Name: simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology
Uploaded: 2018-03-21T13:00:00
Description: 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

We thank all the medical and ancillary staff of the hospital and the patients for consenting to participate. We thank Gabrielle White Wolf, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this report. This study was supported by a Grant-in-Aid for Genome Research Project from the Yamanashi Prefecture (Y.H. and M.O.) and a grant from The YASUDA Medical Foundation (Y.H.).

1. TIC Preparation for Quick Microscopic Assessment Using Normal Glass Slides
<ol>
	<li>Perform the TIC preparation as soon as possible after clinical pathological tissue materials are available. If TIC specimens cannot immediately be prepared, keep the tissue materials covered with saline moistened sterile-gauze and store in the refrigerator to prevent drying of tissues.</li>
	<li>Prepare 5 mm3 tissue material such as solid tumors (e.g., liver, lung, and breast tissues) clinically obtained by surge...

<ol>
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In this study, we presented an alternative method for obtaining tumor DNA from clinical pathological specimens using TIC. TIC preparation is very simple and needs less time compared with FFPE methods, without the requirement for special instruments10. All procedures from the TIC preparation to DNA extraction can be completed within two days (Figure 1). This method thus shortens the turnaround time for performing genetic testing. Notably, this provides a significant ad...

Next generation sequencing technology has provided researchers significant advancements in analyzing genome information in genetic variations, Mendelian disease, hereditary predisposition, and cancer 1,2,3. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have pursued the identification of genetic alterations in several types of common cancers4. Hundreds of essential cancer driver genes have been successfully identified, and some of these molecules are being targeted for drug development1,5,6.
In the clinical setting, FFPE specimens are commonly used for pathological diagnosis and molecular testing for various diseases, including cancer. However, during the fixation process with formalin, DNA-protein or DNA-DNA cross-linking occurs and DNA fragmentation is induced. Thus, FFPE DNA samples are not always suitable for genetic analysis because of low quality and quantity of DNA7,8,9. Additionally, it takes several days to prepare FFPE specimens, and technical skill is necessary to accurately prepare the sections. Therefore, it is desirable to develop a simple and rapid method for obtaining high-quality intact DNA.
Cytology is an alternative method for pathological diagnosis. Cytological sample preparation is a simpler, less expensive, and more rapid approach compared with FFPE preparation10. The TIC technique has been performed on sentinel lymph nodes and marginal tissues from breast cancer patients for intraoperative rapid diagnosis for some years11,12. However, there are few reports that have examined whether high-quality genomic DNA can be extracted from TIC specimens and used for subsequent genetic analysis. Cytological specimens are commonly stained with Papanicolaou (Pap) or Giemsa staining, and we previously reported that the amount and quality of DNA extracted from TIC specimens (especially Giemsa-stained samples) are superior to samples obtained from FFPE tissues13. Compared with Pap staining, Giemsa staining has an advantage in requiring less staining procedures. In Pap staining, after the samples have been fixed and stained, they must be mounted with mounting medium (e.g., Malinol) for distinguishing sample contents, such as tumor cells, normal cells, and inflammatory cells under a microscope. If the Pap specimen is prepared without the mounting step, it is almost impossible to observe the cells under a microscope because the specimen is dried. In comparison, Giemsa staining can be observed in the dried state, therefore, the mounting step is not necessary for quick cellular evaluation. For microdissection, Giemsa staining is more suitable because it requires dry specimens.
In this report, we introduce a simple and rapid method for preparing TIC specimens with Giemsa staining and demonstrate that TIC is a better source for DNA compared with FFPE specimens.

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FINE FROST white20 micro slide glass</td>
			<td style="width:191px;">Matsunami Glass ind, Ltd</td>
			<td style="width:143px;">SFF-011</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;">Arcturus PEN Membrane Glass Slides&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>LCM0522</td>
			<td></td>
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			<td height="41" style="height:41px;">Cyto Quick A solution</td>
			<td>Muto Pure Chemicals</td>
			<td>20571</td>
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			<td>Muto Pure Chemicals</td>
			<td>20581</td>
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			<td height="41" style="height:41px;">May-Grunwald Solution</td>
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			<td>15053</td>
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			<td height="41" style="height:41px;">Giemsa solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">QIAamp DNA FFPE tissue kit</td>
			<td>Qiagen</td>
			<td>56404</td>
			<td></td>
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			<td height="41" style="height:41px;">TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444557</td>
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			<td height="41" style="height:41px;">TaqMan RNase P Detection Reagents Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316831</td>
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			<td height="41" style="height:41px;">TaqMan Assay from FFPE DNA QC Assay v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>4324034</td>
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			<td height="41" style="height:41px;">MicroAmp Fast Optical 96-Well Reaction Plate&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4346907</td>
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			<td height="41" style="height:41px;width:283px;">MicroAmp optical Adhesive Film</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4311971</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">MicroMixer E36</td>
			<td style="width:191px;">TITEC</td>
			<td style="width:143px;">0027765-000</td>
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			<td height="41" style="height:41px;width:283px;">ViiA 7 Real-Time PCR System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">VIIA7-03</td>
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			<td height="41" style="height:41px;width:283px;">Himac CF16RXII</td>
			<td style="width:191px;">Hitachi-koki</td>
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			<td height="41" style="height:41px;width:283px;">Ion Library TaqMan Quantitation Kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4468802</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion AmpliSeq Cancer Hotspot Panel v2</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4475346</td>
			<td style="width:180px;"></td>
		</tr>
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			<td height="41" style="height:41px;width:283px;">Ion AmpliSeq Library Kit 2.0</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4480442</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion Xpress Barcode Adapters 1-16 Kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4471250</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM Hi-Q View Sequencing Kit (200 base)</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">A30044</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion Chef System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4484177</td>
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			<td height="41" style="height:41px;width:283px;">Veriti 96-well Thermal Cycler</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>Veriti200</td>
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			<td height="41" style="height:41px;width:283px;">Ion 318 Chip Kit v2 BC</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4488150</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>PGM11-001</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM Wash 2 Bottle kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>A25591</td>
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			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:143px;">A63881</td>
			<td style="width:180px;"></td>
		</tr>
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			<td height="41" style="height:41px;width:283px;">16-position Magnetic Stand</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4457858</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">AM12450</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Nuclease-free water</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">AM9938</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Optical 96-well Reaction Plates</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4306737</td>
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			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Clear Adhesive Film</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4306311</td>
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			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:143px;">A63881</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ethanol(99.5)</td>
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			<td height="41" style="height:41px;width:283px;">Sodium hydroxide (10M)</td>
			<td style="width:191px;">Sigma</td>
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			<td height="41" style="height:41px;width:283px;">DTU-Neo</td>
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			<td height="41" style="height:41px;width:283px;">E-36&#160;</td>
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			<td>0027765-000</td>
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			<td height="41" style="height:41px;width:283px;">ECLIPSE Ci-L</td>
			<td style="width:191px;">Nikon</td>
			<td style="width:143px;">704354</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-2XLS+</td>
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			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-10XLS+</td>
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		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014392</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-100XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014384</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014391</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014382</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">petit-change</td>
			<td>WAKEN</td>
			<td>MODEL8864</td>
			<td style="width:180px;">Mini centrifuge</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">petit-incubator</td>
			<td>WAKEN</td>
			<td>WKN-2290</td>
			<td style="width:180px;">Air incubator</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">SensiCare Powder-free Nitrile Exam Gloves</td>
			<td style="width:191px;">MEDLINE</td>
			<td>SEM486802</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Sterile gauze</td>
			<td style="width:191px;">Osaki</td>
			<td style="width:143px;">11138</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Refrigerator MediCool</td>
			<td style="width:191px;">SANYO</td>
			<td style="width:143px;">MPR-312DCN-PJ</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.130</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.260</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER&#160; S</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">FA-10</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Vortex Genius 3</td>
			<td style="width:191px;">IKA</td>
			<td>41-0458&#160;</td>
			<td style="width:180px;">Vortex mixer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Pincette</td>
			<td style="width:191px;">NATSUME</td>
			<td style="width:143px;">A-5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">1.5 mL microtube</td>
			<td style="width:191px;">BIOBIK</td>
			<td style="width:143px;">RC-0150</td>
			<td style="width:180px;"></td>
		</tr>
	</tbody>
</table>

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

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Figure 1 shows the entire process from preparing TIC specimens to DNA extraction. Notably, the procedure takes only two days to obtain genomic DNA from TIC samples. We evaluated any effects of tumor storage before the slide processing. We found that tumor cells were attached onto the glass slide when tissue specimens were immediately touched onto the slide, and when tissues were kept in saline moistened sterile-gauze for 1 h (Figure 2</st...

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

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

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

The overall goal of this protocol is to obtain high-quality tumor DNA from clinical samples. This method can help answer key questions in basic and clinical science such as cancer research, diagnosis, and treatment decisions. The main advantage of this technique is that we can obtain tumor DNA more simply and quickly than with previous methods.To begin the protocol, touch the tumor surface of a previously prepared and resected tissue onto a normal glass slide several times with gloved hands. Visually confirm that the touched area is covering more than 80%of the glass slide. Lightly press the normal glass slide against a Polyethylene Naphthalate or PEN membrane slide and rub it gently two to three times with gloved hands.Visually confirm that the cells are transferred from the normal glass slide to the PEN membrane slide. Air dry both the glass and PEN membrane slides for five minutes at room temperature. After drying, stain the normal glass slide for direct cytological examination.Dip the glass slides into fixative solution for five seconds and then stain with Giemsa staining solution for 15 seconds. Assess and screen the tumor contents and cellularity on the normal glass slide entirely with a microscope for quick assessment. Evaluate tumor cells based on several criteria.If the sample shows tumor cellularity over 60%by quick microscopic assessment from the previous section, cut the tumor-touched film of the PEN membrane slides for DNA extraction with a knife and gloved hands. Transfer the cut film to a sterile microcentrifuge tube with a pin set and gloved hands. Store the film-containing microcentrifuge tube at four degrees Celsius until DNA extraction.Open the software and plan the run condition and set the run parameter within the software. Click the plan tab and select template, then select the appropriate run method. Select the application and technique type and click next.Select the instrument, sample preparation kit, library kit type, template kit, sequencing kit, base calibration mode, chip type, control sequence, and barcode set and click next. Select plugins and click next. Select project and click next.Then select default reference and BED files of the targeted region. Type the sample name, select the barcode, and click plan run. Next, perform template preparation and chip loading in an automated instrument according to the manufacturer's instructions.Thaw the reagent cartridge at room temperature for 45 minutes before use. Dilute the undiluted library with nuclease-free water according to the library concentration calculated in step 6.8 and make 20 picomolar libraries. Prepare a pooled library for sequencing and store it on ice.Add 25 microliters of the pooled library with a 100 microliter pipette to the bottom of the sample tube. Turn on the power and open the cover of the automated instrument. Place the sequencing chip, chip adapter, enrichment cartridge, tip cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge to the appropriate position of the automated instrument.Touch set up run and step by step on the screen. Close the cover and touch start check on the screen. After the deck scan process, touch next.Check the display contents, kit type, chip ID, sample ID, chip type, plans. Set the time and touch OK.After finishing the chip loading, touch next and open the cover. Unload the sequencing chip from the chip adapter with gloved hands.Place the chip into the chip container, wrap it with parafilm, and store the chip at four degrees Celsius until the sequencing reaction. Then remove the enrichment cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge from the appropriate position of the automated instrument with gloved hands. Transfer an empty tip cartridge to the waste tip position of the automated instrument with gloved hands.Touch next and close the cover. Then touch start and clean the automated instrument with ultraviolet rays for four minutes. Dissolve a sodium chlorite tablet in 1, 000 milliliters of ultra-pure water and filter the solution with a 0.22 micron filter.Once the sequencing instrument has warmed up, touch clean and next on the screen of the sequencing instrument. Clean the sequencing instrument with 250 milliliters of filter-sterilized sodium chlorite solution, then 250 milliliters of ultra-pure water. Then touch initialize and select the appropriate sequencing kit on the screen.Install a gray shipper with gloved hands. Initialize the sequencing instrument with the wash solution, the pH adjustment solution, and the pH standard solution. Next, add 20 microliters of DATP, DGTP, DCTP, and DTTP nucleotides in 50 milliliter tubes with a 100 microliter pipette.Install a gray shipper at the appropriate location with gloved hands. Load the 50 milliliter tube and screw it onto the sequencing instrument. Select next on the screen to start the initialization step.After completing the initialization step, touch run on the screen and select the appropriate library preparation instrument. Scan the two-dimensional barcode of the chip. Insert the sequencing chip in the appropriate position.Close the chip clamp and instrument door and touch chip check, next, and OK on the screen to start the sequencing run. After the sequencing reaction, transfer the data and perform data analyzing pipeline on the sequencing server. The absolute DNA quantities and the RQ value, an indicator of the degradation level of genomic DNA, show that a higher DNA yield is achieved using the TIC specimens compared with the FFPE specimens.In addition, the RQ values from the TIC DNA were significantly higher compared with that of the FFPE DNA across different tumor types. APC Q1367*was identified in both FFPE and TIC DNA extracted from site one and APC S1356*KRAS G12D, and TP53 M237i were detected in both FFPE and TIC DNA extracted from site two, three, and four suggesting that the identical somatic mutations were identified in paired FFPE and TIC DNA samples prepared from the same tumor site. Notably, the same somatic mutations were detected between primary colorectal cancer and two metastatic liver cancer samples suggesting that the tumor clones from site two metastasized to liver.After its development, this technique paved the way for researchers in the field of molecular and medical research to identify genetic cancers.

simple-rapid-method-to-obtain-high-quality-tumor-dna-from-clinical

Research

JoVE Journal

Cancer Research

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

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.

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

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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.

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

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

Isolation of Nuclei for Multiome Sequencing in Solid Tumor Specimen

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

Author Spotlight: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...