S.W. is a scholar of and is supported by the Cancer Prevention and Research Institute of Texas (RR210034)

This research protocol was approved by the Institutional Review Board (IRB) of the University of Texas Southwestern Medical Center. Informed consent was obtained from all patients prior to surgery.
1. Reagents and materials preparation
<ol>
	<li>Prepare the 0.2 N sodium chloride (HCl) solution in a fume hood by slowly adding 8.212 mL of HCl (37% w/w or 12.1 N) to 491.788 mL of ddH2O. Store at room temperature (RT). 
	CAUTION: Slowly add acid to water. Do not add water to acid.</li>
	<li>Prepare 10 mM citric acid solution (pH 6.0) by dissolving 1.47 g of Tri-sodium citrate (dihydrate) in 400 mL of ddH2O. Use HCl to adjust to pH 6.0, and then bring the final volume to 500 mL with ddH2O. Store the buffer at RT.</li>
	<li>Prepare the 10% Tween-20 solution by adding 100 &#181;L of Tween-20 to 900 &#181;L of ddH2O. Store it at RT.</li>
	<li>Prepare the 10% IGEPAL solution by adding 5 mL of IGEPAL CA-630 to 45 mL of ddH2O. Store it at RT.</li>
	<li>Prepare the 20x SSC (pH 7.0, 3 M NaCl, 0.3 M Sodium Citrate) solution by dissolving 44.1 g of Tri-sodium citrate (dihydrate) and 87.65 g of sodium chloride (NaCl) in 900 mL of ddH2O. Use HCl to adjust to pH 7.0, and then bring the final volume to 1000 mL with ddH2O. Store the buffer at RT.</li>
	<li>Prepare the 2x SSC solution by adding 100 mL of 20x SSC to 900 mL of ddH2O. If necessary, add 0.5 mL of preservative (Table of Materials). Store it at RT.</li>
	<li>Prepare the probe hybridization buffer by mixing 910 &#181;L of ddH2O, 500 &#181;L of 20x SSC, 50 &#181;L of 10% Tween-20, 40 &#181;L of RNase A, 1 mL of 50% dextran sulfate, and 2.5 mL of formamide. Aliquot into 1 mL and store them at -20 &#730;C.</li>
	<li>Prepare the 0.4x SSC with 0.3% IGEPAL solution by mixing 100 mL of 2x SSC, 15 mL of 10% IGEPAL, and 385 mL of ddH2O. Store it at RT.</li>
	<li>Prepare the 2x SSC with 0.1% IGEPAL solution by adding 5 mL of 10% IGEPAL to 495 mL of 2x SSC solution. Store it at RT.</li>
	<li>Freshly prepare the Proteinase K digestion buffer before use by adding 1 &#181;L of Proteinase K to 99 &#181;L of Tris-EDTA buffer.</li>
	<li>Prepare 1 mg/mL DAPI storage stock by dissolving 1 mg of DAPI into 1 mL of ddH2O. Store it at -20 &#176;C and away from light. Prepare the DAPI working solution by adding 1 &#181;L of the DAPI storage stock to 999 &#181;L of 2x SSC solution. Keep away from light till use.</li>
</ol>
2. Sample pretreatment
NOTE: The slide used here contains the specimen.
<ol>
	<li>Age the slide at 60-90&#176;C for 20 min or overnight (Figure 1). 
	NOTE: This step facilitates paraffin melting. Usually, 20 min of heating is sufficient. It can be extended to overnight to accommodate the schedule.</li>
	<li>Deparaffinize the slide by immersing it into xylene or its substitutes in a Coplin jar for 10 min. Repeat this step with fresh xylene or its substitutes. Perform all the following pretreatment and washing steps in a Coplin jar. 
	NOTE: Xylene substitutes are safe and eco-friendly alternatives to xylene. One of the substitutes (see Table of Materials) performs as well as xylene, if not better. Although xylene substitutes produce less odor than xylene, using it inside a fume hood is recommended. If xylene substitutes are not accessible, all procedures are compatible with xylene-based deparaffinization without any changes.</li>
	<li>Wash off the xylene substitute with 100% ethanol for 5 min.</li>
	<li>Rehydrate the slide with 70% ethanol immersion for 5 min.</li>
	<li>Immerse the slide into 0.2 N hydrochloric acid (HCl) at RT for 20 min. 
	NOTE: HCl effectively extracts acid-soluble proteins, such as basic nuclear proteins, to improve DNA accessibility to FISH probes11.</li>
	<li>Immerse the slide into 10 mM of hot citric acid solution and incubate at 90-95 &#176;C for 20 min. 
	NOTE: Citric acid treatment under high temperatures similarly extracts acid-soluble proteins. Both acid treatments are thought to extract extracellular matrix proteins to decrease autofluorescence12. It is recommended that the citric solution be preheated to the desired temperature range before being applied to the slide. Microwave can be a convenient way to do so. A water bath, such as with a sous vide cooker, is the most effective and economical solution for high-temperature incubation.</li>
	<li>Rinse the slide briefly in 2x SSC to neutralize pH.</li>
	<li>Digest the tissue by adding 100-200 &#181;L (enough to completely cover the tissue depending on the size of the section) of Proteinase K digestion buffer and incubate at RT for 1 min. 
	NOTE: Proteinase K digestion further increases the accessibility for FISH probes and reduces autofluorescence. The time of digestion should be optimized based on the tissue types. In most cases, 1 min of digestion is sufficient. Over-digestion leads to halo-shaped nuclei morphology, and the digestion time should be reduced.</li>
	<li>Immediately stop Proteinase K digestion and dehydrate the slide by immersing it into 70% ethanol for 2 min, followed by 85% and 100% ethanol treatment for 2 min each.</li>
</ol>
3. FISH and imaging
<ol>
	<li>Prepare the FISH hybridization mix by diluting 2 &#181;L of FISH probe stock with 8 &#181;L of hybridization buffer, then apply it to the slide. Cover the sample with a coverslip. 
	NOTE: The total FISH probe stock used ranges from 0.5-4 &#181;L, depending on the image quality. If the signal is too low, increase the probe input. Reduce the FISH probe input if the background is too high, especially when fluorescent debris outside the nuclei is observed. The hybridization buffer can either be the one provided with the commercially purchased probes or prepared as in the section 1.</li>
	<li>Place the slides onto a hot plate, such as a slide moat hybridization system, to denature DNA at 75 &#176;C for 2-5 min. Then, transfer the slide onto another hot plate set at 37 &#176;C to hybridize overnight. 
	NOTE: If the hot plate has a water tray or reservoir to maintain the humidity during hybridization, sealing the coverslip with rubber cement is unnecessary.</li>
	<li>After hybridization, dip the slide into 40-60 &#176;C warmed 0.4x SSC with 0.3% IGEPAL CA-630 washing buffer, then carefully remove the coverslip. Continue the washing twice for 5 min each in the dark, with agitation for the first 10-15 s. 
	NOTE: Dipping the slide into the washing buffer helps gently release the coverslip.</li>
	<li>Wash the slide by in SSC with 0.1% IGEPAL CA-630 for 5 min at RT in the dark, with agitation for the first 10-15 s.</li>
	<li>To quench autofluorescence, treat the slide with the autofluorescence quenching kit (see Table of Materials) by applying 150 &#181;L of reagent (50 &#181;L + 50 &#181;L + 50 &#181;L of reagents A, B, C) for 2-5 min, then wash it with 2x SSC for 5 min. 
	NOTE: This is an optional step. Tissue autofluorescence primarily originates from extracellular matrix components, such as collagen and elastin. It is also significantly influenced by lysosomes and mitochondria due to their lipofuscin, NADPH, and flavin coenzyme content. Aldehyde fixation and blood cell presence may also increase autofluorescence well13,14.</li>
	<li>Stain the slide with DAPI for 10 min. Rinse the slide with 2x SSC buffer for 5 min. 
	NOTE: If the slide is not treated by the autofluorescence quenching kit, DAPI staining can be reduced to 2 min.</li>
	<li>Quickly dip the slide into deionized water for no more than 1 s, then quickly dry it by absorbing extra moisture with a paper towel. 
	NOTE: This is an optional step. Deionized water treatment effectively prevents salt crystal deposition of SSC buffer on the slide and improves imaging quality. However, under such low ion conditions, hydrogen bonds between the FISH probe and targeted DNA are weakened, leading to probe dissociation and signal loss. Therefore, maintaining the water treatment step for a very short time is crucial.</li>
	<li>Dry the slide, then mount it with antifade mounting media. Seal the coverslip with nail polish before imaging. 
	NOTE: If the slide is treated by autofluorescence quenching reagent, mount the slide with an antifade mounting medium per the manufacturer&#39;s instruction. Moreover, depending on the type of mounting media, hardening or non-hardening, the sample must be cured for 1-24 h before sealing and imaging. It is recommended that the sample be cured at least overnight to achieve the best refractive index for imaging.</li>
	<li>Use a 60&#215; oil lens to capture fluorescence signal. Use the DAPI channel to adjust the focus. Ensure to obtain multiple Z-stacks. Typically, 5-10 z-stacks with a 1-&#181;m interval are sufficient. Perform a maximum 3D projection to achieve the best resolution. Apply deconvolution or other background-clearing algorithms to further improve the image quality.</li>
</ol>

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

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	<li>ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-cancer+analysis+of+whole+genomes.">Pan-cancer analysis of whole genomes.</a> Nature. 578 (7793), 82-93 (2020).</li><li>Wu, S., Bafna, V., Chang, H. Y., Mischel, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrachromosomal+DNA:+An+emerging+hallmark+in+human+cancer.">Extrachromosomal DNA: An emerging hallmark in human cancer.</a> Annu Rev Pathol. 17, 367-386 (2022).</li><li>Luebeck, J., 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=Extrachromosomal+DNA+in+the+cancerous+transformation+of+Barrett's+oesophagus.">Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.</a> Nature. 616 (7958), 798-805 (2023).</li><li>Nathanson, D. 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=Targeted+therapy+resistance+mediated+by+dynamic+regulation+of+extrachromosomal+mutant+EGFR+DNA.">Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA.</a> Science. 343 (6166), 72-76 (2014).</li><li>Kim, H., 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=Extrachromosomal+DNA+is+associated+with+oncogene+amplification+and+poor+outcome+across+multiple+cancers.">Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers.</a> Nat Genet. 52 (9), 891-897 (2020).</li><li>Pal Choudhuri, 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=Acquired+cross-resistance+in+small+cell+lung+cancer+due+to+extrachromosomal+DNA+amplification+of+MYC+paralogs.">Acquired cross-resistance in small cell lung cancer due to extrachromosomal DNA amplification of MYC paralogs.</a> Cancer Discov. 14 (5), 804-827 (2024).</li><li>Greytak, S. R., Engel, K. B., Bass, B. P., Moore, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+molecular+data+generated+with+FFPE+biospecimens:+Lessons+from+the+literature.">Accuracy of molecular data generated with FFPE biospecimens: Lessons from the literature.</a> Cancer Res. 75 (8), 1541-1547 (2015).</li><li>Chrzanowska, N. M., Kowalewski, J., Lewandowska, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorescence+in+situ+hybridization+(FISH)+in+diagnosis+and+tailored+therapies+in+solid+tumors.">Use of fluorescence in situ hybridization (FISH) in diagnosis and tailored therapies in solid tumors.</a> Molecules. 25 (8), 1864 (2020).</li><li>Cui, C., Shu, W., Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization:+Cell-based+genetic+diagnostic+and+research+applications.">Fluorescence in situ hybridization: Cell-based genetic diagnostic and research applications.</a> Front Cell Dev Biol. 4, 89 (2016).</li><li>Finn, E. H., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+DNA+FISH+protocol+to+visualize+genome+regions+in+human+cells.">A high-throughput DNA FISH protocol to visualize genome regions in human cells.</a> STAR Protoc. 2 (3), 100741 (2021).</li><li>Watters, A. D., Bartlett, J. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+in+paraffin+tissue+sections.">Fluorescence in situ hybridization in paraffin tissue sections.</a> Mol Biotechnol. 21 (3), 217-220 (2002).</li><li>Richardson, S. O., 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=One-fits-all+pretreatment+protocol+facilitating+fluorescence+in+situ+hybridization+on+formalin-fixed+paraffin-embedded,+fresh+frozen+and+cytological+slides.">One-fits-all pretreatment protocol facilitating fluorescence in situ hybridization on formalin-fixed paraffin-embedded, fresh frozen and cytological slides.</a> Mol Cytogenet. 12, 27 (2019).</li><li>Monici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+autofluorescence+research+and+diagnostic+applications.">Cell and tissue autofluorescence research and diagnostic applications.</a> Biotechnol Annu Rev. 11, 227-256 (2005).</li><li>Davis, A. 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=Characterizing+and+diminishing+autofluorescence+in+formalin-fixed+paraffin-embedded+human+respiratory+tissue.">Characterizing and diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue.</a> J Histochem Cytochem. 62 (6), 405-423 (2014).</li><li>Cancer Genome Atlas Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+portraits+of+human+breast+tumours.">Comprehensive molecular portraits of human breast tumours.</a> Nature. 490 (7418), 61-70 (2012).</li><li>Liang, Z., 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=Chromatin-associated+RNA+dictates+the+ecDNA+interactome+in+the+nucleus.">Chromatin-associated RNA dictates the ecDNA interactome in the nucleus.</a> bioRxiv. , (2023).</li><li>Lange, J. 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=The+evolutionary+dynamics+of+extrachromosomal+DNA+in+human+cancers.">The evolutionary dynamics of extrachromosomal DNA in human cancers.</a> Nat Genet. 54 (10), 1527-1533 (2022).</li><li>Hung, K. L., 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=ecDNA+hubs+drive+cooperative+intermolecular+oncogene+expression.">ecDNA hubs drive cooperative intermolecular oncogene expression.</a> Nature. 600 (7890), 731-736 (2021).</li><li>Storlazzi, C. 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=Gene+amplification+as+double+minutes+or+homogeneously+staining+regions+in+solid+tumors:+origin+and+structure.">Gene amplification as double minutes or homogeneously staining regions in solid tumors: origin and structure.</a> Genome Res. 20 (9), 1198-1206 (2010).</li><li>Guerin, T. M., Marcand, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakage+in+breakage-fusion-bridge+cycle:+an+80-year-old+mystery.">Breakage in breakage-fusion-bridge cycle: an 80-year-old mystery.</a> Trends Genet. 38 (7), 641-645 (2022).</li><li>Nguyen, H. 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=3D+mapping+and+accelerated+super-resolution+imaging+of+the+human+genome+using+in+situ+sequencing.">3D mapping and accelerated super-resolution imaging of the human genome using in situ sequencing.</a> Nat Methods. 17 (8), 822-832 (2020).</li><li>Deshpande, V., 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=Exploring+the+landscape+of+focal+amplifications+in+cancer+using+AmpliconArchitect.">Exploring the landscape of focal amplifications in cancer using AmpliconArchitect.</a> Nat Commun. 10 (1), 392 (2019).</li><li>Rajkumar, U., 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=EcSeg:+Semantic+segmentation+of+metaphase+images+containing+extrachromosomal+DNA.">EcSeg: Semantic segmentation of metaphase images containing extrachromosomal DNA.</a> iScience. 21, 428-435 (2019).</li></ol>

S.W. is a member of the scientific advisory board of Dimension Genomics Inc.

FISH is a fast and affordable option for cytogenetic diagnosis. Especially in determining whether ecDNA is present in cancer, FISH evidence remains the gold standard1. FISH in FFPE tissue allows rapid determination of gene status in a patient&#39;s biopsy specimens, allowing for quicker diagnosis and tracking changes throughout the disease&#39;s progress. This technique is particularly valuable for testing clinical samples that have already been collected for pathology.
This protocol involves several critical steps. The first step is thorough deparaffinization. Residual paraffin can disrupt FISH hybridization. If the sample still appears waxy after step 2, it should be treated again with fresh xylene or its substitutes.
Second, protein extraction and digestion are critical. These processes not only enhance the DNA&#39;s accessibility to the FISH probe but also significantly reduce auto-fluorescence. This protocol includes three deproteinization steps. While the treatment with 0.2 N HCl and 10 mM citric acid is straightforward, the proteinase K digestion may require optimization. Over-digestion is the most common error when using proteinase K, resulting in halo-shaped nuclei. Shortening the digestion time will improve the nuclei morphology. Additionally, it is recommended not to digest more than four samples simultaneously to minimize the time difference between the first and the last sample. It is important to note that even an intact nucleus may appear as a halo under high-magnification and high-resolution microscopy. This is because the nucleus is not on the same focal plane. Therefore, it is suggested to take multiple Z-stacks and perform a max projection to inspect the nuclear morphology.
Lastly, quenching autofluorescence is recommended. Although acid extraction and proteinase K digestion can significantly reduce protein-derived background, fluorescent metabolites may still affect the imaging quality.
While FISH offers unparalleled spatial resolution in identifying focal gene amplification, it has limitations. First, the content and throughput are low compared to PCR or next-generation sequencing (NGS) based approaches. Typically, one to three FISH probes of different colors can be applied to a single slide without specialized equipment. Nonetheless, advancements in automation technologies have made high-content and high-throughput FISH, such as in situ sequencing21, feasible. Second, the FISH probe design requires prior information. The ongoing efforts to identify recurrent focal amplification events in cancer have enabled the creation of pre-designed FISH panels for laboratory and clinical applications. For instance, MYC-family oncogenes are frequently amplified as ecDNA in small-cell lung cancer to mediate chemotherapy resistance. Therefore, a FISH panel targeting MYC, MYCL, and MYCN genes can expedite the determination of treatment responses in biopsies. In comparison, NGS allows a more unbiased screening of genes of interest. However, among NGS-based technologies, only whole-genome sequencing with computation-expensive analysis22 can characterize ecDNA.
In summary, we present robust and comprehensive instructions for investigating focal gene amplification in FFPE samples. By examining the FISH signal pattern, it becomes unequivocally clear whether and how a gene locus is amplified. We anticipate the integration of machine learning into the image analysis23 of interphase nuclei to extract cytogenetic information regarding copy number and the form of amplification (chromosome or ecDNA), thereby streamlining the molecular diagnosis process and enhancing our understanding of pathogenetic mechanisms in cancer.

The study of focal oncogene amplification is crucial as it drives cancer formation, progression, and therapy resistance1. Importantly, oncogenes and immunoregulatory genes may amplify as extrachromosomal DNAs (ecDNA), whose asymmetric inheritance promotes genetic heterogeneity in cancer2,3. ecDNA has been linked to therapy resistance and unfavorable clinical outcomes4,5,6.
Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a vast archival resource in pathology laboratories, offering abundant information for retrospective studies. However, extracting molecular data from FFPE specimens through PCR or sequencing is challenging due to nucleic acid fragmentation, degradation, and cross-linking during fixation7. Among the array of techniques available for molecular analysis of FFPE tissues, fluorescent in situ hybridization (FISH) has proven effective for visualizing specific DNA sequences8.
Despite the advancement of modern molecular diagnostic techniques, the ability of FISH to visualize and quantify gene amplification at the single-cell level provides valuable insights into the molecular mechanisms underlying tumorigenesis and clinical outcomes. By using fluorescently labeled probes complementary to the target gene of interest, FISH can conveniently resolve the localization of an oncogene and may infer the form of oncogene amplification (such as ecDNA) within individual cells, which is otherwise impossible or expensive through other technologies. Therefore, FISH offers an economical way to assess tumor heterogeneity and clonal evolution9. Furthermore, advances in automation, imaging, and computational analysis have facilitated high-throughput analysis of FISH data, enabling robust quantification of gene amplification across large tissue cohorts10.
However, applying FISH to FFPE tissue presents inherent challenges, including cross-linking artifacts and background autofluorescence. Overcoming these obstacles requires careful optimization of each procedure to ensure accurate and reproducible results. This paper provides a step-by-step, fully optimized protocol for applying FISH to investigate gene amplification in FFPE tissue samples. Using a probe targeting the ERBB2 (HER2) gene locus, we demonstrate that FISH can robustly detect ERBB2 amplification status in FFPE samples from breast cancer patients. It is even possible to estimate whether ERBB2 is amplified as ecDNAs. By synthesizing existing literature and our experimental findings, we elucidate the methodological considerations, technical challenges, and potential pitfalls of FISH-based analysis. We also discuss the clinical relevance of gene amplification profiling in various cancer types, highlighting its prognostic significance and potential for personalized therapeutic strategies.
In summary, this paper underscores the importance of FISH as a valuable tool for studying gene amplification in FFPE tissue specimens, offering unparalleled insights into tumor biology and guiding clinical decision-making in oncology. With continued refinement and integration with complementary molecular assays, FISH-based analysis stands poised to further enhance our understanding of cancer pathogenesis and improve patient outcomes in the era of precision medicine.

<table><tbody><tr><td>DAPI</td><td>Tocris Bioscience</td><td>5748</td><td>Nucleus staining</td></tr><tr><td>Dextran sulfate 50% solution</td><td>EMD Millipore Sigma</td><td>S4030</td><td>Probe hybridization buffer</td></tr><tr><td>ERBB2 (HER2) FISH Probe</td><td>Empire Genomics</td><td>ERBB2-20-RE</td><td>FISH probe</td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>2716</td><td>Dehydrating and hydrating tissue</td></tr><tr><td>Formamide</td><td>Thermo Scientific Chemicals</td><td>205821000</td><td>Probe hybridization buffer</td></tr><tr><td>Formula 83 (Xylene substitute)</td><td>CBG Biotech</td><td>CH0104A</td><td>Removing paraffin</td></tr><tr><td>Hydrochloric acid</td><td>Fisher Chemical</td><td>A144-500</td><td>Sample pretreatment</td></tr><tr><td>IGEPAL CA-630&amp;nbsp;</td><td>Thermo Scientific Chemicals</td><td>J19628K2</td><td>Slide washing</td></tr><tr><td>Proclin 300</td><td>Sigma-Aldrich</td><td>48914-U</td><td>Preservative for SSC buffer (optional)</td></tr><tr><td>Proteinase K (800 units/mL)</td><td>New England Biolabs</td><td>P8107S</td><td>Protein digestion</td></tr><tr><td>RNase A (20 mg/mL)</td><td>New England Biolabs</td><td>T3018L</td><td>Probe hybridization buffer</td></tr><tr><td>Slide Moat Hybridization System</td><td>Boekel Scientific</td><td>280001</td><td>Sample denature and hybridization. Alternative hot plates are acceptable.</td></tr><tr><td>Sodium chloride</td><td>Fisher Chemical</td><td>S2713</td><td>SSC buffer</td></tr><tr><td>Sodium citrate dihydrate</td><td>Fisher BioReagents</td><td>FLBP3271</td><td>SSC buffer and sample pretreatment</td></tr><tr><td>Tris-EDTA (TE) buffer</td><td>Fisher BioReagents</td><td>BP2473500</td><td>Proteinase K digestion buffer</td></tr><tr><td>Tween-20</td><td>Fisher BioReagents</td><td>BP337-500</td><td>Probe hybridization buffer</td></tr><tr><td>Vectashield antifade mounting media</td><td>Vector Laboratories&amp;nbsp;</td><td>H190010</td><td>Slide mounting</td></tr><tr><td>Vector TrueVIEW</td><td>Vector Laboratories&amp;nbsp;</td><td>SP8400</td><td>Autofluorescence quenching kit</td></tr></tbody></table>

robust detection of gene amplification in formalin-fixed paraffin-embedded samples by fluorescence in situ hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

We used FFPE samples from both HER2-positive and negative breast cancers to demonstrate the result of FISH imaging. Amplification of HER2 (encoded by the ERBB2 gene) is a favorable marker due to the availability and effectiveness of HER2 molecular targeting therapies. On the contrary, patients with triple-negative breast cancers, which lack expression of HER2, estrogen receptor (ER), and progesterone receptor (PR), face poor outcomes due to limited therapeutic options. Therefore, determining the HER2 status is crucial in breast cancer research and treatment15.
In the triple-negative breast cancer sample, most nuclei display two distinct dots representing HER2/ERBB2 FISH signals. Some nuclei may only have one dot due to sectioning bias (Figure 2, left). In contrast, HER2-positive samples present abundant FISH signals with two different patterns. One pattern shows scattered dots throughout the nucleus (Figure 2, middle). This pattern is a characteristic of ecDNA morphology, as ecDNAs may not occupy a unique and organized nuclear territory16. Furthermore, ecDNAs&#39; asymmetric segregation during mitosis drives copy number variation, leading to signal heterogeneity among nuclei17. Some nuclei may show occasional clusters, indicative of ecDNA hubs18 (Figure 2, right). The other type of HER2 amplification primarily displays rod-shaped, condensed aggregates. This morphology likely indicates chromosome-based amplification, such as homogeneously staining regions (HSR)19 or through the breakage-fusion-bridge (BFB) cycle20. Notably, ecDNA, HSR, and BFB amplification can co-exist in the same nucleus. Therefore, examining multiple nuclei is recommended to infer the form of focal amplification.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66978/66978fig01.jpg" /> 
Figure 1: Schematic for FISH in FFPE samples. <a href="https://www.jove.com/files/ftp_upload/66978/66978fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66978/66978fig02.jpg" /> 
Figure 2: Representative FISH image in breast cancer FFPE samples. Magnification: 600x; Scale bar: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66978/66978fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

robust-detection-gene-amplification-formalin-fixed-paraffin-embedded

Research

JoVE Journal

Cancer Research

1.1K Views.  University of Texas Southwestern Medical Center. This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens. 

Video: Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is presented.

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Genetics

FISH for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

Author Spotlight: RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells 

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain &#954; or &#955;. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of ...

Medicine

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These ...

FFPE Tissue Pretreatment for RNA CISH: A Procedure to Process Formalin-Fixed Paraffin-Embedded Tissue Sections for RNA Chromogenic In Situ Hybridization

Source: Outh-Gauer, S. et al. <a href="https://www.jove.com/video/59422" target="_blank">Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.</a> J. Vis. Exp. (2019)
This video demonstrates the steps involved in the pretreatment of FFPE tissue specimens for RNA chromogenic in situ hybridization. The pretreatment process helps maintain nucleic acid integrity within the cells and overall tissue morphology.

Source: Outh-Gauer, S. et al. Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis. J. Vis. Exp. (2019)
This video ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

Assays & techniques

Probe Hybridization and Signal Amplification in RNA In Situ Hybridization: A Technique for Detecting Specific RNA Sequences in Tissue Sections

Source: Outh-Gauer, S. et al. <a href="https://www.jove.com/video/59422" target="_blank">Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.</a> J. Vis. Exp. (2019)
This video describes a sequential hybridization strategy that involves hybridizing mRNA probes to specific target mRNA. Following probe hybridization, signal amplification is performed to enhance resolution via reduction of signal-to-noise ratio during RNA-CISH of histological samples.

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Summary

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Chapters in this video

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

FFPE Tissue Pretreatment for RNA CISH: A Procedure to Process Formalin-Fixed Paraffin-Embedded Tissue Sections for RNA Chromogenic In Situ Hybridization

Probe Hybridization and Signal Amplification in RNA In Situ Hybridization: A Technique for Detecting Specific RNA Sequences in Tissue Sections