参加に同意すること、病院と患者のすべての医療および補助的なスタッフに感謝しますガブリエル白狼、エダンズ ・ グループ (www.edanzediting.com/ac) このレポートの下書きを編集してから、博士に感謝いたします。本研究は、ゲノム研究プロジェクト (容湖とミズーリ州) は、山梨県からの助成金から、安田医療財団 (容湖) 補助金によって支えられました。

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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+artefacts+in+a+prospective+series+of+formalin-fixed+tumours+tested+for+mutations+in+hotspot+regions+by+massively+parallel+sequencing.">Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing.</a> BMC Med Genomics. 13 (7), 23 (2014).</li></ol>

著者が明らかに何もありません。

本研究ではチックを使用して臨床病理標本から腫瘍 DNA を取得するための代替方法を提案します。TIC 準備非常にシンプルかつ FFPE のメソッドは、特別な楽器10の要件なしと比較してより少ない時間を必要があります。(図 1) 2 日以内は、DNA の抽出に TIC 準備からすべての手順を完了できます。このメソッド従って遺伝学的検査を行うため所要時間が短く?...

次世代シーケンシング技術提供している研究者の遺伝の変化、メンデルの病気、遺伝的素因、がん1,2,3 のゲノム情報を解析の飛躍的な発展.がんゲノム アトラス (TCGA) と国際がんゲノム コンソーシアム (ICGC) は、一般的な癌4のいくつかの種類の遺伝子変異の同定を追求してきた。何百もの重要なドライバー遺伝子は正常に識別されているといくつかこれらの分子の薬物開発1,5,6のターゲットにされています。
臨床場面における FFPE 標本は病理診断とがんを含むさまざまな病気の分子テストのため通常使用されます。ただし、ホルマリンで固定過程では、DNA 蛋白質または DNA のクロスリンクが発生した、DNA 断片化を誘導しました。したがって、FFPE DNA サンプルは常に遺伝学的解析に適した DNA7,8,9の低質と量のため。また、FFPE 標本を準備するまでに数日かかるし、技術的なスキルのセクションを正確に準備する必要があります。したがって、高品質そのままな DNA を求める簡便かつ迅速な手法を開発することをお勧めします。
細胞診、病理組織学的診断のための代替方法です。細胞サンプル準備は簡単、以下の FFPE 準備10と比較して高価で、かつ高速なアプローチです。TIC 手法は、センチネル リンパ節と周辺組織のいくつかの年11,12術中迅速診断乳がん患者に行われています。ただし、チックの標本から高品質の DNA を得ることができるかどうかを検討して、その後の遺伝学的解析のため使用するほとんどの報告がありません。細胞診標本が一般的パパニコロー (Pap) またはギムザ染色、染色し、我々 は以前報告量と TIC 標本 (特にギムザ染色サンプル) から抽出した DNA の品質は FFPE から採取した試料よりも優れて組織13。Pap 染色に比べると、少ない汚損プロシージャを必要とする利点を有するギムザ染色.Pap 染色の後、サンプルを固定されているし、ステンド グラス、彼らマウントしますする必要がある媒体 (例えばMalinol)、腫瘍細胞、正常細胞、炎症細胞を顕微鏡など、サンプルの内容を区別するためのマウント。Pap の標本は、取付手順なし準備だない試料を乾燥するために顕微鏡下で細胞を観察することはほとんど不可能。比較では、乾燥した状態で観察することができますギムザ染色、したがって、取り付け手順は迅速に携帯電話評価する必要ありません。レーザーマイクロダイ セクション、ギムザ染色より適切な乾燥標本が必要なため。
本報告では TIC ギムザ染色標本を準備するための簡易迅速法を導入し、チックが dna の FFPE 標本と比較してより良いソースであることを示す.

<table><tbody><tr><td>FINE FROST white20 micro slide glass</td><td>Matsunami Glass ind, Ltd</td><td>SFF-011</td><td></td></tr><tr><td>Arcturus PEN Membrane Glass Slides</td><td>Thermo Fisher Scientific</td><td>LCM0522</td><td></td></tr><tr><td>Cyto Quick A solution</td><td>Muto Pure Chemicals</td><td>20571</td><td></td></tr><tr><td>Cyto Quick B solution</td><td>Muto Pure Chemicals</td><td>20581</td><td></td></tr><tr><td>May-Grunwald Solution</td><td>Muto Pure Chemicals</td><td>15053</td><td></td></tr><tr><td>Giemsa solution</td><td>Muto Pure Chemicals</td><td>15002</td><td></td></tr><tr><td>QIAamp DNA FFPE tissue kit</td><td>Qiagen</td><td>56404</td><td></td></tr><tr><td>TaqMan Fast Advanced Master Mix</td><td>Thermo Fisher Scientific</td><td>4444557</td><td></td></tr><tr><td>TaqMan RNase P Detection Reagents Kit</td><td>Thermo Fisher Scientific</td><td>4316831</td><td></td></tr><tr><td>TaqMan Assay from FFPE DNA QC Assay v2</td><td>Thermo Fisher Scientific</td><td>4324034</td><td></td></tr><tr><td>MicroAmp Fast Optical 96-Well Reaction Plate&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>4346907</td><td></td></tr><tr><td>MicroAmp optical Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4311971</td><td></td></tr><tr><td>MicroMixer E36</td><td>TITEC</td><td>0027765-000</td><td></td></tr><tr><td>ViiA 7 Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>VIIA7-03</td><td></td></tr><tr><td>Himac CF16RXII</td><td>Hitachi-koki</td><td>CF16RII</td><td></td></tr><tr><td>Ion Library TaqMan Quantitation Kit</td><td>Thermo Fisher Scientific</td><td>4468802</td><td></td></tr><tr><td>Ion AmpliSeq Cancer Hotspot Panel v2</td><td>Thermo Fisher Scientific</td><td>4475346</td><td></td></tr><tr><td>Ion AmpliSeq Library Kit 2.0</td><td>Thermo Fisher Scientific</td><td>4480442</td><td></td></tr><tr><td>Ion Xpress Barcode Adapters 1-16 Kit</td><td>Thermo Fisher Scientific</td><td>4471250</td><td></td></tr><tr><td>Ion PGM Hi-Q View Sequencing Kit (200 base)</td><td>Thermo Fisher Scientific</td><td>A30044</td><td></td></tr><tr><td>Ion Chef System</td><td>Thermo Fisher Scientific</td><td>4484177</td><td></td></tr><tr><td>Veriti 96-well Thermal Cycler</td><td>Thermo Fisher Scientific</td><td>Veriti200</td><td></td></tr><tr><td>Ion 318 Chip Kit v2 BC</td><td>Thermo Fisher Scientific</td><td>4488150</td><td></td></tr><tr><td>Ion PGM System</td><td>Thermo Fisher Scientific</td><td>PGM11-001</td><td></td></tr><tr><td>Ion PGM Wash 2 Bottle kit</td><td>Thermo Fisher Scientific</td><td>A25591</td><td></td></tr><tr><td>Agencourt&amp;trade; AMPure&amp;trade; XP Kit</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>16-position Magnetic Stand</td><td>Thermo Fisher Scientific</td><td>4457858</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td><td>Thermo Fisher Scientific</td><td>AM12450</td><td></td></tr><tr><td>Nuclease-free water</td><td>Thermo Fisher Scientific</td><td>AM9938</td><td></td></tr><tr><td>MicroAmp&amp;trade; Optical 96-well Reaction Plates</td><td>Thermo Fisher Scientific</td><td>4306737</td><td></td></tr><tr><td>MicroAmp&amp;trade; Clear Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4306311</td><td></td></tr><tr><td>Agencourt&amp;trade; AMPure&amp;trade; XP Kit</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Ethanol(99.5)</td><td>Nacalai Tesque</td><td>08948-25</td><td></td></tr><tr><td>Sodium hydroxide (10M)</td><td>Sigma</td><td>72068</td><td></td></tr><tr><td>DTU-Neo</td><td>TAITEC</td><td>0063286-000</td><td></td></tr><tr><td>E-36</td><td>TAITEC</td><td>0027765-000</td><td></td></tr><tr><td>ECLIPSE Ci-L</td><td>Nikon</td><td>704354</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-2XLS+</td><td>METTLER TOLEDO</td><td>17014393</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-10XLS+</td><td>METTLER TOLEDO</td><td>17014388</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-20XLS+</td><td>METTLER TOLEDO</td><td>17014392</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-100XLS+</td><td>METTLER TOLEDO</td><td>17014384</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-200XLS+</td><td>METTLER TOLEDO</td><td>17014391</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-1000XLS+</td><td>METTLER TOLEDO</td><td>17014382</td><td></td></tr><tr><td>petit-change</td><td>WAKEN</td><td>MODEL8864</td><td>Mini centrifuge</td></tr><tr><td>petit-incubator</td><td>WAKEN</td><td>WKN-2290</td><td>Air incubator</td></tr><tr><td>SensiCare Powder-free Nitrile Exam Gloves</td><td>MEDLINE</td><td>SEM486802</td><td></td></tr><tr><td>Sterile gauze</td><td>Osaki</td><td>11138</td><td></td></tr><tr><td>Refrigerator MediCool</td><td>SANYO</td><td>MPR-312DCN-PJ</td><td></td></tr><tr><td>FEATHER TRIMMING BLAD</td><td>FEATHER</td><td>No.130</td><td></td></tr><tr><td>FEATHER TRIMMING BLAD</td><td>FEATHER</td><td>No.260</td><td></td></tr><tr><td>FEATHER S</td><td>FEATHER</td><td>FA-10</td><td></td></tr><tr><td>Vortex Genius 3</td><td>IKA</td><td>41-0458</td><td>Vortex mixer</td></tr><tr><td>Pincette</td><td>NATSUME</td><td>A-5</td><td></td></tr><tr><td>1.5 mL microtube</td><td>BIOBIK</td><td>RC-0150</td><td></td></tr></tbody></table>

simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology

管理およびがん患者のための特定の分子標的薬の治療前にがんの突然変異の状態を確認する重要です。臨床設定では、ホルマリン固定パラフィン包埋 (FFPE) 組織は、遺伝学的検査のため活躍しています。ただし、FFPE の DNA は、ホルマリン固定過程における断片化や破損は、一般的に。したがって、FFPE DNA は時々 低品質と量の DNA のための遺伝学的検査の適切ではないです。顕微鏡下で観察することができますタッチ捺印細胞診がん細胞からゲノム DNA を取得する (TIC) の方法をご紹介します。細胞形態と癌細胞数は、チックの標本を使用して評価できます。さらに、2 日以内 TIC サンプルからゲノム DNA の抽出を完了ことができます。合計金額、このメソッドを使用して得られた TIC DNA の品質 FFPE DNA のそれよりも高かった。この迅速かつ簡単な方法では、研究者 (例えば、次世代シーケンス解析、デジタル PCR および量的なリアルタイム PCR) 遺伝学的検査の高品質 DNA を取得し、結果をレポートのターンアラウンド時間を短縮することができます。

図 1は、DNA の抽出に TIC 標本作製から全体のプロセスを示しています。特に、TIC サンプルからゲノム DNA を取得するのみ 2 日間を要します。スライド処理の前に腫瘍ストレージの効果を評価しました。組織検体はすぐにスライドに触れられたとき、および組織は、生理食塩水湿らせた滅菌・ ガーゼ 1 h (図 2) のために保たれ...

Watch this Scientific Journal Video about Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology at JoVE.com

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

腫瘍組織から高品質 DNA を取得は、次世代シーケンシングによる遺伝子の変異を分析するための重要な第一歩です。この記事では腫瘍細胞を豊かにし、押印細胞診標本のタッチから無傷の DNA を取得する簡単かつ迅速な方法を提案する.

タッチ捺印細胞診を用いた臨床病理標本から質の高い腫瘍 DNA を取得する簡易法

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

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JoVE Journal

Cancer Research

10.8K ビュー.  Yamanashi Central Hospital. 腫瘍組織から高品質 DNA を取得は、次世代シーケンシングによる遺伝子の変異を分析するための重要な第一歩です。この記事では腫瘍細胞を豊かにし、押印細胞診標本のタッチから無傷の DNA を取得する簡単かつ迅速な方法を提案する.

ビデオ: Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

固体と液体の腫瘍におけるアクショナブル変異の検出のための次世代シーケンシング

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

がん研究

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

循環腫瘍細胞を取り込むため医療用ワイヤー線を用いた腫瘍細胞の特性解析: 蛍光抗体法や DNA 魚に基づく 3 D アプローチ

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

患者の血液サンプルから生存循環腫瘍細胞の急速な分離

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

生物工学

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者の生体流体における腫瘍関連循環DNAの検出とモニタリング

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

目標シーケンシングアプローチによる比較病変分析

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

行動学

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

簡単かつ迅速なプロトコル非酵素的に浸潤リンパ球の分析のための新鮮なヒト組織を解離

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

免疫・感染学

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

エクソンキャプチャと超並列配列決定することにより腫瘍標本における体細胞遺伝子変異を検出する

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

生物学

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCRは、ホルマリン固定、パラフィン包埋された生検組織中のサイトメガロウイルスDNAの検出のための高感度かつ迅速な方法であり、

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &#8216;Touch DNA&#8217; Evidence

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a &#8216;blind-swabbing&#8217; approach will co-sample cellular material from the different individuals, even if the individuals&#8217; cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim&#8217;s DNA may be found in significant excess thus masking any potential perpetrator&#8217;s DNA.
In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, &#8216;smart analysis&#8217; method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 &#181;l microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., &#8220;clumps&#8221;) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in &#8216;touch DNA&#8217; samples, together with an enhanced amplification protocol involving a one-step 5 &#181;l micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).

法医学インチタッチDNA」の証拠から簡体マイクロマニピュレーションにより回収する単一のヒト生体粒子の強化された遺伝解析

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for ...

Enhancing Tumor Content through Tumor Macrodissection

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we describe macrodissection, a method designed to augment the percentage tumor content of a tissue specimen by removing and eliminating unwanted tissue prior to performing downstream nucleic acid extractions. FFPE tissue blocks were sectioned to produce 4-5 &#181;m slide-mounted tissue sections. A representative section was submitted for hematoxylin and eosin (H&#38;E) staining and subsequently reviewed by a board-certified pathologist. During the review, the pathologist identified and marked the regions of tumor tissue in the H&#38;E. Once complete, the demarked H&#38;E was used to guide resection of the serial unstained sections from the same tissue block. To demonstrate the effects of macrodissection, RNA extracted from matched macrodissected and non-dissected Diffuse Large B-Cell Lymphomas (DLBCL) were run on a digital gene expression assay capable of determining DLBCL subtype and BCL2 translocation status. The results showed that macrodissection changed the subtype or BCL2 translocation status calls in 60% of the samples examined. In conclusion, macrodissection is a simple and effective method for performing tumor enrichment prior to nucleic acid extractions, the product of which can then be confidently used in downstream genomic studies.

This protocol presents a method to increase the percent tumor content of formalin-fixed paraffin-embedded tissue samples.

腫瘍マクロ解剖による腫瘍含量の増加

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we ...