi, Siemens; Liaison, DiaSorin; Cobas e601, Roche) and one manual immunoassay (ELISA classic Toxo IgG, Virion Serion) were performed on the following: individual patient serum samples, the WHO international standards, control samples, and calibrators provided by 5 immunoassay manufacturers. Statistical analysis was used to illustrate the results. Results No perfect correlation (slope = 1.0) was found between any 2 assays. Large differences in anti-Toxoplasma IgG titers were observed among the 5 immunoassays using serum samples from individual patients. Using IS 01/600 as a calibrator minimized the inter-assay variability of anti-Toxoplasma IgG values Conclusions There is still significant effort needed towards standardization of anti-Toxoplasma IgG quantitative immunoassays.

Background The early detection of type 1 diabetes (T1D) largely depends on a reliable approach to monitor β cell loss. An effective way to evaluate the decline of β cell mass would allow early preventative intervention to preserve insulin secretion. Main body Recent progress in the development of novel biomarkers, based on tissue-specific methylation patterns, has inspired relevant studies in T1D. In this review, we focus on the application of circulating β cell-derived unmethylated insulin (INS) DNA. Circulating β cell-derived unmethylated INS DNA has a potential clinical value for the early detection of T1D, surveillance of islet transplantation rejection, and evaluation of response to therapy. Utilizing differentiated methylation patterns in different organs and employing a wide variety of molecular technologies also provide insights into the interrogation of biomarkers in other diseases with massive tissue-specific cell loss. Conclusion Circulating unmethylated INS DNA is a promising molecular biomarker for the early detection of T1D.

// Guojing Wang 1, 2, 4 , Tingting Jia 5 , Xixia Xu 3 , Le Chang 1, 2, 4 , Rui Zhang 1, 2 , Yu Fu 1, 2, 4 , Yulong Li 1, 2, 4 , Xin Yang 1, 2, 4 , Kuo Zhang 1, 2 , Guigao Lin 1, 2 ,Yanxi Han 1, 2 , Jinming Li 1, 2, 4 1 National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, People’s Republic of China 2 Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, People’s Republic of China 3 Central Research Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China 4 Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing People’s Republic of China 5 Department of Clinical Laboratory, Beijing Chaoyang Hospital, Capital Medical University, Beijing, People’s Republic of China Correspondence to: Jinming Li, email: jmli@nccl.org.cn Keywords: MS2 VLP, phage surface display, TAT peptide, miR-122 Received: May 24, 2016 Accepted: July 01, 2016 Published: July 18, 2016 ABSTRACT Current treatments for hepatocellular carcinoma (HCC) have shown inadequate. MicroRNA-122 (miR-122) mediated RNA interference brings new prospects. A safe, efficient miRNA delivery system is an indispensable assurance. Previously, we developed an MS2 bacteriophage virus-like particle (VLP)-based microRNA delivery system crosslinked with the HIV TAT peptide, which served as an effective inhibitor in the treatments of systemic lupus erythematosus and osteoporosis. However, defects, such as low crosslinking efficiency, high cost, and potential toxicity of the crosslinking agent, needed to be confronted. Therefore, TAT peptide was designed to display on the surface of MS2 VLPs, instead of being chemically crosslinked, using the platform of phage surface display. The results reflected that MS2 VLPs displaying TAT could effectively penetrate the cytomembrane and deliver miR-122. Additionally, its inhibitory effects on HCC were significant in Hep3B, HepG2, and Huh7 cells and Hep3B related animal models. Thus, we have established a novel miR-122 delivery system based on MS2 VLPs surface displaying TAT peptide, which could effectively perform the function of penetrating cytomembrane and the inhibition of HCC.

An external quality assessment for detection of trisomy 21, 18, and 13 by massively parallel sequencing was implemented by the National Center for Clinical Laboratories of People's Republic of China in 2014. Simulated samples were prepared by mixing fragmented abnormal DNA with plasma from non-pregnant women. The external quality assessment panel, comprising 5 samples from pregnant healthy women, 2 samples with sex chromosome aneuploidies, and 13 samples with different concentrations of fetal fractions positive for trisomy 21, 18, and 13, was then distributed to participating laboratories. In total, 55.6% (47 of 84) of respondents correctly identified each of the samples in the panel. Seventeen false-negative and 87 gray zone results were reported, most [102 of 104 (98.1%)] of which were derived from for trisomy samples with effective fetal fractions4%. No laboratories generated false-positive results. In addition, we observed varied diagnostic capabilities of different assays, with the assay on the basis of NextSeq CN500 performing better than others, whereas Z values generated by BGISEQ-100 fluctuated greatly. There were no significant correlations between the numbers of unique sequence reads and Z values from any trisomy sample generated by BGISEQ-100. Overall, most clinical laboratories detected samples containing effective fetal fractions4%. Our study shows need for further laboratory training in the management of samples with low fetal fractions. For some assays, precision of Z values needs to be improved.

Background KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. Methods We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. Results We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. Conclusion The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.

Tamoxifen has been successfully used for treating breast cancer and preventing cancer recurrence. Cytochrome P450 2D6 (CYP2D6) plays a key role in the process of metabolizing tamoxifen to its active moiety, endoxifen. Patients with variants of the CYP2D6 gene may not receive the full benefit of tamoxifen treatment. The CYP2D6*10 variant (the most common variant in Asians) was analyzed to optimize the prescription of tamoxifen in China. To ensure referring clinicians have accurate information for genotype-guided tamoxifen treatment, the Chinese National Center for Clinical Laboratories (NCCL) organized a national proficiency testing (PT) to evaluate the performance of laboratories providing CYP2D6*10 genotyping. Ten genomic DNA samples with CYP2D6 wild-type or CYP2D6*10 variants were validated by PCR-sequencing and sent to 28 participant laboratories. The genotyping results and pharmacogenomic test reports were submitted and evaluated by NCCL experts. Additional information regarding the number of samples tested, the accreditation/certification status, and detecting technology was also requested. Thirty-one data sets were received, with a corresponding analytical sensitivity of 98.2% (548/558 challenges; 95% confidence interval: 96.7–99.1%) and an analytic specificity of 96.5% (675/682; 95% confidence interval: 97.9–99.5%). Overall, 25/28 participants correctly identified CYP2D6*10 status in 10 samples; however, two laboratories made serious genotyping errors. Most of the essential information was included in the 20 submitted CYP2D6*10 test reports. The majority of Chinese laboratories are reliable for detecting the CYP2D6*10 variant; however, several issues revealed in this study underline the importance of PT schemes in continued external assessment and provision of guidelines.