Name: building up a high-throughput screening platform to assess the heterogeneity of her2 gene amplification in breast cancers
Uploaded: 2017-12-05T15:00:00
Description: Watch this Scientific Journal Video about Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers at JoVE.com

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이 연구는 IRCCS Ca에서 제도적 검토 보드에 의해 승인 되었다 ' Granda 재단, 폴 병원, 밀라노, 이탈리아.
1입니다. 다양 한 환자와 조직 표본
<ol><li>모든 제공 되며 고 오신 (H & E) 및 원래 진단에서 IHC 슬라이드 포함 하 여 분석, 모든 경우의 보관 슬라이드를 검색 하 고, 있으면, 하나의 H & E의 일치 비 종양 약 유 방 조직 (예를 들어, 수술 여백) .</li>
	<li>
케이스 검토를...

<ol>
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여기, 우리가 heterogeneously 처리 유방암의 높은 수율 TMAs HER2 유전자와 그 해당 centromere SISH 분석을 수행 하는 실험실 전략 세부. 이 방법은 비용 효율적 이며 유 방 암 검색 양식 병 리 기록 보관소의 큰 동료에서 HER2 유전자 증폭이 연구에 대 한 대부분 실험실에서 실시 될 수 있습니다.
때문에 HER2의 임상 중요성 유방암과 그것의 다른 유형의 식에 의해 생성 된 과제...

HER2 proto-oncogene overexpressed 이며 모든 침략 적인 유 방 암1,2의 15-30%에서 증폭 이다. HER2 overexpression의 존재에 의해 유추 됩니다 > 얼룩 (3 +), 유전자 증폭 HER2/centromere 비율은 ≥2 또는 유전자 복사 번호는 ≥6 때 평가 될 수 있다 동안에 계산에 강한 막 immunohistochemical (IHC)와 10%의 셀 제자리 교 잡 (ISH)3에 의해 최소 20 셀.
내부 종양 유전자이 바이오 마커의 평가 및 치료 응답4잠재적으로 불리 한 기여자 되 고 유방암에서 널리 설명 되었습니다. 에 따르면 대학의 미국 병리학 자 (모자), HER2 이종 존재 HER2 에 증폭 하는 경우 > 5%와 < 종양 침투의 50% 세포5. 유감 스럽게도, 유방암에서 HER2 공간이 성분의 실제 발생률 남아 병리학 사이 논쟁의 주제, 몇몇 저자는 유지 그것은 대단히 드문 이벤트와 최대를 제안 하는 다른 경우의 40%는 HER2 이기종1,5,6,7,8,,910. 이 상태를 지탱 하는 생물 학적 메커니즘에도 불구 하 고는 하지 아직 완전히 명백 하 게, 내부 종양 HER2 이종의 전조 및 임상에 미치는 영향은 유 방 암 환자11에 대 한 중요 한.
최근, 밝은 분야 분자 기술이, 발 틱 (CISH) 실버 틱 (SISH), 형광 분 (물고기)12에 비해 몇 가지 장점을 함께 FFPE 직물에서 HER2이 감지 하는 신뢰할 수 있는 방법으로 등장 했습니다. 유감 스럽게도, 단일 경우의 대량 분석 대형 코 호트 연구에서 허무 남아 있습니다. 여러 그룹 histochemistry, IHC와 ISH TMA 기술과 조합 수 암 생물학13,14,,1516의 연구에 귀중 한 전략을 나타내는 제안 했다. 이 널리 채택 된 방법으로 다른 환자에서 조직 샘플 분석할 수 있습니다 동시에, 조직 및 고용 하 고 그로 인하여 경우14의 큰 시리즈의 유니폼 분석을 육성 시 약을 최소화. 그러나, 아무 프로토콜 다른 처리 시 약, 고정 배, 및 고용, 같은 보존 방법으로 보관을 받았다 여러 조직 샘플 동시 처리량이 높은 분자 특성에 대 한 사용할 수 있습니다. 블록입니다.
유방암에서 HER2 공간이 성분의 전조와 임상 의미를 감안할 때, 우리 heterogeneously 처리 경우의 큰 시리즈에 그것을 평가 하는 통합된 분자 플랫폼을 개발 했다. 여기, 우리는 생성 하 고 SISH 통해 유방암의 높은 수율 TMAs에 HER2 증폭의 내 종양이 분석 실험실 전략 묘사. 다음 프로토콜은 측정 하는 종양에 대 한 개발 되었습니다 > 5 m m (> TNM 2017에 따르면 pT1b)17. 작은 병 변, 탈모 직렬 섹션에 분석을 수행 하는 것이 좋습니다. 우리의 절차 각각의 경우에 대 한 6 가지 영역 (범위 4-8)의 의미를 포괄 최대 30 유방암의 동시 IHC 및 SISH 분석 할 수 있습니다. 모두 180 조직 코어 직경, 코어, 그리고 그리드 가장자리 사이 2 밀리미터 사이 500 µ m에서에서 1 m m의 각 TMA 블록에 대해 생성 됩니다.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Surgipath Paraplast</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">39601006</td>
			<td style="width:383px;">Tissue embedding medium, 56 &#176;C melting point</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Eosin Y&#160; 1% water solution</td>
			<td style="width:328px;">Bio Optica, Milan, Italy, EU</td>
			<td style="width:120px;">510002</td>
			<td style="width:383px;">Eosin yellowish, water-soluble</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Carazzi&#8217;s hematoxylin</td>
			<td style="width:328px;">Bio Optica, Milan, Italy, EU</td>
			<td style="width:120px;">506012</td>
			<td style="width:383px;">Alum hematoxylin ripened using potassium iodate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Diamond quality</td>
			<td style="width:328px;">Laboindustria, Arzergrande, Italy, EU</td>
			<td style="width:120px;">33533</td>
			<td style="width:383px;">26x76 mm microscope slides</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Leica CV Mount</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">14046430011</td>
			<td style="width:383px;">Mounting medium, with no monomers, based on polymers of butylmethacrylate in xylene</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">FLEX IHC microscope slides</td>
			<td style="width:328px;">Agilent Technologies (Dako),&#160; Santa Clara, CA, USA</td>
			<td style="width:120px;">K8020</td>
			<td style="width:383px;">Coated microscope slides for adhesion of FFPE for use in IHC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">BenchMark ULTRA</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">N750-BMKU-FS</td>
			<td style="width:383px;">Slide staining system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">CONFIRM anti-Estrogen Receptor (ER) (SP1) Rabbit Monoclonal Primary Antibody</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-4324</td>
			<td style="width:383px;">Primary antibody, ready-to-use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">CONFIRM anti-Progesterone Receptor (PR) (1E2) Rabbit Monoclonal Primary Antibody</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-2223</td>
			<td style="width:383px;">Primary antibody, ready-to-use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-4286</td>
			<td style="width:383px;">Primary antibody, ready-to-use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">PATHWAY HER2 (4B5) Rabbit Monoclonal Primary Antibody</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-4493</td>
			<td style="width:383px;">Primary antibody, ready-to-use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">ultraView Universal DAB Detection Kit</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">760-500</td>
			<td style="width:383px;">Indirect, biotin-free system for detecting mouse IgG, mouse IgM and rabbit primary antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">INFORM HER2 Dual ISH DNA Probe Cocktail</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">780-4422</td>
			<td rowspan="3" style="width:383px;">INFORM HER2 Dual ISH assay - Dual color in situ hybridization FDA approved automated assay for determining HER2 gene status in breast cancer patients&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ultraView Silver ISH DNP Detection Kit</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">800-098</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ultraView Red ISH DIG Detection Kit</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">800-505</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">ISH Protease 3</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">780-4149</td>
			<td style="width:383px;">Used in the ISH process to remove protein that surrounds the target DNA sequences of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Hematoxylin</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">760-2021</td>
			<td style="width:383px;">Modified Gill&#39;s hematoxylin counterstain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Hematoxylin II Counterstaining</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-2208</td>
			<td style="width:383px;">Modified Meyer&#39;s hematoxylin counterstain reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Bluing reagent</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">760-2037</td>
			<td style="width:383px;">Aqueous solution of buffered lithium carbonate for bluing hematoxylin stained sections on glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">HybReady</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">780-4409</td>
			<td style="width:383px;">Formamide-based buffer for ISH assays</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">EZ Prep (10x)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-102</td>
			<td style="width:383px;">Concentrate solution for paraffin removal from tissue samples during IHC and ISH reactions, and to dilute 1:10.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:297px;">SSC Buffer (10X)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-110</td>
			<td style="width:383px;">Sodium Chloride Sodium Citrate buffer solution is used for stringency washes and to rinse slides between staining steps and provide a stable aqueous environment for the in situ hybridization reactions. Dilute 1:5.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:297px;">ULTRA LCS</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">650-210</td>
			<td style="width:383px;">Prediluted (ready-to-use) coverslip solution used as a barrier between the aqueous reagents and the air to prevent evaporation in the IHC and ISH reactions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Reaction Buffer (10x)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-300</td>
			<td style="width:383px;">Tris based buffer solution (pH 7.6 &#177; 0.2) to rinse slides between staining steps during IHC and ISH. Dilute 1:10.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ULTRA Cell Conditioning (ULTRA CC2)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-223</td>
			<td rowspan="2" style="width:383px;">Pretreatment steps in the processing of tissue samples during IHC and ISH. Ready to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ULTRA Cell Conditioning (ULTRA CC1)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-224</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">ultraView Silver Wash II</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">780-003</td>
			<td style="width:383px;">Ready-to-use solution to rinse slides between IHC and ISH staining steps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Microtome</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">RM 2255</td>
			<td style="width:383px;">Automated rotary microtome</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Multistainer</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">ST 5020</td>
			<td style="width:383px;">Workstation for automated staining and coverslipping</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Minicore 1</td>
			<td style="width:328px;">Alphelys, Plaisir, France, EU</td>
			<td style="width:120px;">00-MICO-1</td>
			<td style="width:383px;">Semi-automatic arrayer for TMA contruction with TMA Designer 2 embedded software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Aperio ScanScope CS2</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">K080254</td>
			<td style="width:383px;">Image capture device - digital pathology scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue-Tek III Uni-Cassette</td>
			<td>Sakura Finetek Europe B.V</td>
			<td style="width:120px;">4135</td>
			<td>Cassette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Tissue-Tek Paraform Standard Base Mold</td>
			<td>Sakura Finetek Europe B.V</td>
			<td style="width:120px;">7055</td>
			<td>Stainless-Steel base metal mold</td>
		</tr>
	</tbody>
</table>

building up a high-throughput screening platform to assess the heterogeneity of her2 gene amplification in breast cancers

인간 표 피 성장 인자 수용 체 2 (HER2)에 대 한 표적으로 한 치료 HER2-양성 유방암 환자의 결과 근본적으로 변경. 그러나,의 경우 소수 주요 임상 과제 생성 HER2 양성 세포의 유형이 다른 분포를 표시 합니다. 날짜 하려면, 특성화 및 큰 동료에서 HER2 이기종 유전자 증폭의 정량화에 더 안정적이 고 표준화 된 프로토콜 제안 되었습니다. 여기, 우리는 동시에 여러 개의 유방암의 다른 지형 지역에 걸쳐 HER2 상태를 평가 하는 높은 처리량 방법 제시. 특히, 우리는 향상 된 조직 microarrays (TMAs) 종양의 대상된 매핑 통합을 구성 하는 실험실 절차를 설명 합니다. 모든 TMA 매개 변수는은 제자리에서 교 잡 (SISH)의 포 르 말린 고정 파라핀 포함 (FFPE) 유 방 조직에 대 한 특별히 최적화 되었습니다 했습니다. (즉, ER, PR, Ki67, 그리고 HER2) 전조 및 예측 생체의 Immunohistochemical 분석 자동화 된 절차를 사용 하 여 수행 되어야 한다. 사용자 지정된 SISH 프로토콜은 다른 고정, 처리 및 저장 프로시저를 받았다 여러 조직에 걸쳐 높은-품질 분자 분석을 수 있도록 구현 되었습니다. 이 연구에서 우리는 증거--방침의 특정 DNA 시퀀스 heterogeneously 처리 되 고 여러 가지 지형 영역에 동시에 지역화 된 유방암 효율적이 고 비용 효율적인 방법을 사용 하 여 수를 제공 합니다.

전반적으로, 444 침략 적인 유 방 암 15 TMAs 분 분석 최적화에 반영 되었습니다. 샘플링 2,664 명소 중 2,651 (99.5%) 이전에 선택한 분야의 대표 되었고 따라서 후속 분석에 대 한 순종으로 간주. 내 종양이 종양의 독특한 지형 지역에 HER2 양성 클론의 유형이 다른 배포판에 특정 초점 IHC 및 SIH, 결정 되었다. 표 3 은 다른 histotypes에서 ER, PR, Ki67, HER2 상태에 초점을 맞추?...

Watch this Scientific Journal Video about Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers at JoVE.com

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

HER2 양성 세포의 다른 유형의 배포 유방암의 하위 집합에서 관찰 될 수 있다 고 임상 딜레마를 생성 합니다. 여기, 정의 계량, HER2 내부 종양 유전자이 heterogeneously 처리 유방암의 큰 시리즈에 비교 하는 안정적이 고 비용 효율적인 프로토콜을 소개 합니다.

유방암에서 HER2 유전자 증폭의이 질을 평가 하기 위해 높은 처리량 검열 플랫폼을 구축

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

Research

JoVE Journal

Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

<em&gt; 체외</em인간 대식 세포의 분화와 양극화의 이질성을 연구&gt; 모델

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

연구

면역학 및 감염병학

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

고처리량/고함량 스크리닝에 적용된 세포내 진균의 정량화를 위한 현미경 표티픽 분석

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA 바이러스의 넓은 스펙트럼 화학 억제를위한 높은 처리량 검사

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

High-throughput Gene Tagging in Trypanosoma brucei

High-throughput Gene Tagging in Trypanosoma brucei

높은 처리량 유전자 태그에<em&gt; 트리파노소마 (Trypanosoma) brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

작은 분자의 높은 처리량 스크리닝 적합한 형광 계 분석 림프구

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

살 모 넬 라 종의 심사에 대 한 높은 처리 플랫폼 /Shigella 종

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

포유류 세포에서 효율 Resealing 원형질 막의 높은 처리량 측정

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

평가 하 고 호 중구 Extracellular 함정 대형 계량 높은 처리량 분석 결과

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

세균성 지속성에 미치는 영향을 밝히기 위해 화학 화합물의 높은 처리량 선별

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

호스트-병원체 상호 작용을 조사하기 위한 고처리량 전사 분석

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of ...