DHPLC和DDF检测散发性卵巢癌p53基因突变的应用

目的：借助变性高效液相色谱仪（DHPLC）和荧光双脱氧指纹印迹法（F-DDF）检测散发性卵巢癌p53基因突变。方法：提取50例散发性卵巢癌标本DNA，使用DHPLC和F-DDF检测p53基因外显子5～8突变，其中测序证实18例p53基因外显子5～8有突变，F-DDF使用ddGTP和ddTTP扩增，Cy5标记其引物，DHPLC使用美国Transgenomic公司的WAVE系统。结果：18例突变都可以使用DHPLC的方法发现（敏感性100％），无假阳性和假阴性，FDDF检测出16例突变标拳（敏感性78％）。就肿瘤的异质性而言（未知的突变型／野生型DNA比率），即使突变性DNA被野生性DNA稀释，DHPLC仍然能发现突变，说明这一方法的高度敏感性，根据等位基因丢失（LOH）判断，即使只有5％的突变DNA，DHPLC仍能发现异常，但这一异常难以被测序证实。结论：对于异质性肿瘤而言，DHPLC检测突变是可靠的，F-DDF的敏感性和特异性相时较低，这两种方法都能检测基因突变，DHPI上的缺点是费用昂贵，操作较复杂，而F-DDF优势在于不需要特辣的仪器及操作简便。可作为临床筛查基因突变的方法．     

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung,Melanoma, and Gastrointestinal Malignancies

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.In modern oncologic practice, patients with advanced-stage non-small cell lung cancer (NSCLC),^{1, 2} melanoma,^{3, 4} and colorectal adenocarcinoma^{5, 6} are often treated with targeted therapies as standard of care or after enrollment in clinical trials. Molecular mutation analysis is the preferred testing modality to guide therapeutic decision making and/or eligibility for biological studies. Therefore, laboratory-developed mutation assays require robust workflows that produce high-quality sequence information from routine clinical specimens, namely formalin-fixed, paraffin-embedded (FFPE) samples. As molecular testing transitions from an ancillary tool to a seminal requirement for optimal oncologic patient management, multiplex sequencing assays with clearly defined content and bioinformatics workflows are essential for accurate and consistent results, reporting, and patient management.Published guidelines endorse which genes to test in a particular tumor type and provide timeframes for receipt of actionable results, but they also grant individual laboratories autonomy to perform mutation testing using any suitable validated method.² Historically at our institution, single-gene mutation analysis for clinically relevant genes was performed either in-house or at a Clinical Laboratory improvement Amendment–certified reference laboratory. Depending on the result, reflex testing was performed for additional genes per mutation frequency or designated algorithms. Unfortunately, this approach introduced considerable turn-around time delays and unnecessary cost, particularly when send-out testing was required. Therefore, we sought testing modalities that could analyze multiple clinically relevant mutations simultaneously, accurately, and expeditiously.Next-generation sequencing (NGS) technologies have revolutionized genomic medicine by allowing high-throughput, parallel sequencing of the human genome.⁷ Currently, however, a large proportion of clinical NGS endeavors are supported by larger academic institutions with shared access to established genomic and bioinformatics research infrastructures, and routine clinical implementation of NGS is complicated by mitigating factors, such as clinical performance, laboratory expertise, lengthy turn-around times, and cost.⁸ Thus, we investigated affordable methods to detect clinically relevant somatic mutations in NSCLC, melanoma, and gastrointestinal (GI) malignancies that generated high-quality sequencing data from FFPE samples, and offered manageable turn-around times. Targeted amplicon-based library preparation methods combined with parallel sequencing offered a practical solution, and recent studies have demonstrated the utility of this approach.^{9, 10}Reversible terminal dideoxynucleotide sequencing chemistry by Illumina (San Diego, CA) consistently generates accurate and reproducible sequencing data.^{11, 12} To use this chemistry for clinical testing, we purchased the bench-top NGS sequencer, the Illumina MiSeq, and paired it with the MiSeq-compatible Illumina TruSight tumor (TST) 26-target amplicon-based library preparation kit. TST targets 26 genes and 174 amplicons selected from College of American Pathologists/National Comprehensive Cancer Network guidelines, relevant publications, and late-phase pharmaceutical clinical trials (Supplemental Table S1). TST offered several advantages over other commercially available mutation testing kits, such as bidirectional targeting of the positive and negative DNA strands, full-exon coverage as opposed to hotspot analysis, and robust vendor-supplied bioinformatics techniques optimized for somatic variant detection. More important, TST library preparation is optimized for FFPE samples, multiple safeguards exist to detect FFPE variant artifacts, and deep sequencing of TST libraries consistently yields high depths of coverage of targeted regions.Somatic mutation testing for many of the TST genes has clinical utility in a wide variety of solid tumors. For example, testing for CTNNB1 exon 3 is performed clinically for diagnostic and prognostic purposes in pediatric desmoid tumors, select PIK3CA hotspot mutations are positive prognostic factors for breast carcinoma, and multiple exons in PDGFRA and KIT are routinely tested in GI stromal tumors to predict response to targeted therapies. More important for our intended validation purposes, all of the clinically relevant genes and regions mutated in NSCLC, melanoma, and colonic adenocarcinoma that were tested in our routine clinical practice were represented. In addition, we could easily incorporate the TST NGS into a 5 business day workflow model, and a cost-analysis demonstrated a reasonable cost per test.Last, TST NGS data are processed from raw sequence (FASTQ) to called variants with on-board MiSeq Reporter software version 2.3, and variant annotations can be performed with Illumina''s VariantStudio software version 2.1 software using standard desktop and laptop computers. The ease of library preparation, sequencing, and data analysis with tools provided by a single vendor best fit our clinical priorities and the resources available at our academic molecular pathology laboratory.Herein, we present our results from the clinical validation of TST NGS using 80 sequenced samples that were selected from 100 FFPE patient samples (40 NSCLCs, 30 melanomas, and 30 GI malignancies). During our validation, we achieved high depths of coverage for multiple clinically relevant variants when multiplexing 8 to 12 samples on a single MiSeq flow cell. TST NGS consistently demonstrated sensitivities comparable to reference assays, showed 100% concordance with known variants, detected novel variants in many samples, and uncovered variants missed by less-sensitive testing modalities. The TST variant-calling pipeline was robust and showed high concordance when compared with an alternative analysis pipeline, and we used an in-house custom Java program to assess laboratory-defined quality control (QC) metrics and streamline clinical reporting (developed by G.H.S., Emory University, http://github.com/ghsmith/coverageQc). More important, although the results detailed herein represent the experience of a single institution, the data and validation strategies shown herein are broadly applicable to most clinical molecular laboratories interested in offering NGS for NSCLCs, melanomas, and GI malignancies as well as many other solid tumors.     

D—Dimer PLUS测定血浆D-二聚体浓度性能评价

目的 验证D-Dimer PLUS测定血浆D-二聚体的各项性能参数.方法 参照美国临床实验室标准化委员会(NC-CLS)的相关文件,对D-Dimer PLUS在Sysmex CA7000型全自动血凝仪上测定血浆D-二聚体的性能进行全面的评价.结果 检测下限83.86μg/L;线性范围0～2 000 μg/L;回收率93%～113%;低值和高值批内不精密度(变异系数CV%)5.31%和2.67%,低值和高值总不精密度4.69%和4.14%;与mini-VIDAS(酶联免疫吸附实验)的测定结果 进行比对,相关系数r=0.915,平均百分偏差634.6%;内源性干扰物结合胆红素、乳糜微粒、溶血血红蛋白对D-二聚体测定结果 产生影响的浓度值分别为299 mg/L,882 FTU和7.8 g/L,游离胆红素和类风湿因子浓度达362 mg/L和540,000 IU/L时,D-二聚体测定结果 没有显著变化;验证参考值范围0～398 μg/L,高于厂家提供的参考值范围0～324μg/L,需重新建立参考值范围.结论 D-Dimer PLUS测定血浆D-二聚体浓度具有符合临床要求的精密度、准确度和灵敏度,可用于临床诊断实验.     

ARMS法检测肺腺癌EGFR基因突变及临床意义

目的 探讨扩增阻滞突变系统(ARMS)法在肺腺癌中表皮生长因子受体(EGFR)基因突变检测的应用及其临床意义。方法 收集2015年1月～2016年8月西安交通大学第一附属医院病理科的肺腺癌标本566例作为研究对象。其中,胸腔积液细胞块标本34例,肺活检标本401例,手术切除标本131例,采用ARMS法进行石蜡标本EGFR基因突变检测,分析EGFR基因突变与肺腺癌患者临床资料的相关性。结果 肺腺癌标本566例中,吸烟腺癌患者239例,非吸烟腺癌患者327例。吸烟患者中,EGFR突变率与患者年龄、性别、手术方式等无明显关联(P>0.05),而与肺癌原发部位关系密切(P<0.05); 非吸烟患者中,EGFR突变率与性别、年龄、标本类型及肺癌原发病灶部位无明显相关性(P>0.05)。结论 ARMS法可有效应用于肺腺癌临床病理石蜡标本中EGFR基因突变检测。吸烟是EGFR突变率的重要影响因子,且对左、右肺均有影响,但是对右肺的影响较大。     

Somatic Mutation Affecting the Rhesus and Duffy Blood Group Systems

A normal blood donor has been encountered whose red cell grouping tests showed a mixed cell population of CDe/cde, Fy(a+) and cde/cde, Fy(a—) cells. A family study revealed that the cde/cde, Fy(a—) cell population could not have arisen through a normal genetic pathway and reasons are given for suggesting that a gene mutation had occurred. As both Rhesus and Duffy genes are affected, it is concluded that the mutation has modified a common gene involved at some stage in the development of both blood group antigens.     

间接免疫荧光抗体法快速检测脓液中的厌氧菌

用脆弱、多形和不解糖类杆菌3种常见厌氧菌标准株的免疫血清,以间接免疫荧光抗体法(IFA)检测75份脓液中的厌氧菌,与厌氧培养结果比较,IFA3种菌的敏感性为72.7％,阳性符合率为88.9％,阴性符合率为81.4％.少数IFA阳性与培养鉴定结果不完全一致。3份IFA阳性培养阴性.IFA方法简便,2小时内可得结果,是一种快速、特异和敏感的厌氧菌检测方法.     

Validation and Implementation of Targeted Capture and Sequencing for the Detection of Actionable Mutation,Copy Number Variation,and Gene Rearrangement in Clinical Cancer Specimens

Recent years have seen development and implementation of anticancer therapies targeted to particular gene mutations, but methods to assay clinical cancer specimens in a comprehensive way for the critical mutations remain underdeveloped. We have developed UW-OncoPlex, a clinical molecular diagnostic assay to provide simultaneous deep-sequencing information, based on >500× average coverage, for all classes of mutations in 194 clinically relevant genes. To validate UW-OncoPlex, we tested 98 previously characterized clinical tumor specimens from 10 different cancer types, including 41 formalin-fixed paraffin-embedded tissue samples. Mixing studies indicated reliable mutation detection in samples with ≥10% tumor cells. In clinical samples with ≥10% tumor cells, UW-OncoPlex correctly identified 129 of 130 known mutations [sensitivity 99.2%, (95% CI, 95.8%–99.9%)], including single nucleotide variants, small insertions and deletions, internal tandem duplications, gene copy number gains and amplifications, gene copy losses, chromosomal gains and losses, and actionable genomic rearrangements, including ALK-EML4, ROS1, PML-RARA, and BCR-ABL. In the same samples, the assay also identified actionable point mutations in genes not previously analyzed and novel gene rearrangements of MLL and GRIK4 in melanoma, and of ASXL1, PIK3R1, and SGCZ in acute myeloid leukemia. To best guide existing and emerging treatment regimens and facilitate integration of genomic testing with patient care, we developed a framework for data analysis, decision support, and reporting clinically actionable results.The era of precision oncology began in 1998 with the approval of the anti- human epidermal growth factor receptor 2 (HER2) monoclonal antibody, trastuzumab, for the treatment of HER2-positive breast cancer.¹ At the same time, an immunohistochemistry-based diagnostic test (HercepTest; Dako, Glostrup, Denmark) was approved for the identification of tumors that express HER2, necessary to ascertain which patients are eligible for trastuzumab treatment. This advance was followed by the introduction of erlotinib, a small molecule tyrosine kinase inhibitor against epidermal growth factor receptor (EGFR), which has proven useful in patients with non-small cell lung cancer with activating EGFR mutations.^2–4 More recently, two U.S. Food and Drug Administration–approved drugs that also require a genomic sequence-based companion diagnostic have advanced into late-stage clinical trials: vemurafenib, which targets metastatic malignant melanoma harboring the BRAF V600E mutation⁵ , and crizotinib, which has shown efficacy against non-small cell lung cancers that have ALK rearrangements.⁶ Clinical trials for additional agents directed against specific genes or mutations are currently underway, and are expected to progressively increase the repertoire of targeted cancer therapies available.These successes and accumulated discoveries of potential cancer driver mutations through the use of exome and whole-genome sequencing^7–12 raise important questions about the long-term practicality of existing clinical diagnostics for the molecular characterization of cancers. As new targeted therapies are approved for molecular subtypes, and more genes with prognostic value are identified, the number of single-gene tests needed to adequately classify a tumor subtype increases, with the consequences of potentially exhausting available tissue specimens and of driving up health care costs. Yet, despite the concerns for increased risk and health care expense associated with additional tissue acquisition for molecular testing, validated clinical diagnostics suitable for assaying multiple genes and different classes of mutations in a multiplexed fashion remain lacking. Most currently available multiplexed clinical assays examine only a limited number of specific sites in a relatively small number of genes.^13,14 More recently, next-generation sequencing assays have been developed for detecting cancer-associated mutations in clinical specimens in a more comprehensive manner, but these assays have only been validated on a small number of tumor types (breast, colon, and prostate).^15–17 As assays of this type become more widespread, a framework for identifying, interpreting, and reporting actionable variants will be required for this technology to reach its full potential as a clinical diagnostic test.Here, we describe our development and clinical validation of a targeted massively parallel sequencing assay for 194 cancer-relevant genes, UW-OncoPlex, designed as a comprehensive diagnostic test for mutational events of all types in an efficient and cost-effective manner. The assay is intended to allow the most complete and informative molecular characterization of a wide variety of clinical specimens, and is scalable to large numbers of additional genes in the future. Our assay improves on earlier approaches, most importantly by expanding the spectrum of mutations detectable to include complex genomic rearrangements and copy number variants (CNVs), in addition to greater sensitivity for all variants. We also develop an accompanying data interpretation and decision support network to inform patient prognoses and therapeutic options.     

核酸薄膜技术快速检测结核分枝杆菌katG突变的研究

目的研制一种新的核苷酸薄膜,通过导流杂交技术检测结核分枝杆菌katG基因突变类型,以快速判断结核分枝杆菌对异烟肼的耐受性,寻找一种快速简便检测耐异烟肼结核病的方法。方法根据结核分枝杆菌katG基因序列设计1个野生型及2个常见突变型寡核苷酸特异探针,探针5’末端连接亚甲基(-CH2)。同时设计1个人类基因组探针以及1个生物素探针。制作低密度核苷酸薄膜微阵距列,通过导流杂交技术进行杂交,将杂交结果与DNA测序法对照。结果核酸测序显示,突变位点分别出现在包括315位点在内的7个位点,315位点突变占75.0%(18/24),最常见突变形式为AGC315ACC所致Ser315Thr氨基酸改变。杂交结果与测序结果符合率为93.9%(31/33),与DNA测序相比,其阳性预测值、阴性预测值、敏感度、特异度分别为90.0%、100%、100%、86.7%。结论核酸薄膜导流杂交技术能快速、简便、高效地检测临床标本中有无结核分枝杆菌,并可提示结核分枝杆菌有无异烟肼耐药性,指导临床用药。     

A Pyrosequencing-Based Assay for the Rapid Detection of IDH1 Mutations in Clinical Samples

Mutations of both the IDH1 and IDH2 (isocitratedehydrogenase enzyme 1 and 2) genes have recently been described in cases of human glioma. Since IDH1 mutations have been associated with better clinical outcome, they are suitable predictive markers for adult glioma patients. We have developed a pyrosequencing assay that allows both the sensitive and rapid detection of mutant IDH1 alleles in DNA extracted from formalin-fixed, paraffin-embedded tissues. PCR products that span exon 4 of IDH1 were used as a template for pyrosequencing. For validation, PCR products were additionally cloned and sequenced conventionally by Sanger sequencing. Sensitivity was measured by titration of wild-type and mutant sequences. PCR kinetic experiments were performed to investigate the influences of PCR cycle number on the accuracy of the assay. We found that a minimum of 5% of mutant IDH1 alleles can easily be detected with the pyrosequencing approach. So far, there are few data regarding IDH1 mutation status in high-grade gliomas of childhood. Therefore, we applied this assay to 47 pediatric high-grade glioma samples (age range 6 weeks to 23 years). Mutations were found in 5/14 astrocytoma III and in 6/33 glioblastomas. In conclusion, we have developed a pyrosequencing-based assay for the detection of mutations at the hotspot regions of IDH1 and provide proof for its applicability as a molecular diagnostic assay for clinical samples.Gliomas are the most common type of brain tumors and range from benign low grade gliomas to aggressive glioblastomas. Glioblastomas are the most common and most malignant tumors of the brain. They may manifest at any age, but preferentially affect adults, with a peak incidence between 45 and 70 years.^1,2 Glioblastomas are highly invasive and aggressively growing tumors that respond poorly to radiation therapy and most forms of chemotherapy. The prognosis is correspondingly poor, with most patients dying within one year after diagnosis. Adults glioblastomas may develop from diffuse astrocytomas World Health Organization grade II or anaplastic astrocytomas,³ but more frequently, they manifest after a short clinical history de novo without evidence of a less malignant precursor lesion (primary glioblastoma). Glioblastomas carry complex genetic and epigenetic alterations. Primary glioblastomas of adults frequently show loss of heterozygosity on chromosome 10q (70% of cases), EGFR amplification (36%), p16(INK4a) deletion (31%), and PTEN mutations (25%).³ Secondary glioblastomas as well as their lower grade precursor lesions exhibit frequent mutations of the TP53 gene and epigenetic inactivation of DUSP4.^3,4 Pediatric diffuse high-grade gliomas (HGG) differ from GBM of adults. While they are believed to occur de novo, only very few pediatric high grade gliomas display EGFR amplifications^5,6,7,8 although the receptor protein is detectable in many cases.⁸ Inactivation of the p53/MDM2/p14 pathway, in contrast, is also frequently present in pediatric tumors, similar to adult GBM.⁶ In particular, the TP53 mutational rate is in a similar range.⁹ The RB pathway seems to be affected less frequently in pediatric HGG.¹⁰ Recently somatic mutations at codon 132 of IDH1 gene have been reported in a screening approach of 20,661 protein coding genes in glioblastomas.^11,12 Interestingly, all mutations were located at amino acid residue 132 (position 395) an evolutionarily conserved position located within the isocitrate binding site¹³ with the substitution of Arg -> His (G->A).¹¹ In rare cases mutations of IDH2 have also been described in the homologous region in gliomas lacking IDH1 mutations.¹ Isoforms of the enzyme isocitrate dehydrogenase- IDH1 and IDH2 catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate using either NAD or NADP as cosubstrates.^14,15 Isocitrate dehydrogenase enzyme isoform 1 is located in peroxisomes, whereas IDH2 is present in mitochondria.¹⁶IDH1 is mainly involved in metabolic processes and its role in cancer biology is largely unknown. Enzymatic studies with substitution of arginine at residue 132 of IDH1 with a different amino acid (glutamate) have reported that this mutation renders the enzyme catalytically inactive, suggesting a critical role for this residue.¹⁷ More recent data, however suggest that the mutant form of the enzyme leads to the production of an alternative metabolite 2-hydroxyglutarate.Many of the glioblastomas carrying IDH1 mutations were secondary glioblastomas and contained TP53 mutations.^1,3,11 Moreover these mutations were associated with increased overall survival. Therefore, IDH1 mutation status might be a valuable prognostic marker for GBM patients. The assays published so far comprise the use of PCR and direct Sanger sequencing of PCR products. In a clinical environment, it is mandatory to optimize the approach for best sensitivity and accuracy, especially if a clinical decision or prognostic evaluation is desired. Heterogeneity of tumor tissue is a critical aspect for molecular diagnostic approaches, since mutations may only be present in a subset of tumor cells, and the wild type allele is still present in those tumor cells carrying IDH1 mutation. In addition, gliomas often contain significant amounts of other cells from different types. Therefore, a sensitive and quantitative method for the detection of mutant IDH alleles in DNA extracted from fresh frozen as well as formalin-fixed, paraffin-embedded tumor tissues is needed. So far there are few data regarding IDH1 mutation status in high grade gliomas of childhood.¹ Therefore, we applied this assay on 47 pediatric HGG samples to detect mutations in IDH1.