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 surgery or endoscopy.
	<ol>
		<li>Gently wipe the tissue with sterile-gauze coated with physiological saline and remove blood, if the tissue surface has a lot of blood.</li>
		<li>For microscopic specimens such as biopsy material, keep the sample moistened with sterile-gauze soaked in physiological saline.</li>
	</ol>
	</li>
	<li>Cut and trim the normal tissue with a trimming knife and expose the surface of the tumor lesion, if the tumor masses are not visible grossly.</li>
	<li>Touch the tumor surface of the resected specimens onto a normal glass slide several times with gloved hands. Visually confirm the touched area is over 80% of the normal glass slide.</li>
	<li>Lightly press the normal glass slide against a polyethylene naphthalate (PEN) membrane slide and rub gently 2 - 3 times with gloved hands. Visually confirm the cells are transferred from the normal glass slide to the PEN membrane slide.</li>
	<li>Air-dry both the glass and PEN membrane slides for 5 min at room temperature.</li>
	<li>Stain the normal glass slide for direct cytological examination. Dip the glass slides with fixative solution for 5 s, and then stain with Giemsa staining solution for 15 s.</li>
	<li>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; nuclear enlargement, abnormal karyotype, abundant chromatin, unequal distribution of cells, ratio of nuclear component/cytoplasmic component, cell size, and cell polarity.</li>
</ol>
2. Preparation of the PEN Membrane Slide Film for Genetic Testing
<ol>
	<li>If the sample shows tumor cellularity over 60% by quick microscopic assessment (step 1.8), 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 pincette and gloved hands.</li>
	<li>If the tumor cellularity was determined as low (less than 60% of the tumor contents) by quick microscopic assessment (step 1.8), use laser capture microdissection and obtain tumor samples.
	<ol>
		<li>Perform Giemsa staining to assess the tumor cells using standard protocols.</li>
		<li>Cut the film of the PEM membrane slide by appropriate laser capture microdissection.</li>
		<li>Transfer the cut film to a sterile microcentrifuge tube with a pincette and gloved hands.</li>
		<li>Store the film-containing microcentrifuge tube at 4 &#176;C until DNA extraction (the protocol can be paused here).</li>
	</ol>
	</li>
</ol>
3. DNA Extraction
<ol>
	<li>Perform DNA extraction from the TIC or FFPE tissue samples using a FFPE DNA extraction kit according to the manufacturer&#39;s instructions with minor modifications. An equivalent kit is available for the FFPE DNA extraction step.</li>
	<li>Add 180 &#181;L of tissue lysis buffer (pH = 8.3) to the film-containing microcentrifuge tube with a manual 200-&#181;L pipette. Add 20 &#181;L of proteinase K with a manual 20-&#181;L pipette and mix by vortexing with a vortex mixer at maximum speed (approximately 2,500 rpm) for 5 s.</li>
	<li>Incubate the samples at 56 &#176;C overnight with an air incubator.</li>
	<li>Incubate the FFPE and TIC samples at 90 &#176;C in a heat block for 1 h and 10 min, respectively. Briefly spin down the microcentrifuge tube at 1,500 x g for 5 s at room temperature with a mini centrifuge.</li>
	<li>Add 200 &#181;L of lysis buffer to the sample with a 200-&#181;L pipette and mix thoroughly by vortexing at maximum speed for 5 s.</li>
	<li>Add 200 &#181;L of ethanol (96 - 100%) with a 200-&#181;L pipette and mix thoroughly by vortexing at maximum speed for 5 s. Briefly spin down the microcentrifuge tube at 1,500 x g for 5 s at room temperature with a mini centrifuge.</li>
	<li>Carefully transfer the entire lysate to the spin column with a 1,000-&#181;L pipette and centrifuge at 6,000 x g for 1 min at 25 &#176;C.</li>
	<li>Place the spin column in a clean 2-mL collection tube with gloved hands, and discard the collection tube containing the flow-through into a plastic disposal box.</li>
	<li>Add 500 &#181;L of wash buffer to the spin column with a 1,000-&#181;L pipette and centrifuge at 6,000 x g for 1 min at 25 &#176;C.</li>
	<li>Place the spin column in a clean 2-mL collection tube with gloved hands, and discard the collection tube containing the flow-through into a plastic disposal box.</li>
	<li>Add 500 &#181;L of wash buffer to the spin column with a 1,000-&#181;L pipette and centrifuge at 6,000 x g for 1 min at 25 &#176;C.</li>
	<li>Discard the collection tube containing the flow-through into a plastic disposal box. Place the spin column in a clean 1.5-mL microcentrifuge tube with gloved hands and centrifuge at 20,000 x g for 3 min at 25 &#176;C to dry the membrane.</li>
	<li>Place the spin column in a DNA-low binding tube with gloved hands.
	<ol>
		<li>Add 40 - 50 &#181;L of elution buffer to the center of the membrane with a 100-&#181;L pipette.</li>
		<li>Incubate at room temperature for 5 min and centrifuge at 20,000 x g for 1 min at 25 &#176;C.</li>
		<li>Store the DNA samples at -20 &#176;C until the next step (the protocol can be paused here).</li>
	</ol>
	</li>
</ol>
4. Estimation of DNA Quality by Quantitative Real Time PCR
<ol>
	<li>Prepare the master mix in a sterile microcentrifuge tube with a pipette, as follows: 10 &#181;L of 2x Real-Time PCR Master Mix, 1 &#181;L of 20x RNase P Primer-Probe Mix (amplicon size: 87 bp), and 8 &#181;L of sterile nuclease-free water.</li>
	<li>Prepare the second master mix in one sterile microcentrifuge tube as follows: 10 &#181;L of 2x Real-Time PCR Master Mix, 1 &#181;L of 20x RNase P Primer-Probe Mix (amplicon size: 268 bp), and 8 &#181;L of sterile nuclease-free water.</li>
	<li>Perform serial dilutions of human control genomic DNA (supplied in the kit) 4 times for a five-point standard curve and determine the absolute DNA concentrations13.</li>
	<li>Add 19 &#181;L of the two prepared master mixes (prepared in step 4.1 and 4.2) into separate wells of an optical 96-well reaction plate with a 20-&#181;L pipette.</li>
	<li>Add 1 &#181;L of FFPE DNA or TIC DNA to separate wells containing the reaction mix with a 2-&#181;L pipette. Add 1 &#181;L of nuclease-free water into a separate well containing the reaction mix for the no template control.</li>
	<li>Hold the non-adhesive side of an optical adhesive film and peel back the protective backing from the center of the film. Gently drag the applicator over the film and seal the film over the 96-well plate.</li>
	<li>Gently mix the 96-well plate using a 96-well plate mixer for 10 s at room temperature at 2,000 rpm. Centrifuge the plate briefly at 1,000 x&#160;g for 3 min at room temperature.</li>
	<li>Power on the real-time PCR instrument and insert the 96-well plate. Run the PCR reactions using the following protocol: 95 &#176;C for 20 s, followed by 45 cycles of 95 &#176;C for 1 s and 60 &#176;C for 20 s. Use &#34;standard curve&#34; and &#34;fast mode.&#34;</li>
	<li>Assess DNA fragmentation with the ratio of DNA (relative quantification; RQ) obtained for the long amplicon (268 bp) to the short amplicon (87 bp). RQ is the mean value of the long amplicon/the mean value of the short amplicon13.</li>
</ol>
5. Preparation of the Next Generation Sequencing Library
<ol>
	<li>Prepare the sequencing library for next generation sequencing according to the manufacturer&#39;s instructions.</li>
	<li>Prepare the multiplex PCR master mix in a sterile microcentrifuge tube per sample as follows: 4 &#181;L of 5x Multiplex PCR reaction solution, 4 &#181;L of 5x primer pool, &#8804;6 &#181;L TIC or FFPE DNA (1 - 100 ng), and add nuclease-free water up to 20 &#181;L.
	<ol>
		<li>Add the multiplex PCR master mix to a PCR tube and mix gently by tapping the tube.</li>
		<li>Briefly spin down the microcentrifuge tube at 1,500 x g for 5 s at room temperature with a mini centrifuge.</li>
	</ol>
	</li>
	<li>Run the PCR reactions using the following protocol: 99 &#176;C for 2 min, followed by 20 cycles of 99 &#176;C for 15 s and 60 &#176;C for 4 min, and holding step 10 &#176;C. Briefly spin down the PCR tube with a mini centrifuge at 1,500 x g for 5 s at room temperature. 
	NOTE: Determine the number of cycles based on the number of primer pairs.</li>
	<li>Open the lid of the PCR tube, and add 2 &#181;L of the restriction enzyme with a 2-&#181;L pipette. Close the lid of PCR tube and mix gently by tapping the PCR tube. Briefly spin down the PCR tube with a mini centrifuge at 1,500 x g for 5 s at room temperature.</li>
	<li>Run the PCR reactions using the following protocol: 50 &#176;C for 10 min, 55 &#176;C for 10 min, 60 &#176;C for 20 min, and holding step 10 &#176;C. Briefly spin down the PCR tube with a mini centrifuge at 1,500 x g for 5 s at room temperature.</li>
	<li>Add the adaptor ligation master mix into the each well containing the digested PCR amplicons with a pipette as follows: 4 &#181;L of adaptor ligation solution, 0.5 &#181;L of barcode, 0.5 &#181;L of adaptor, 2 &#181;L of nuclease-free water, and 2 &#181;L of DNA ligase. Close the lid of PCR tube and mix gently by tapping. Briefly spin down the PCR tube with a mini centrifuge at 1,500 x g for 5 s at room temperature.</li>
	<li>Run the PCR reactions using the following protocol: 22 &#176;C for 30 min, 68 &#176;C for 5 min, 72 &#176;C for 5 min, and holding step 10 &#176;C.</li>
	<li>Purify the sequencing library with magnetic beads according to the manufacturer&#39;s instructions.</li>
	<li>Transfer the adaptor-ligated library solution into the 1.5-mL DNA low-binding tube. Add 45 &#181;L of magnetic beads into the DNA low-binding tube for the 1st purification. Mix gently by tapping the tube and incubate for 5 min at room temperature.</li>
	<li>Place the DNA-low binding tube in a magnetic rack, then incubate for 2 min at room temperature until the solution is clear. Carefully discard the supernatant with a 200-&#181;L pipette without disturbing the magnetic beads.</li>
	<li>Add 150 &#181;L of freshly prepared 70% ethanol with a 200-&#181;L pipette, then move the tube side-to-side of the magnet to wash the beads. Carefully discard the supernatant without disturbing the magnetic beads.</li>
	<li>Repeat step 5.11 for a second wash.</li>
	<li>Briefly spin down the tube with mini centrifuge at 1,500 x g for 5 s at room temperature. Place the DNA low-binding tube in a magnetic rack, and carefully discard the ethanol droplets with a 10-&#181;L pipette.</li>
	<li>Add 50 &#181;L of Low TE into the DNA low-binding tube containing the magnetic beads pellet to disperse the beads. Incubate for 2 min at room temperature.</li>
	<li>Place the DNA low-binding tube in a magnetic rack, and incubate at room temperature for 2 min until the solution is clear.</li>
	<li>Transfer the 50 &#181;L of supernatant into the new DNA low-binding tube and add 75 &#181;L of magnetic beads with a 100-&#181;L pipette for the 2nd purification. Mix gently by tapping the tube and incubate for 5 min at room temperature.</li>
	<li>Place the DNA low-binding tube in a magnetic rack, then incubate for 2 min at room temperature until the solution is clear. Carefully discard the supernatant with a 200-&#181;L pipette without disturbing the magnetic beads.</li>
	<li>Add 150 &#181;L of freshly prepared 70% ethanol with a 200-&#181;L pipette, then move the tube side-to-side of the magnet to wash the beads. Carefully discard the supernatant without disturbing the magnetic beads.</li>
	<li>Repeat step 5.18 for a second wash.</li>
	<li>Briefly spin down the tube with a mini centrifuge at 1,500 x g for 5 s at room temperature. Place the DNA low-binding tube in a magnetic rack, and carefully discard the ethanol droplets with a 10-&#181;L pipette.</li>
	<li>Add 50 &#181;L of low TE into the DNA low-binding tube containing the magnetic beads pellet to disperse the beads. Incubate for 2 min at room temperature.</li>
	<li>Place the DNA low-binding tube in a magnetic rack, and incubate at room temperature for 2 min until the solution is clear.</li>
	<li>Transfer the 45 &#181;L of supernatant containing purified library into the new DNA low-binding tube with a 100-&#181;L pipette.</li>
</ol>
6. Quantify the Library Concentration by Quantitative Real Time PCR
<ol>
	<li>Determine the concentration of each library according to the manufacturer&#39;s instructions13.
	<ol>
		<li>Prepare a 20-fold dilution solution as follows: mix 2 &#181;L of purified library and 38 &#181;L of nuclease-free water in a DNA low-binding tube with a 2-&#181;L and 100-&#181;L pipette.</li>
		<li>Store the undiluted libraries at -20 &#176;C until step 7.3.</li>
	</ol>
	</li>
	<li>Prepare a 200-fold dilution solution as follows: mix 5 &#181;L of the 20-fold diluted purified library (prepared in step 6.1) and 45 &#181;L of nuclease-free water in a DNA low-binding tube.</li>
	<li>Prepare a 2,000-fold dilution solution as follows: mix 5 &#181;L of the 200-fold diluted purified library (prepared in step 6.2) and 45 &#181;L of nuclease-free water in a DNA low-binding tube.</li>
	<li>Prepare the reaction master mix as follows: mix 10 &#181;L of 2x master mix solution and 1 &#181;L of 20x primer-probe assay solution in sterile microcentrifuge tube with a pipette, then mix by tapping the tube. Add 11 &#181;L of the reaction master mix into the wells of an optical 96-well reaction.</li>
	<li>Add 9 &#181;L of the 2,000-fold diluted library, 9 &#181;L of each standard control, or 9 &#181;L of nuclease-free water to each well with a 10-&#181;L pipette.</li>
	<li>Hold the non-adhesive side of optical adhesive film and peel back the protective backing from the center of the film.
	<ol>
		<li>Gently drag the applicator over the film and seal the film over the 96-well plate.</li>
		<li>Gently mix the 96-well plate using a 96-well plate mixer for 10 s at room temperature.</li>
		<li>Centrifuge the plate briefly at 1,000 x&#160;g for 3 min at room temperature.</li>
	</ol>
	</li>
	<li>Power on the real-time PCR instrument and insert the 96-well plate. Run the PCR reactions using the following protocol: 50 &#176;C for 2 min, 95 &#176;C for 20 s, followed by 40 cycles of 95 &#176;C for 1 s and 60 &#176;C for 20 s. Use &#34;standard curve&#34; and &#34;fast mode.&#34;</li>
	<li>Calculate the undiluted library concentration by multiplying the concentration determined with qPCR by 2,000.</li>
</ol>
7. Next Generation Sequencing
<ol>
	<li>Plan the run condition and set the run parameter within the software.
	<ol>
		<li>Click [Plan tab] and [Template], and select the appropriate run method.</li>
		<li>Select the application and technique type, and click [Next].</li>
		<li>Select the instrument, sample preparation kit (optional), library kit type, template kit, sequencing kit, base calibration mode, chip type, control sequence (optional), and barcode set, and click [Next].</li>
		<li>Select plugins and click [Next].</li>
		<li>Select project and click [Next].</li>
		<li>Select default reference and BED files of the targeted region.</li>
		<li>Type the sample name, select the barcode, and click [Plan Run].</li>
	</ol>
	</li>
	<li>Perform template preparation and chip loading in an automated instrument according to the manufacturer&#39;s instructions. Thaw the reagent cartridge at room temperature for 45 min before use.</li>
	<li>Dilute the undiluted library with nuclease-free water according to the library concentration calculated in step 6.8 and make 20 pM libraries.
	<ol>
		<li>Prepare a pooled library for sequencing and store on ice.</li>
		<li>Add 25 &#181;L of the pooled library with a 100 &#181;L pipette to the bottom of the sample tube. Use the pooled library within 48 h.</li>
	</ol>
	</li>
	<li>Power on and open the cover of the automated instrument.
	<ol>
		<li>Place the sequencing chip, chip adaptor, enrichment cartridge, tip cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge to the appropriate position of the automated instrument.</li>
		<li>Touch the [Set up Run] and [Step by step] on the screen.</li>
		<li>Close the cover and touch [Start check] on the screen.</li>
		<li>After the deck scan process, touch [Next] on the screen.</li>
		<li>Check the display contents (kit type, chip type, chip ID, sample ID, plans), set the time, and touch [OK] on the screen.</li>
	</ol>
	</li>
	<li>After finishing the chip loading:
	<ol>
		<li>Touch [Next] on the screen and open the cover.</li>
		<li>Unloaded the sequencing chip from chip adaptor with gloved hands.</li>
		<li>Place the chip into the chip container, rap with parafilm and store at 4 &#176;C until the sequencing reaction.</li>
		<li>Removed 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.</li>
		<li>Transfer an empty-tip cartridge to the waste tip position of the automated instrument with gloved hands.</li>
		<li>Touch [Next] and close the cover.</li>
		<li>Touch [Start] and clean the automated instrument by ultraviolet rays for 4 min.</li>
	</ol>
	</li>
	<li>Dissolve a sodium chlorite tablet in 1,000 mL of ultrapure water and filter solution with a 0.22-&#181;m filter flow filter unit.
	<ol>
		<li>Power on the sequencing instrument.</li>
		<li>Touch [Clean] and [Next] on the screen of the sequencing instrument.</li>
		<li>Clean the sequencing instrument with 250 mL of filter-sterilized sodium chlorite solution and subsequently 250 mL of ultrapure water.</li>
	</ol>
	</li>
	<li>Touch [Initialize] and select the appropriate sequencing kit on the screen.
	<ol>
		<li>Install a gray shipper at the appropriate location with gloved hands.</li>
		<li>Initialize the sequencing instrument with the wash solution (provided in the kit), the pH adjustment solution (containing 350 &#181;L of 100 mM sodium hydroxide), and the pH standard solution (provided in the kit).</li>
	</ol>
	</li>
	<li>Add 20 &#181;L of dATP, dGTP, dCTP, and dTTP nucleotides (provided in the kit) in 50-mL tubes (provided in kit) with a 100-&#181;L pipette.
	<ol>
		<li>Install a gray shipper at the appropriate location with gloved hands.</li>
		<li>Load the 50-mL tube and screw onto the sequencing instrument.</li>
		<li>Touch the [Next] on the screen to start the initialization step, which takes approximately 25 min.</li>
	</ol>
	</li>
	<li>After completing the initialization step:
	<ol>
		<li>Touch [Run] on the screen and select the appropriate library preparation instrument.</li>
		<li>Scan the two-dimensional barcode of the chip.</li>
		<li>Insert the sequencing chip on the appropriate position.</li>
		<li>Close the chip clamp and instrument door.</li>
		<li>Touch [Chip check], [Next], and [OK] on the screen to start the sequencing run.</li>
	</ol>
	</li>
	<li>After the sequencing reaction, transfer the data and perform data-analyzing pipeline on the sequencing server13,17.</li>
</ol>

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A., Rossi, J., Cook, N., Wu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fixation+on+the+amplification+of+nucleic+acids+from+paraffin-embedded+material+by+the+polymerase+chain+reaction.">Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.</a> J Histochem Cytochem. 39 (3), 351-354 (1991).</li><li>Srinivasan, M., Sedmak, D., Jewell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fixatives+and+tissue+processing+on+the+content+and+integrity+of+nucleic+acids.">Effect of fixatives and tissue processing on the content and integrity of nucleic acids.</a> Am.J.Pathol. 161 (6), 1961-1971 (2002).</li><li>Adhya, A. K., Mohanty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+touch+imprint+cytology+in+the+preoperative+diagnosis+of+malignancy+in+low+resource+setting.">Utility of touch imprint cytology in the preoperative diagnosis of malignancy in low resource setting.</a> Diagn Cytopathol. 45 (6), 507-512 (2017).</li><li>Lumachi, F., Marino, F., Zanella, S., Chiara, G. B., Basso, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Touch+Imprint+Cytology+and+Frozen-section+Analysis+for+Intraoperative+Evaluation+of+Sentinel+Nodes+in+Early+Breast+Cancer.">Touch Imprint Cytology and Frozen-section Analysis for Intraoperative Evaluation of Sentinel Nodes in Early Breast Cancer.</a> Anticancer Research. 32 (8), 3523-3526 (2012).</li><li>Sumiyoshi, K., 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=Usefulness+of+intraoperative+touch+smear+cytology+in+breast-conserving+surgery.">Usefulness of intraoperative touch smear cytology in breast-conserving surgery.</a> Exp Ther Med. 1 (4), 641-645 (2012).</li><li>Amemiya, K., 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=Touch+imprint+cytology+with+massively+parallel+sequencing+(TIC-seq):+a+simple+and+rapid+method+to+snapshot+genetic+alterations+in+tumors.">Touch imprint cytology with massively parallel sequencing (TIC-seq): a simple and rapid method to snapshot genetic alterations in tumors.</a> Cancer Med. 5 (12), 3426-3436 (2016).</li><li>Goto, T., 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=Mutational+analysis+of+multiple+lung+cancers:+Discrimination+between+primary+and+metastatic+lung+cancers+by+genomic+profile.">Mutational analysis of multiple lung cancers: Discrimination between primary and metastatic lung cancers by genomic profile.</a> Oncotarget. 8 (19), 31133-31143 (2017).</li><li>Goto, T., 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=Detection+of+tumor-derived+DNA+dispersed+in+the+airway+improves+the+diagnostic+accuracy+of+bronchoscopy+for+lung+cancer.">Detection of tumor-derived DNA dispersed in the airway improves the diagnostic accuracy of bronchoscopy for lung cancer.</a> Oncotarget. 8 (45), 79404-79413 (2017).</li><li>Hirotsu, Y., 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=Targeted+and+exome+sequencing+identified+somatic+mutations+in+hepatocellular+carcinoma.">Targeted and exome sequencing identified somatic mutations in hepatocellular carcinoma.</a> Hepatol Res. 46 (11), 1145-1151 (2016).</li><li>Hirotsu, Y., 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=Comparison+between+two+amplicon-based+sequencing+panels+of+different+scales+in+the+detection+of+somatic+mutations+associated+with+gastric+cancer.">Comparison between two amplicon-based sequencing panels of different scales in the detection of somatic mutations associated with gastric cancer.</a> BMC Genomics. 17 (1), 833 (2016).</li><li>Hirotsu, Y., 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=Intrinsic+HER2+V777L+mutation+mediates+resistance+to+trastuzumab+in+a+breast+cancer+patient.">Intrinsic HER2 V777L mutation mediates resistance to trastuzumab in a breast cancer patient.</a> Med Oncol. 34 (1), 3 (2017).</li><li>Hirotsu, Y., 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=Detection+of+BRCA1+and+BRCA2+germline+mutations+in+Japanese+population+using+next-generation+sequencing.">Detection of BRCA1 and BRCA2 germline mutations in Japanese population using next-generation sequencing.</a> Mol Genet Genomic Med. 3 (2), 121-129 (2015).</li><li>Hirotsu, Y., 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=Multigene+panel+analysis+identified+germline+mutations+of+DNA+repair+genes+in+breast+and+ovarian+cancer.">Multigene panel analysis identified germline mutations of DNA repair genes in breast and ovarian cancer.</a> Mol Genet Genomic Med. 3 (5), 459-466 (2015).</li><li>Li&#232;vre, A., 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=KRAS+mutation+status+is+predictive+of+response+to+cetuximab+therapy+in+colorectal+cancer.">KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer.</a> Cancer Res. 66 (8), 3992-3995 (2006).</li><li>Mok, T. S., 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=Osimertinib+or+Platinum-Pemetrexed+in+EGFR+T790M-Positive+Lung+Cancer.">Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer.</a> N Engl J Med. 376 (7), 629-640 (2017).</li><li>Wong, S. 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=Targeted-capture+massively-parallel+sequencing+enables+robust+detection+of+clinically+informative+mutations+from+formalin-fixed+tumours.">Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours.</a> Sci rep. 13 (3), 3493 (2013).</li><li>Wong, S. 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>

The authors have nothing to disclose.

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><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

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. ...

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

Research

JoVE Journal

Cancer Research

10.8K Views.  Yamanashi Central Hospital. 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.

Video: 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.

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

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 ...

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.

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

Bioengineering

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 ...

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.

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

Behavior

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.

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

Immunology and Infection

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.

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

Biology

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.

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).

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.

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