Name: automated dissection protocol for tumor enrichment in low tumor content tissues
Uploaded: 2021-03-29T14:00:00
Description: Watch this Scientific Journal Video about Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues at JoVE.com

The authors would like to thank Carmina Espiritu and Robin E. Taylor for their support in automated dissection development as well as the Genentech Pathology Core Laboratory staff that supported this work.

Prior to initiation, obtain appropriate tissue specimens according to Institutional Review Board (IRB) protocols. All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Genentech, Inc.
1. Tissue and slide preparation
<ol>
	<li>Select FFPE or fresh frozen tissue blocks and utilize the corresponding processing method below.</li>
	<li>Cut tissue block sections onto positively charged glass slides at the desired thickness. Serially section the...

<ol>
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Presented here is a protocol for the application of digital annotation and automated dissection to dissect tumor regions from low tumor content FFPE or fresh frozen tissues for tumor enrichment and use in WES. Combining digital annotation and mask creation with automated dissection significantly reduces the required hands-on time and expertise common to classical methods of tumor enrichment inclusive of manual macrodissection and LCM. The protocol demonstrates a potentially important mid-range tumor enrichment option tha...

Next generation sequencing (NGS) is increasingly utilized for both patient care and in cancer research to help guide treatments and facilitate scientific discovery. Tissue is often limited and small specimens with variable tumor content are routinely used. Tumor adequacy and integrity, therefore, remain a barrier to obtaining meaningful data. Samples with lower tumor percentages may cause difficulty in distinguishing true variants from sequencing artifacts and are often ineligible for NGS1. Tumor enrichment of low tumor content cases, those below 20%, has been shown to help yield sufficient material in order to generate reproducible sequencing data and ensure low frequency variants are not missed2,3. However, limits will vary depending on the platforms utilized and planned use of the data generated.
Traditionally, enrichment of tumor regions for extraction is performed by manual macrodissection or laser capture microdissection (LCM) of formalin fixed paraffin embedded (FFPE) slides. Manual macrodissection, or scraping specified tissue areas from slides, allows tumor regions to be removed for use in downstream assays with relatively low cost, but with low accuracy and low precision2,4. Minimal technical accuracy can be very effective with higher tumor content cases where large swaths of tumor are present and/or minimal tissue loss does not significantly impact results, but low tumor content cases or cases with more dispersed tumor require increased precision. LCM was therefore invented in the 1990s and became a valuable way to precisely remove small, defined, microscopic regions of tissue from formalin fixed paraffin embedded (FFPE) slides5,6,7,8. LCM can be utilized to collect single cell populations when complex heterogeneity of the sample exists9 allowing for collection of previously difficult to separate cell populations. However, LCM requires costly machinery that requires extensive technical expertise and hands-on time10,11,12,13,14.
The instrument used for automated tissue dissection has precision in between that of LCM (~10 &#181;m) and macrodissections (~1 mm)15. Additionally, it exhibits both cost and technical expertise requirements between that of macrodissection and LCM and is designed to perform rapid tissue enrichment from sequential FFPE slides to alleviate the disadvantages of previous methods15. Automated dissection in this fashion utilizes digital annotations or on-stage slide reference image overlays onto serially sectioned unstained tissue slides for dissecting and enriching regions of interest. The instrument uses plastic spinning blade milling tips, 1.5 mL collection tubes and can be used with a number of different fluids for dissection to collect regions of interest for downstream assays inclusive of nucleic extraction and sequencing. The spinning plastic milling tip utilizes inner and outer syringe barrel reservoirs and a plunger to collect buffer, then mills and collects tissue16. The variable milling tip size diameter (250 &#181;m, 525 &#181;m, 725 &#181;m) can allow for dissection of separate tissue areas for comparison, multifocal regions that can be pooled or individual small areas from single or multiple FFPE slides. Section thicknesses used for harvest can be adjusted based on individual experiment needs and users can ensure regions of interest have not been depleted by performing an additional H&#38;E on one serial section immediately after the last section used for harvest.
Automated dissection was identified as a way to enrich tumor content in low tumor content cases and we tested and expanded the intended functionality of an automated tissue dissection instrument, which is currently marketed for use on FFPE clinical specimens up to 10 &#181;m in thickness. The work shows that automated dissection can be applied to both FFPE and fresh frozen human or animal tissue sections up to 20 &#181;m in thickness for research purposes. The protocol also demonstrates an approach to digitally annotate and automate dissection for tumor enrichment in tissues with low tumor content and/or cases with nested, dispersed tumor where meaningful macrodissection is challenging or not feasible and show both quality and yield of nucleic acid sufficient for NGS. Automated dissection can therefore provide mid-level precision and increased throughput for tumor enrichment and could also be applied to enrich other regions of interest or combined with other platforms to answer research or clinical questions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent SureSelectXT</td>
			<td>Agilent</td>
			<td>G9611A</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect Fill Station</td>
			<td>Roche</td>
			<td>8106533001</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect Instrument, Base</td>
			<td>Roche</td>
			<td>8106568001</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect Instrument, Head</td>
			<td>Roche</td>
			<td>8106550001</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect Milling Tips Small</td>
			<td>Roche</td>
			<td>8106509001</td>
			<td></td>
		</tr>
		<tr>
			<td>AVENIO Millisect PC</td>
			<td>Roche</td>
			<td>8106495001</td>
			<td></td>
		</tr>
		<tr>
			<td>BioAnalyzer</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5427R</td>
			<td>Eppendorf</td>
			<td>22620700</td>
			<td>Micro-centrifuge</td>
		</tr>
		<tr>
			<td>Incubation Buffer</td>
			<td>Promega</td>
			<td>D920D</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Autostainer XL</td>
			<td>Leica</td>
			<td>ST5010</td>
			<td>Automated stainer</td>
		</tr>
		<tr>
			<td>Molecular Grade Mineral Oil</td>
			<td>Sigma</td>
			<td>M5904-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Promega</td>
			<td>V302B</td>
			<td>Digestion buffer</td>
		</tr>
		<tr>
			<td>Qiagen AllPrep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80284</td>
			<td></td>
		</tr>
		<tr>
			<td>RLT Plus buffer</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus positively charged microscope slides</td>
			<td>Thermo Scientific</td>
			<td>6776214</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated dissection protocol for tumor enrichment in low tumor content tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

FFPE and FF mouse liver sections containing metastatic colorectal cancer in xenografts were selected. Sections were H&#38;E stained (Figure 1A,E,I) and scanned on a whole slide imager at 20x magnification. A pathologist digitally annotated tumor regions of interest and a mask was generated using commercial software and formatted as a digital png reference image (Figure 1B,F,J). Serial 10 &#181;m and 20 &#181;m thick unstained sa...

Watch this Scientific Journal Video about Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues at JoVE.com

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

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