Metallographic in situ hybridization

www.elsevier.com/locate/humpath

Human Pathology (2007) 38, 1145–1159

Progress in pathology

Metallographic in situ hybridizationB

Richard D. Powella,*, James D. Pettayb,c, William C. Powelld, Patrick C. Roched,Thomas M. Grogand, James F. Hainfelda, Raymond R. Tubbsb,c

aNanoprobes, Incorporated, 95 Horseblock Road, Unit 1, Yaphank, NY 11980, USAbCleveland Clinic Foundation, Division of Pathology and Laboratory Medicine, Cleveland, OH 44195, USAcCleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USAdVentana Medical Systems, Incorporated, 1910 Innovation Park Drive, Tucson, AZ 85755, USA

Received 23 March 2007; revised 30 April 2007; accepted 1 May 2007

☆ This project was supported by SmProgram (STTR) grant 5R42CA83618-0Research (SBIR) grant 1R43CA11118Institute (National Institutes of Health) tand Cleveland Clinic Lerner College ofgrant number 5R44 GM064257-03 fromMedical Sciences (National Institutes ofCleveland Clinic Lerner College of Meresponsibility of the authors and do noviews of the National Cancer Institute oMedical Sciences.

⁎ Corresponding author.E-mail address: rpowell@nanoprobe

0046-8177/$ – see front matter © 2007doi:10.1016/j.humpath.2007.05.004

SummaryMetallographic methods, in which a target is visualized using a probe or antibody that depositsmetal selectively at its binding site, offers many advantages for bright-field in situ hybridization (ISH)detection as well as for other labeling and detection methods. Autometallographically enhanced goldlabeling procedures have demonstrated higher sensitivity than conventional enzyme chromogens.Enzyme metallography, a novel procedure in which an enzymatic probe is used to deposit metal directlyfrom solution, has been used to develop bright-field ISH methods for HER2 gene determination in breastcancer and other biopsy specimens. It provides the highest level of sensitivity and resolution, both forvisualizing endogenous gene copies in nonamplified tissues and for resolving multiple gene copies toallow copy enumeration in amplified tissues without the need for oil immersion or fluorescence optics.An automated enzyme metallography procedure, silver ISH, has been developed for use in slide-staininginstruments. Metallographic staining also provides excellent results for immunohistochemistry and maybe combined with other staining procedures for the simultaneous detection of more than one gene orcombinations of genes and proteins.© 2007 Published by Elsevier Inc.

Keywords:
In situ hybridation;Enzyme metallography;HER2;Breast cancer;Automated slide staining;Autometallography
all Business Technology Transfer3 and Small Business Innovation2-01 from the National Cancero Nanoprobes, Inc., Yaphank, NY,Medicine, Cleveland, OH, and bythe National Institute of GeneralHealth) to Nanoprobes, Inc., anddicine. Its contents are solely thet necessarily represent the officialr the National Institute of General

s.com (R. D. Powell).

Published by Elsevier Inc.

1. Introduction

1.1. Historical development of in situhybridization detection methods

After the determination of the structure of DNA [1], it wasnot until 15 years later that methods for localizing DNA andRNA at the molecular level began to emerge, usingcomplementary sequences as probes. The development ofthe method was driven by the discovery that chemicaldenaturation procedures such as high temperature or sodiumhydroxide treatment could alter the staining properties ofdifferent regions along chromosomes, leading to the

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1146 R. D. Powell et al.

development of banding profiles that could be used toidentify and localize chromosomal features [2]. Differentialstaining with fluorescent dyes was used to elucidate bandingstructures within chromosomes [3]. The genesis of modern insitu hybridization (ISH) was the discovery that specificsequences localized to these bands. Initially, detection usedradioisotopically labeled RNA probes to localize comple-mentary DNA (cDNA) in tissue sections and cell spreadsusing autoradiography: [4-6] in one of the three earliestreports, Pardue and Gall [4] used thymidine-3H–labeledRNA fractions extracted from cultured mouse and Xenopuscellular extracts to localize cDNA in denatured tissuesections. Although cumbersome and limited in resolutionby the scattering cone of radioactive emissions, these earlyexperiments effectively demonstrated the localization ofspecific DNA sequences to chromosomal regions.

The technique of ISH achieved wider use as a number ofenabling technologies were developed. Improved probepreparation and labeling methods, including random primelabeling, nick translation reaction, polymerase chain reaction(PCR)–based labeling, and improved methods for cloningtarget sequences, made probe preparation more convenientand affordable [2]. The critical development, however, wasthe introduction of fluorescent labels, which provide higherresolution and simpler visualization and avoid the hazards ofradioactive probes and the long delay required to develop theautoradiographic signal. These enabled the technique offluorescence ISH (FISH) [7]. Labeling may be direct, inwhich the fluorescent label is linked directly to the nucleicacid hybridization probe [8], or indirect, in which thishybridization probe is bound in turn by a labeled secondaryprobe such as fluorescently labeled antibody or proteintargeted to a unique feature or hapten incorporated into theprobe [9]. Another important advantage of FISH is thecapability of imaging multiple targets: the development of avariety of spectrally distinct fluorescent labels enabled thesimultaneous detection of multiple genetic targets usingdifferently labeled probes [10]. More recently, sensitivity hasbeen increased by the application of target or signalamplification protocols [11] such as tyramide signalamplification (TSA), in which either fluorescently labeledtyramides [12] or biotin-tyramide, which is then detectedwith fluorescently labeled avidin or streptavidin [13], isdeposited by an enzyme targeted to the gene of interest. FISHhas been used successfully for mapping repetitive and single-copy DNA sequences on metaphase chromosomes, inter-phase nuclei, chromatin fibers, and naked DNA molecules,and for chromosome identification and karyotype analysisthrough the localization of large repeated families, typicallythe ribosomal DNAs and major tandem array families [2].However, one of the most important applications has beenFISH of single-copy DNA sequences, in particular disease-related genes in humans and other eukaryotic model species[14], and the detection of infectious agents [15-17]. Theacceptance of FISH as a clinical molecular diagnosticmethod [18] has established it as an important tool for the

diagnosis of cancer and other medical conditions. However,despite these advantages, FISH is not always ideal forclinical diagnostic practice, and in this article, we willdiscuss alternative methods for ISH using enzyme metallo-graphy (EnzMet), which can visualize single genes in abright field, requires less complex equipment, enables fasterthroughput, and can provide more information for thepracticing pathologist.

1.2. The HER2 gene: significance and statusassessment

The detection methods presented in this article areapplicable to the visualization of many genetic targets.However, because of its medical significance, their initialapplication has been directed to the evaluation of the HER2gene in breast cancer. Breast cancer is the second leadingcause of cancer death in women: the American CancerSociety predicts that 178,480 new cases of invasive breastcancer and 62,030 new cases of carcinoma in situ will bediagnosed in the United States in 2007, and 40,060 womenand 450 men will die from the disease [19]. A woman'slifetime chance of developing invasive breast cancer is about1 in 8 [19]. Tumors from individual patients differ in theirbiologic behavior and present the pathologist with 2important challenges: (a) to identify smaller tumors of lowstage, which, although they appear to have a relativelyfavorable clinical prognosis, possess phenotypic and geno-typic characteristics that portend aggressive malignantbehavior; and (b) to select the most efficacious therapeuticregimen [20].

Only about half of breast cancer patients are cured bylocal treatment [21], and therefore, indicators that predictaggressive malignant behavior are important. The HER2(ERBB2) proto-oncogene encodes a transmembrane receptortyrosine kinase of Mr185 kDa (p185), which is structurallyand functionally analogous to the epidermal growth factorreceptor; [22] the oncogene is known to stimulate thetranscription of N-acetylglucosaminyltransferase V, resultingin a 3-fold increase in enzyme activity in neu-transformedNIH3T3 cells [23]. Amplification of the HER2 oncogene(also referred to as HER2/neu) and overexpression of thecorresponding HER2-encoded protein are found in about20% to 30% of invasive breast carcinomas [24] and correlateclinically with aggressive malignant behavior in tumors andpoor patient outcomes [25,26], including higher rates ofmetastasis [27,28], higher mortality [29], more rapidprogression, and increased likelihood of recurrence [30].Genomic amplification of the HER2 gene in lymph node–negative invasive breast carcinoma has been associated witha significant reduction in disease-free survival [31] andpoorer prognosis in metastatic breast cancer [32]. Amplifica-tion of the HER2 gene correlates with reduced levels ofestrogen and progesterone receptors, lower natural killer cellactivity [33], and significantly higher proliferative activity[34]. A recent study demonstrated that proliferation index

1147Metallographic in situ hybridization

and HER2 overexpression were the biologic markers withthe highest predictive prognostic value in minimal breastcarcinoma [35].

HER2 gene amplification is also associated withresistance to chemotherapy [31], possibly due to theincreased proliferation [36]. In these cases, humanizedmonoclonal antibodies against p185 (Herceptin, trastuzu-mab) have been shown to be highly effective in both themetastatic and adjuvant settings [37,38] and even more soin combination with the antitumor drugs cisplatin [39] orpaclitaxel [40]. However, in the absence of HER2 over-expression, antibody therapy is significantly less effective[40] and can be hazardous. In combination with conven-tional chemotherapy, it has been shown to cause cardio-toxicity: in one study, 28% of patients receiving bothtrastuzumab and an anthracycline developed heart failure[41]. Combination therapies have also been linked tobleeding [42]. Therefore, determination of HER2 status isimportant both for prognosis and therapy selection.

HER2 status in single cells or cell extracts can bedetermined by quantitative methods including dot [43] andslot blots [25], Southern blotting with autoradiographicdetection [44], and competitive or differential PCR relativeto a reference gene that is not amplified [45]. However, theprimary approach used by pathologists is to examine theexpression and distribution of cancer markers in paraffin-embedded biopsy sections using either immunohistochem-istry (IHC) or ISH with labeled probes visualized by lightor fluorescence microscopy. This allows the pathologist tocompare the distribution pattern of cancer markers withother morphological features of the tissues and make acomplete diagnosis.

At this time, EnzMet has been developed for applicationin breast carcinoma. However, the opportunity for extensionto in situ detection of other clinically important genes, suchas the EGFR gene copy in lung cancer, is readily apparent.

1.3. IHC or ISH?

HER2 status may be evaluated by assessment of either theHER2 gene or the degree of expression of the encoded protein.HER2 protein overexpression is assessed by staining theexpressed protein (p185) in cells and tissues [46]. Imageanalysis has been used to quantitate overexpression, and theresults were found to correlate closely with those obtained byWestern blotting [47] and DNA analysis [48]. An immunohis-tochemical assay using a polyclonal antibody to the encodedgene protein ofHER2with a grading system (DakoHercepTest,Carpinteria, CA) has now been approved by the Food and DrugAdministration (FDA) [49]. Although useful, this approachpresents problems with interpretive and technical variability. Ina comparison between quantitative PCR, HercepTest, andmonoclonal antibody (CB11 and TAB250) staining, only 80%concordance was found. Most of the errors were false-positive,in which staining indicated apparent overexpression of theencoded protein, but the gene was not amplified. Of 254 cases,

61 were found to be amplified by PCR; 23% (93%concordance) were positive with CB11, 27% (90% concor-dance) with TAB250, and 37% with HercepTest [50].

ISH offers the significant advantage of enabling quanti-tative evaluation of HER2 gene amplification: each HER2gene copy generates a separate signal, and quantitation can beaccomplished by counting the number of signals per nucleus[51,52]. FISH studies showed that HER2 amplification wasthe strongest indicator of recurrence of breast cancer in youngwomen [53] and a stronger discriminator than tumor size inpredicting poor clinical outcome [54]. Two manual FISHdiagnostic assays to detect amplification of the HER2 gene(Ventana Inform, Ventana Medical Systems Inc, Tucson, AZ,and Abbott Molecular/Vysis PathVysion, Abbott Molecular/Vysis, Des Plaines, IL) have been approved by the FDA;PathVysion has been FDA-approved as a stand-alone test toselect patients for trastuzumab (Herceptin) therapy. Fluores-cently-labeled DNA gene probes are used to identifyindividual HER2 genes in nuclei of formalin-fixed, paraf-fin-embedded specimens; diagnosis is made by counting thenumber of signals observed by fluorescence microscopy.

Comparison of gene amplification, measured by FISH,with immunohistochemical staining yields significant dis-cord, which has furthered debate on the significance ofHER2 gene amplification and protein overexpression. Thishas important implications for therapy selection [55],especially given the approval of Herceptin for use inadjuvant setting in combination with docetaxel [56-59]. Arecent study found that although CB11 monoclonal antibodyIHC results showed concordance with FISH (κ = 0.83),polyclonal antibodies may not be sufficiently accurate andconsistent for reliable assessment of HER2 protein over-expression [60]. Currently, at least 10 different antibodies areavailable for commercial purchase, with differing perfor-mance characteristics in terms of cell conditioning require-ments (antigen retrieval) [61] and reported interpretivepatterns (cytoplasmic versus cell membrane or both) [62]:a more reliable indicator must be included for full andaccurate diagnosis and informed therapeutic decisionsregarding the use of Herceptin. Two comparison studiesfound that the most significant differences between IHC andFISH were found for cases with no gene amplification butsignificant positive staining for protein overexpression. Inthe first, the difference was attributed to an alternativemechanism for overexpression, such as transcriptionalactivation [63]. In a second study of 52 cases, the 2 FISHassays gave 98% concordance, with 14 cases showingamplification and 37 no amplification, whereas the IHCassay detected HER2 overexpression in a larger proportionof cases: 13 high-positive and 13 medium-positive. Of the 13high-positive cases, 10 also showed gene amplification, but 9of the 13 medium-positive cases did not [64], confirming thatIHC tends to produce false-positive results. The possibilitythat HER2 protein was being overexpressed without geneamplification was tested using messenger RNA (mRNA)hybridization [65]. Four hundred breast biopsies were


evaluated using monoclonal (CB11) and polyclonal (Her-cepTest) antibodies; 145 were subsequently evaluated byFISH and 144 by mRNA. Expression of mRNA was highlyconcordant with FISH and digoxigenin-based FISH ampli-fication. Immunohistochemical false-positive cases (appar-ent protein overexpression with no HER2 geneamplification) occurred with both HercepTest (23%) andCB11 (17%); most false-positive results (34 of 44) werescored as 2+. All 2+ false-positive cases were mRNA-negative, and only 17% had increased gene copy. Therefore,it was concluded that the 2+ score as defined in theguidelines for the FDA-approved HercepTest should not beused as a criterion for trastuzumab therapy unless confirmedby FISH.

The concordance between HER2 gene amplification andprotein overexpression and trastuzumab response has alsobeen investigated. Overall, both 2-color FISH and the moreaccurate of the IHC assays have similar predictive value. In astudy on 88 patients receiving weekly trastuzumab andpaclitaxel therapy for metastatic breast cancer, both IHC (2+or 3+ score was scored as amplified) and ISH results showedconcordance with response. Overall response rate in patientswith normal HER2 status ranged from 41% to 46%, whereasresponses in HER2-amplified (FISH) and overexpressing(IHC) patients ranged from 67% to 81%. Tests using theCB11 and TAB250 antibodies and FISH showed thestrongest significance. Good concordance of the resultsbetween HercepTest and FISH was found for the 78 patientsfor whom data from both assays were available, with a κvalue of 0.72 (95% CI, 0.56-0.87); a higher degree ofconcordance, indicating excellent agreement, was foundbetween FISH and the CB11 antibody, with a κ of 0.80 (95%CI, 0.66-0.93). Of note, 10 patients had tumors categorizedas HER2 overexpressing by the HercepTest and HER2normal by the CB11 antibody, suggesting that the lessaccurate HercepTest antibody tends to produce false-positiveresults [66]. In a second study on 26 women receivingweekly docetaxel with trastuzumab therapy for metastaticbreast cancer, patients with tumors that were either HER2 3+by HercepTest or FISH-positive had response rates in the63% to 65% range; all HER2 3+ patients were also FISH-positive. Patients with tumors that were HER2 2+ or FISH-negative had response rates ranging from 0% to 25%. Theonly HER2 2+ patient who achieved a positive response waspositive by FISH. These data suggest that tumors found to beHER2 2+ by HercepTest should be tested for HER2amplification by FISH: only if the tumor is positive byFISH should the patient subsequently receive trastuzumabbecause FISH-negative patients are unlikely to respond [67].

Determination of HER2 gene status by FISH may be themore accurate and reproducible method for selecting patientseligible for trastuzumab therapy. However, a panel recentlyconvened by the American Society of Clinical Oncology(ASCO) and the College of American Pathologists (CAP)affirmed that a firm conclusion as to the superiority of FISHover IHC could not be drawn from the studies performed

thus far, primarily because most studies have not measuredclinical outcome, and the few clinical outcome–basedstudies published did not use IHC reagents that are incurrent common use and FDA-approved [68].

ISH assessment of HER2 status can yield 3 results:nonamplified, with 2 or fewer gene copies per cell;amplified, with more than 6 or 8 gene copies; and low-level amplified, generally with 3 to 6 copies per cell.Interpretation of low-level amplification requires considera-tion of polysomy of chromosome 17, on which the HER2gene resides, which is a significant cause of false-positiveresults. Genuine amplification is therefore defined not interms of the number of copies of the HER2 gene but by theratio of HER2 to chromosome 17 centromere copies. This isestablished by using either a reflexed second test or aninternal, visually distinct centromeric chromosome 17 probe[69]. The PathVysion system includes a second colorcentromeric chromosome 17 probe as an internal control:HER2 amplification is based on a ratio of HER2 tochromosome 17 of equal to or greater than 2.0 [17]. Arecent study compared HER2 status from IHC and bothFISH assays for 2279 cases and found discordant FISHresults in 89 tumors: this was due to chromosome 17polysomy in 57 cases classified as positive by Inform butnegative by PathVysion, and chromosome 17 monosomy in32 cases classified as positive by PathVysion but negative byInform [70]. Although chromosome 17 polysomy in suchcases is usually regarded as a false-positive result, the fullclinical and prognostic significance of these conditions andof genuine low-level amplification is not fully understood:for example, it has been reported that a net increase in HER2gene copy number consecutive to polysomy 17 in theabsence of specific gene amplification might lead to a strongprotein overexpression in a small subset of breast carcinomas[71]. Therefore, in cases with apparent low-level amplifica-tion, this control is an important requirement [72-74].

1.4. Bright-field ISH methods

Although the fluorescence-based PathVysion FISH pro-cedure is FDA-approved and reliable, it has disadvantagesfor the pathologist. It requires fluorescent optics, whichrequire dark adaptation on the part of the observer and are notimmediately available in many laboratories. The need forhigh magnification and, sometimes, oil immersion add to thecomplexity of the procedure. Furthermore, morphologicalchanges and standard counterstained morphology cannotreadily be visualized simultaneously, making it difficult toevaluate the amplification in a cellular context. Fluorescentlylabeled specimens are subject to fading, hindering permanentarchival. A reliable, sensitive bright-field colorimetric orchromogenic modification of this assay would be highlydesirable for most practicing pathologists.

In response to this need, chromogenic ISH (CISH)procedures were developed, using enzymatic labeling withconventional organic chromogens. Tanner et al [75] used a


digoxigenin-labeled HER2 probe detected with fluorescein-labeled antidigoxigenin followed by peroxidase-labeled anti-fluorescein developed with diaminobenzidine (DAB) forHER2 gene status in a series of 157 breast carcinomas;amplification of HER2 was identified with CISH in 27(17.2%) cases. However, 10 tumors in that series were FISH-positive but CISH-negative. Sensitivity was increased by theintroduction of polymerized antibody–horseradish peroxidase(HRP) reagents [76-78] or peroxidase-avidin-biotin com-plexes [79,80] for detection. A series of 119 cases wasanalyzed using CISH with polymerized secondary detection:cases were classified as nonamplified (1-5HER2 gene copies),low-level amplified (6-10 copies), or high-level amplified(more than 10 copies). A repeated staining procedure using achromosome 17 centromeric probe was used to confirmamplification for cases with 3 to 10 copies.With the exceptionof 1 discordant case that was classified as low-level amplifiedby FISH but amplified by CISH [81], the FISH and CISHresults agreed completely. A second study used a tissuemicroarray of a series of 113 cases selected from amuch largerpopulation (2000) to include a reasonable number of low-levelor borderline amplified cases where differences might besignificant [82]. One hundred two cases were successfullyanalyzed by both methods. A 100% concordance was foundbetween FISH and CISH in classifying tumors as HER2amplified or nonamplified: 63 cases were scored as amplifiedand 39 as not amplified. The amplified tumors were classifiedfurther into 3 categories: borderline amplification (HER2-to-CEP17 ratio, 2.0-2.5), low-level amplification (ratio, 2.6-3.9),and high-level amplification (ratio, 4.0 or higher). Of 63 cases,22 (35%) were scored as borderline (6 cases) or low-level (16cases) amplification by FISH.Of the 6 borderline cases, 3werealso borderline by CISH, whereas 2 were scored low-level(ratios, 2.7 and 3.2), and 1 case was high-level (ratio, 4.1). Ofthe 16 low-level amplification cases by FISH, 6 also wereconsidered low-level by CISH, whereas 1 case was borderline(ratio, 2.5), and 9 cases were high-level (ratio range, 4.0-6.8)by CISH. Overall, the FISH and CISH ratios were correlatedhighly (r = 0.825). A procedure using TSA has been run onan automated platform [83]. However, the characteristics ofthe staining are not ideal: diffusion and the continuous natureof the stain can make the spots difficult to resolve andobscure the underlying ultrastructure. In one recent compar-ison, FISH was still occasionally able to detect low-levelamplification missed using a chromogenic method [79].

A recent report presents an efficient protocol for dual-color CISH (dc-CISH), using cohybridization of a digox-igenin-labeled HER2 probe and biotinylated chromosome 17centromeric probe [84]. The chromosome 17 centromericprobe was detected first, by means of incubation with mouseantibiotin antibody followed by alkaline phosphatase poly-mer kit developed with new fuchsin to give a red color; afterwashing with distilled water, detection of the digoxigenin-labeled HER2 probe was performed with antidigoxigeninantibody and HRP polymer secondary developed withtetramethyl benzidine chromogen to give a blue-green signal.

In a comparison of the results for 4 cell lines and 40 tumorsamples with those obtained using FISH (PathVysion), theresults of FISH and dc-CISH showed high concordance(91%, κ = 0.82), indicating that dc-CISH, a new alternativeto FISH, enables the assessment of copy number ratio(HER2/17 centromere) in conjunction with proper histo-pathologic evaluation and the ease of bright-field micro-scopy. The features and performance characteristics of FISHand CISH are compared with those of the metallographicmethods that are the subject of this article in Table 1.

2. Application of metallographic methodsto ISH

2.1. Colloidal gold and Nanogold

Colloidal gold labeling, in which antibodies or otherproteins conjugated to gold nanoparticles 1 to 30 nm indiameter are used to label targets of interest, was firstintroduced as an immunoelectron microscopic method[85,86] and later adapted for ISH electron microscopicchromosome [87], satellite [88], and gene localization [89].The subsequent discovery that gold nanoparticles catalyzethe highly selective deposition of silver from mixtures ofsilver (I) salts and reducing agents to give specific, highlyresolved, and visually distinct optical staining [90], a processcalled autometallography or silver enhancement, enabled anew light microscopy method, immunogold-silver staining[91]. This proved effective for histopathology applications,where it was found to give superior or at least equal results tothose obtained with the peroxidase antiperoxidase technique;in some cases, staining was obtained with immunogold-silver staining method when the peroxidase antiperoxidasetechnique gave no result [92]. Streptavidin-colloidal goldwith silver acetate autometallography proved to be asensitive and specific method for ISH detection [93].

Improved results were enabled by Nanogold, a highlymonodisperse 1.4-nm gold particle, exclusively manufac-tured by Nanoprobes, Incorporated (Yaphank, NY). Nano-gold may be covalently linked to a wide variety of molecules,including antibodies, antibody fragments, proteins, peptides,oligonucleotides, and lipids [94]. Developed as an improvedalternative to noncovalently linked colloidal gold [92],Nanogold is a stable compound that does not dissociate[95], interact nonspecifically with biologic systems to causebackground signal [96], or aggregate to give poor samplepenetration [97] as colloidal gold has been found to do.Nanogold-Fab' antibody conjugates penetrate up to 40 μminto specimens; in comparison, 10-nm colloidal gold showedonly surface labeling [98]. When used with silver enhance-ment, Nanogold conjugates may be used to visualize as littleas 0.1 pg (approximately 10-18 mol) of a target immunoglo-bulin G; [99] this is one of the most sensitive detectionmethods available, similar to chemiluminescence.

Table 1 Comparison showing advantages and disadvantagesof FISH with CISH using organic chromogens and usingdifferent metallographic methods

Detectionmethod

Advantages Disadvantages

FISH • Clear signal • Requires darkadaptation

• Straightforwardquantitation

• Requires specializedfluorescent optics

• Multicolorprotocolsestablished

• Signal can fade;must be archiveddigitally

• Adapted forautomated staining

• Does not show tissuemorphology

CISH (usingorganicchromogens)

• Read withstandard bright-fieldlight microscope

• Diffuse signals canreduce ease ofquantitation

• Underlying tissuemorphology can bevisualized

• Signals can bedifficult to distinguishfrom counterstains

• Adaptable forautomated staining

• Some chromogensare hazardous

• 2-Color protocol(internal control)possible

Nanogold withsilverenhancement

• Higher sensitivitythan organicchromogens

• Background signalcan be problematic

• Easilydistinguished fromcounterstains

• Complex protocolwith multipleamplificationprotocols needed forhighest sensitivity

• Read withstandard bright-fieldmicroscope

• Difficult to adapt forautomated staining

• Underlying tissuemorphology easilyvisualized

• Multicolor protocolsnot available

GOLDFISH(Nanogoldwith goldenhancement)

• Highly sensitive • Gene copyquantitation isproblematic

• Easy, rapid visualinterpretation

• Difficult to adapt forautomated staining

• Clean signal withlow background

• Multicolor protocolsnot available

• Easilydistinguished fromcounterstains• Read withstandard bright-fieldmicroscope• Underlying tissuemorphology can bevisualized

Table 1 (continued)

Detectionmethod

Advantages Disadvantages

EnzMet • Highest sensitivity • Multicolor protocolsnot yet available (maybe in future)

• Highest resolutionand signalseparation• Most accuratequantitation• Best visualizationof tissuemorphology andcounterstains• Adaptable forautomation• Simple procedure


When used for ISH detection, silver-enhanced Nanogoldprobes demonstrated higher sensitivity than enzymaticprobes run in parallel experiments, both by direct ISHusing silver-enhanced streptavidin-Nanogold to detect abiotinylated hybridization probe and by an indirect methodin which silver-enhanced streptavidin-Nanogold was usedto detect a biotinylated secondary applied after targetamplification by PCR [100] or to detect deposited biotin-tyramide after TSA, initially known as catalyzed reporterdeposition (CARD) [101,102]. Highly sensitive detectionwas achieved by hybridization with a fluorescein-labeledprobe, then with a biotinylated antifluorescein antibody,followed by 2-step signal amplification with avidin-biotincomplex followed by a TSA procedure in which strepta-vidin-peroxidase was used to deposit biotinylated tyramide.In the in situ detection of the HPV-16 virus in SiHa cells,which in the SiHa cell line carries 1 to 2 integrated copiesof HPV 16 per cell, streptavidin-Nanogold combined withsilver acetate autometallography occasionally producedspeckled nuclear staining even without signal amplification[102]. A panel of 10 cervical cancer specimens, positive bysolution-phase PCR, was examined using both direct andCARD-amplified Nanogold-streptavidin and streptavidin-peroxidase. Without CARD, 5 were positive with silver-enhanced Nanogold but only 2 with peroxidase-DAB. WithCARD, 9 were positive with silver-enhanced Nanogold butonly 5 with DAB (G.W.H. and C.H.-K., unpublishedresults). An example of the results obtained with CARD-amplified Nanogold-streptavidin is shown in Fig. 1.

2.2. Gold enhancement: gold-facilitated ISH

Although the Nanogold-silver enhancement procedureproduced increased sensitivity, the lengthy procedure andrequirement for multiple signal amplification steps made itcumbersome for routine use. Similar sensitivities wereobtained using a simplified procedure in which gold

Fig. 1 CARD–Nanogold-silver ISH. Left, A fluorescein-labeled hybridization probe is detected using an antifluorescein antibody, followedby antibody-peroxidase-ABC complex. This is then treated with biotinylated tyramide: the peroxidase then catalytically converts the tyramideto a reactive free radical species, which quickly reacts with the aromatic ring of a nearby tyrosine residue and anchors the biotin there. Whensufficient biotin is deposited, it is detected with Nanogold-labeled streptavidin and silver acetate autometallography (Society for AppliedImmunohistochemistry; used with permission). Right, Single copies of HPV-16 in SiHa cells detected by CARD-amplified Nanogold-silverISH. Copies of HPV-16 appear as single spots (hematoxylin-eosin counterstain, original magnification ×560).


enhancement replaced silver enhancement. Gold enhance-ment is a novel gold-based autometallographic process,developed at Nanoprobes, in which gold, instead of silver, iscatalytically deposited onto gold particles [103]. It demon-strates several advantages over silver enhancement: it may beused in physiologic buffers, including halides, whichprecipitate silver; it is not light sensitive; and it is moreselective—for example, gold enhancement can be carried outin cells cultured on metal substrates [104]. Gold enhance-ment used with Nanogold for ISH after TSA gave sensitivityequivalent to silver enhancement, with lower background[105]. When the experiment was conducted using acombined Cy3 and Nanogold-labeled streptavidin, signalsfrom HPV16/18 in SiHa cells were visualized by bothfluorescence and bright-field light microscopy. In addition,the high atomic number of the gold allows localization athigher resolution by electron microscopy [106].

A simplified gold-enhanced Nanogold-streptavidinmethod, called Gold-facilitated ISH (GOLDFISH), wasdeveloped to detect amplification of the HER2 gene inparaffin-embedded breast carcinoma sections. Fig. 2 showsa schematic for GOLDFISH, and a comparison of stainingin normal (nonamplified) tissue (2 copies of the HER2gene) and amplified carcinoma tissue (10-20 copies) [107].The GOLDFISH assay demonstrated high interobserverreproducibility: for a series of 66 excisional biopsies orresections, the mean κ among GOLDFISH observers was0.84, and the mean among GOLDFISH observers versusdirect FISH was 0.83, which was at least concordant ofobservers scoring nuclear grade (κ = 0.50) and the presenceof in situ carcinoma (κ = 0.57) by conventional histopathol-ogy. Two specimens that scored 2+ or 3+ by IHC butdemonstrated chromosome 17 polysomy without amplifica-tion by 2-color FISH were appropriately assigned as

nonamplified by GOLDFISH [108]. Staining is opaque,black, and punctated with sharply defined edges. The assaywas designed for qualitative interpretation without the needfor oil immersion: in amplified tissue, large, confluentnuclear signals were generated, whereas endogenous HER2copies in nonamplified tissue produced small, punctuatesignals. Preliminary results have verified that GOLDFISHstaining may be quantitated using automated image analysis[109]. Signal clarity, sensitivity, and reproducibility weresignificantly better than equivalent detection methods basedon enzymatic deposition of organic chromogens [75].The black signal is permanent, does not fade, and iseasily distinguished from overall section color and fromother stains.

2.3. Enzyme metallography

The GOLDFISH assay was configured for simplified,rapid manual interpretation without spot counting: theautometallographic process was optimized to give large,confluent signals in amplified tissue rather than discrete spotsthat could be counted. However, with the elucidation of therole of chromosome 17 aneusomy in producing false-positivegene amplification signals, it became apparent that spotcounting was necessary to differentiate genuine low-levelamplification from chromosome 17 polysomy [70-74,81,82].In addition, the growing significance of image analysis(automated spot counting) rendered a quantitative interpreta-tion method preferable to the more qualitative method usedwith GOLDFISH.

Subsequently, it was discovered that HRP can selectivelydeposit metal from solution even in the absence of aparticulate metal nucleating agent such as Nanogold. This

ig. 3 EnzMet assay. A, Schematic showing reaction sequence.hapten-labeled hybridization probe is bound by a primary

ntibody against the hapten then visualized using a polymerizederoxidase-labeled secondary reagent treated with the enzymeetallographic substrate, which deposits metallic silver at the activeite. B, Endogenous HER2 signals in nonamplified tissue; 1 to 2ER2 signals are clearly visualized per nucleus. C, HER2 in highlymplified tissue, showing multiple, distinct signals correspondingindividual gene copies. B and C both stained using Zymed

olymeric antibody-HRP reagents (Invitrogen, Carlsbad, CA;ematoxylin counterstain, original magnification ×400).

Fig. 2 GOLDFISH HER2 gene amplification assay. A, Sche-matic of assay. A biotinylated HER2 hybridization probe is detectedusing peroxidase-streptavidin followed by biotin-tyramide, which iscatalytically deposited at the target site; the biotin-tyramide isdetected with Nanogold-streptavidin followed by gold enhance-ment. B, GOLDFISH staining of nonamplified tissue: infiltratingductal carcinoma with a normal HER2 gene copy, showing 1 to 2spots per nucleus, corresponding to endogenous HER2 (NFRcounterstain, original magnification ×400). C, GOLDFISH proce-dure in tissue with HER2 gene amplification, showing large,confluent nuclear signals from multiple gene copies in closeproximity (NFR counterstain, original magnification ×400; Amer-ican Society for Investigative Pathology; used with permission).


FAapmsHatoph

resulted in highly sensitive and specific metal deposition ontargets. This new method, termed EnzMet [110,111], wasfound to produce superior results both for detecting HER2


gene amplification [112,113] and for immunohistochemi-cally staining overexpressed HER2 protein in paraffinsections of human breast carcinoma [110].

ISH using a biotin-labeled HER2 probe with mouseantibiotin followed by polymerized goat antimouse–HRP,developed with metallographic substrate, yielded very highsensitivity and resolution. Individual spots corresponding togene copies were clearly visible in both amplified andnonamplified tissues and could be enumerated in an identicalmanner to the fluorescent signals obtained by FISH; singlegenes developed into approximately 1-μm dense metal spots,and background was almost nonexistent [112,113]. Fig. 3shows the method with representative examples. A compar-ison of the staining pattern obtained by EnzMet with thatobserved for DAB is shown in Fig. 3. A series of 101 caseswas assessed using this method versus FISH. Three caseswere low-level amplified by absoluteHER2 copy number butfound to be nonamplified by 2-color FISH because of chro-mosome 17 polysomy; [114] similar cases are easily resolvedby conducting a second ISH procedure on a second slide.

2.4. Adaptation to automated staining systems:silver ISH

EnzMet protocols share many steps with conventionalenzyme chromogenic detection methods, and therefore,adaptation of this chemistry to automated platforms wouldseem relatively straightforward. Differences in technologybetween steps that can be performed manually and those usedin automated assays can cause issues during assay develop-ment. However, once the assay has been adapted toautomation, it yields improvements in efficiency and work-flow advantages that make automated silver ISH (SISH) aroutine assay with increased throughput and high-qualityresults that are easily interpreted. The following descriptionof ISH automation reflects our experience at VentanaMedicalSystems, Inc, with the BenchMark Series of instruments.

3. Practical aspects of automatedmetallographic ISH

The automation of any assay is primarily dependent onthe technology of the instrument used, and these can varysignificantly from manufacturer to manufacturer. The bulkreagent catalog available for the instrument is also anotherconsideration. However, the key aspects of any ISH protocol(independent of manufacturer) fall into 5 main categories:sample preparation, tissue pretreatment, hybridization,detection, and counterstaining.

3.1. Sample preparation

As with any tissue-based assay, the quality of tissuecollection, fixation, and processing can make the difference

between obtaining an appropriately stained and interpre-table slide and one that cannot produce a result. Generally,the time between tissue collection and fixation affects thequality of the morphology: excessive times can havedeleterious effects on the quality of the DNA to behybridized. The type of fixative used can greatly affect theresults. For instance, metal-containing fixatives (B5,Zenker's, and zinc formalin) have the potential to causebackground. Acidic fixatives (Bouin's) can have significanteffects on the intactness of DNA, depending on the lengthof fixation [115]. Formalin-based (non–metal-containing)fixatives have produced the best results. It is also importantthat tissue be completely fixed; in tissue that is too large tofix in the appropriate amount of time, the center mayundergo autolysis, causing loss of morphology and a lossof hybridization.

3.2. Pretreatment

After deparaffinization and rehydration of the tissue onthe slide, access to the DNA is the next criticalparameter. There are 2 primary methods to permeabilizethe tissue: heating the tissue in a retrieval solution andprotease digestion. Most manual commercial assays useboth methods to obtain optimal access to the DNA. Thecorrect amount of tissue conditioning (retrieval andprotease treatment) is important because undercondition-ing can result in weak and patchy signal, whereasoverconditioning can actually strip the DNA from theslide, resulting in little or no signal. The highly controlledconditions of the automated assay remove most of thevariability from this process and result in a consistentsample for probe hybridization.

3.3. Hybridization

Hybridization is almost entirely a function of the probeused, size of the probe fragments, Tm, and hybridizationbuffer. Probe design, labeling, and formulation constitute awide field in themselves; a detailed discussion would bethe subject of another review and will not be addressedhere. The primary drawback to the FDA-approved manualassays is the need for overnight hybridization, which limitsthe throughput to 1 run per day. The new automated HER2SISH assay has decreased the overnight hybridization toapproximately 2 hours, enabling multiple runs per day on asingle instrument.

3.4. Detection

The adaptation of EnzMet to automated instrumenta-tion was relatively straightforward. The most importantconsideration was the specificity of the probe. Because ofthe very high sensitivity of the SISH detection technol-ogy, probe specificity and stringency washes becomecritical to eliminate specific and nonspecific background.


Particularly with detection of single-copy genes, anynonspecific precipitation of silver on the slide can createinterpretation issues for the pathologist. Thus, as wediscuss in the section on interpretation, it is veryimportant that nonspecific background be eliminated.

3.5. Counterstaining

The final step in the automated ISH process is thecounterstain. The deposition of silver on the slide creates ablack signal, and the ease of interpretation is dependent onthe level of contrast between the silver and the counter-stain. We have found that hematoxylin and nuclear fastred (NFR) counterstains produce a slide with the neededcontrast for interpretation. Although it may be suspectedthat the black signal with the light blue hematoxylinnuclear stain might have insufficient contrast, as long asthe hematoxylin is configured as a counterstain and not atthe intensity of a hematoxylin-eosin stain, the results arehighly scorable. Hematoxylin also has the advantage ofproviding nuclear detail for pathologic identification of theneoplastic cells, which is not possible with FISH-basedassays. NFR provides a higher contrast pattern with theSISH signal that allows for rapid identification of nucleiand signal; however, NFR counterstain does lack some ofthe discreetness of hematoxylin with regard to nuclear areaand the potential use of image analysis for automatedinterpretation with an instrument such as the VentanaImage Analysis System (VIAS; Ventana).

4. Interpretation

4.1. Low-level amplification: significance of CEP17control

There is debate in the field of HER2 testing as to theneed for correction of HER2 copy number for chromo-some 17 (CEP17) copy number. The recently publishedASCO/CAP guidelines for HER2 testing include in theirsuggested algorithm protocols for interpretation with andwithout CEP17 [68]. The concern about including CEP17testing is that HER2 testing of a polysomic 17 case canlook like amplified HER2 when, in fact, HER2 has notbeen specifically amplified. This controversy has beenaddressed in multiple publications [72-74]. The HER2SISH assay was developed as a 2-slide assay, 1 for theHER2 probe and a second for the CEP17 probe, and itis the ratio of HER2 to CEP17 that determines theamplification status of the case. It is very important thatcases in the area of the FDA-approved 2.0 cut-off arequantitatively assessed. This analysis was addressed in theASCO/CAP guidelines, and an “equivocal” range was setfor ISH applications to indicate the uncertainty surround-ing the actual cutoff. The ASCO/CAP guidelines direct

that these equivocal cases have additional cells counted orthat the assay be rerun or confirmed by a secondindependent assay such as IHC. There is little informationon the response rates of individuals who fall within theISH equivocal and IHC equivocal ranges. Cases whoexhibit high-level amplification are easily scored by lightmicroscopy at magnifications as low as ×20, whereasnonamplified cases are typically scored at either ×40 or×60 without oil immersion.

4.2. Signal morphology

The HER2 SISH signal has several manifestationsdepending on the presence or absence of amplification.Nonamplified signal is typically represented by small,discreet dots in the nucleus. There can be 1 to 2 per cell,but there will not be signal in every single nucleus because inmany cases, only a portion of a nucleus falls within thesection, and this may not contain the target sequence. Withamplification, 2 patterns of hybridization are typically seen.The first are homogeneously staining regions, which aremanifested by 2 large clusters of SISH signals that indicaterepeated copies of HER2 on a single chromosome [116].These homogeneously staining region–type cases are usuallyscored by an estimation of the copy number based on theSISH signal size in the normal adjacent cells. The secondpattern seen is a more distributed pattern that lends itself tocounting of individual signals. This pattern is indicative of amore random amplification throughout the genome orrestriction of the amplified genes to double-minute chromo-somes [117].

5. Future developments

5.1. Application of metallographic methods to IHC

In addition to its utility for ISH, EnzMet also producesexcellent results when used for immunohistochemicalstaining [110,118]. A comparison between conventionalDAB-peroxidase and EnzMet was conducted using high-complexity tissue midiarrays; 88 solid tumors were evaluatedby automated EnzMet IHC (Nanoprobes and Ventana) [118].Targets were chosen to assess the ability of EnzMet IHC tospecifically localize encoded antigens in the nucleus(estrogen receptor), cytoplasm (cytokeratins), and cytoplas-mic membrane (HER2). Full concordance was foundbetween the EnzMet IHC and conventional IHC; further-more, the EnzMet staining was sharper and more highlyresolved, with many regions assuming a punctuate appear-ance that defined the staining more clearly. In addition, theblack color of the EnzMet reaction product is easilydifferentiated from other stains and counterstains, and thesensitivity of the detection procedure allows additionaldilution of the primary antibodies.


5.2. Combining ISH with IHC: a concomitant geneand protein assay

The feasibility of combining ISH and IHC in a singleprotocol was previously demonstrated in the concomitant

Fig. 4 EnzMet GenePro assay. A, Breast carcinoma case amplified fordenoting HER2 gene copies are present in the nucleus, with associated(hematoxylin counterstain, original magnification ×1000). Arrow denotesnonamplified for HER2 by FISH. Tumor cells demonstrate 1 or 2 nuclearcell surface (hematoxylin counterstain, original magnification ×400; inset,for chromosome 17 but not amplified forHER2 (HER2-to-CEP17 ratio, 1.denoting HER2 gene copy number, and the cellular membrane is devoioriginal magnification ×400; inset, original magnification ×1000). D, Breonly FDA 1+ immunostaining by IHC. Numerous confluent black metnucleus, but associated 1+ cellular membrane expression of the encodecounterstain, original magnification ×1000; Lippincott Williams and Wil

oncoprotein detection with FISH method [119], a 3-colorfluorescence-based system that simultaneously profilesHER2 oncogene copy by FISH and HER2 encoded proteinby the use of a versatile alkaline phosphatase chromogen,fast red K, in either fluorescence or bright-field mode.

HER2 by FISH. Numerous confluent black metallic silver signals3+ cellular membrane expression of the encoded HER2 protein

endogenous HER2 copies in lymphocytes. B, Breast carcinoma caseHER2 signals (black) and absence of HER2 encoded protein on theoriginal magnification ×1000). C, Breast carcinoma case polysomic9). Tumor cells demonstrate 3 to 5 discrete black signals per nucleus,d of HER2-encoded protein expression (hematoxylin counterstain,ast carcinoma case amplified for HER2 by FISH but demonstratingallic silver signals denoting HER2 gene copies are present in thed HER2 protein is negative by FDA scoring criteria (hematoxylinkins; used with permission).


Fast red K protein IHC may be readily combined withenzyme metallographic ISH detection without eithercomponent interfering with the other. For the concomitantgene and protein assay (GenePro), HER2 protein was firststained using CB11 polyclonal antibody followed byalkaline phosphatase-labeled secondary and fast red Kdevelopment. HER2 gene copies were then visualized usinga DNP-labeled cDNA probe, detected with a polymerizedHRP-antibody conjugate, and developed using EnzMetsubstrate. The enzyme metallographic procedure did notaffect the fast red K stain [120]. With the exception of theISH and the enzyme metallographic development, all stepswere automated (Benchmark, Ventana). The method wasvalidated on tissue midiarrays [121], consisting of 94 coresfrom 80 cases of invasive breast carcinoma, including 4cases of infiltrating lobular carcinoma, 1 case of mixedinfiltrating ductal and lobular carcinoma, and 89 cases ofinfiltrating ductal carcinoma.

Staining results are shown in Fig. 4. The mean HER2copy number count, produced from the enzyme metallo-graphic component of the assay when performed in concertwith the IHC component, was compared with the direct FISHHER2 copy number and showed an excellent correlation,with a Pearson coefficient of 0.96 (P b .001). The IHCcomponent of the GenePro assay showed a Pearsoncorrelation of 0.84 (P b .001) in comparison with CB11antibody staining performed on whole tissue sections. Thecombination of gene and protein detection (EnzMetGenePro) gave a specificity of 100% and an accuracy of92.6% (95% confidence interval, 85.3-97.0), using theconsensus diagnosis for each component of the assay.Correlation between the IHC component of the assay andthe HER2 gene amplification status of the assay hadagreement in 98% (92/94) of the cases. Most HER2amplified cases had CB11 staining that was 2+ or 3+,whereas HER2 nonamplified or polysomic cases had CB11staining that was 0 or 1+. The 2 discrepant cases included aCB11-negative and EnzMet-positive case and a CB11-positive and EnzMet-negative case. In a 5-person inter-observer reproducibility study, interobserver κ for eachcomponent was excellent (IHC, κ = 0.94; EnzMet, κ = 0.96).

5.3. Multiple targets and multiple colors

Until recently, the application of multiple stainingprotocols, particularly ISH, to a single specimen has beenprohibitively complex for most applications because of thecomplexity of the procedures involved. However, thedevelopment of automated slide staining systems forapplications such as immunohistochemical slide staining[122] and ISH [123] has relieved their demands on personneltime and enabled the widespread adoption of increasinglycomplex procedures in reliable, standardized formats. There-fore, the incorporation of metallographic protocols intomultianalyte staining systems is now feasible, and our currentresearch is directed toward this goal.

Our longer-term goal is to develop technologies formultiplexed correlated molecular morphological labeling andprofiling of tumors. Evaluation of the relative intensity andspatial distribution of a particular set of tumor markers withindependent predictive or prognostic value will provide aunique profile of the biologic behavior of each tumor, enabling“personalized medicine” and individualized therapy selection.In addition, it may also provide tools for cancer research andapplications such as bright-field spectral karyotyping. Futurestudies may identify additional predictive value, such as acorrelation between a specific result and outcome from acombination therapy. Ultimately, this methodology will beapplicable to may other types of tumors [124,125] and to othermedical conditions, including the recent work of Dr RicardoLloyd on the in situ detection of infection agents [15-17],where differences in biologic behavior or therapeutic responseare related to different markers or infection agents.

References

[1] Watson JD,Crick FHC.Molecular structure of nucleic acids—a structurefor deoxyribose nucleic acids. Nature (London) 1953;171:737-8.

[2] de Jong H. Visualizing DNA domains and sequences bymicroscopy: a fifty-year history of molecular cytogenetics. Genome2003;46:943-6.

[3] Caspersson T, Lindsten J, Lomakka G, Moller A, Zech L. The use offluorescence techniques for the recognition of mammalian chromo-somes and chromosome regions. Int Rev Exp Pathol 1972;11:1-72.

[4] Pardue ML, Gall JG. Molecular hybridization of radioactive DNA tothe DNA of cytological preparations. Proc Natl Acad U S A Sci 1969;64:600-4.

[5] John HA, Birnstiel ML, Jones KW. RNA-DNA hybrids at thecytological level. Nature 1969;223:582-7.

[6] Buongiorno-Nardelli M, Amaldi F. Autoradiographic detection ofmolecular hybrids between RNA and DNA in tissue sections. Nature1970;225:946-8.

[7] Trask BJ. Fluorescence in situ hybridization: applications incytogenetics and gene mapping. Trends Genet 1991;7:149-54.

[8] Bauman JG, Wiegant J, Borst P, van Duijn P. A new method forfluorescence microscopical localization of specific DNA sequencesby in situ hybridization of fluorochromelabelled RNA. Exp Cell Res1980;128:485-90.

[9] Rudkin GT, Stollar BD. High resolution detection of DNA-RNAhybrids in situ by indirect immunofluorescence. Nature 1977;265:472-3.

[10] Nederlof PM, van der Flier S, Wiegant J, et al. Multiple fluorescencein situ hybridization. Cytometry 1990;11:126-31.

[11] Qian X, Lloyd RV. Recent developments in signal amplificationmethods for in situ hybridization. Diagn Mol Pathol 2003;12:1-13.

[12] van de Corput MP, Dirks RW, van Gijlswijk RP, van de Rijke FM,Raap AK. Fluorescence in situ hybridization using horseradishperoxidase–labeled oligodeoxynucleotides and tyramide signalamplification for sensitive DNA and mRNA detection. HistochemCell Biol 1998;110:431-7.

[13] Kerstens HM, Poddighe PJ, Hanselaar AG. A novel in situhybridization signal amplification method based on the depositionof biotinylated tyramine. J Histochem Cytochem 1995;43:347-52.

[14] Tsuda H. HER2 (c-erbB-2) test update: present status and problems.Breast Cancer 2006;13:236-48.

[15] Qian X, Guerrero RB, Plummer TB, Alves VF, Lloyd RV. Detectionof hepatitis C virus RNA in formalin-fixed paraffin-embedded


sections with digoxigenin-labeled cRNA probes. Diagn Mol Pathol2004;13:9-14.

[16] Abdulkarim AS, Petrovic LM, KimWR, Angulo P, Lloyd RV, LindorKD. Primary biliary cirrhosis: an infectious disease caused byChlamydia pneumoniae? J Hepatol 2004;40:380-4.

[17] Hayden RT, Isotalo PA, Parrett T, et al. In situ hybridization for thedifferentiation of Aspergillus, Fusarium, and Pseudallescheria speciesin tissue section. Diagn Mol Pathol 2003;12:21-6.

[18] Masood S, Bui MM, Yung JF, et al. Reproducibility of LSI HER-2/neu SpectrumOrange and CEP 17 SpectrumGreen dual colordeoxyribonucleic acid probe kit. For enumeration of gene amplifica-tion in paraffin-embedded specimens: a multicenter clinical valida-tion study. Ann Clin Lab Sci 1998;28:215-23.

[19] Detailed guide: breast cancer. What are the key statistics for breastcancer? American Cancer Society, http://www.cancer.org/docroot/CRI/content/CRI_2_4_1X_What_are_the_key_statistics_for_breast_cancer_5.asp?sitearea= (Revised September 18, 2006).

[20] Nofech-Mozes S, Spayne J, Rakovitch E, Hanna W. Prognostic andpredictive molecular markers in DCIS: a review. Adv Anat Pathol2005;12:256-64.

[21] McGuire WL. Prognostic factors for recurrence and survival inhuman breast cancer. Breast Cancer Res Treat 1987;10:5-9.

[22] Schechter AL, Stern DF, Vaidyanathan L, et al. The neu oncogene: anerb-B–related gene encoding a 185,000-Mr tumour antigen. Nature1984;312:513-6.

[23] Chen L, Zhang W, Fregien N, Pierce M. The her-2/neu oncogenestimulates the transcription of N-acetylglucosaminyltransferase Vandexpression of its cell surface oligosaccharide products. Oncogene1998;17:2087-93.

[24] Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neuproto-oncogene in human breast and ovarian cancer. Science 1989;244:707-12.

[25] Toikkanen S, Helin H, Isola J, Joensuu H. Prognostic significance ofHER-2 oncoprotein expression in breast cancer: a 30-year follow-up.J Clin Oncol 1992;10:1044-8.

[26] Descotes F, Pavy JJ, Adessi GL. Human breast cancer: correlationstudy between HER-2/neu amplification and prognostic factors in anunselected population. Anticancer Res 1993;13:119-24.

[27] Makar AP, Desmedt EJ, De Potter CR, Vanderheyden JS, SchattemanEA. Neu (C-erbB-2) oncogene in breast cancer and its possibleassociation with the risk of distant metastases. A retrospective studyand review of literature. Acta Oncol 1990;29:931.

[28] Tiwari RK, Borgen PI, Wong GY, Cordon-Cardo C, Osborne MP.HER-2/neu amplification and overexpression in primary humanbreast cancer is associated with early metastasis. Anticancer Res1992;12:419-25.

[29] Seshadri R, Horsfall DJ, Firgaira F, et al. The relative prognosticsignificance of total cathepsin D and HER-2/neu oncogeneamplification in breast cancer. The South Australian Breast CancerStudy Group. Int J Cancer 1994;56:61-5.

[30] Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuireWL. Human breast cancer: correlation of relapse and survival withamplification of the HER-2/neu oncogene. Science 1987;235:177-82.

[31] Allred DC, Clark GM, Tandon AK, et al. HER-2/neu in node-negative breast cancer: prognostic significance of overexpressioninfluenced by the presence of in situ carcinoma. J Clin Oncol 1992;10:599-605.

[32] Chang J, Clark GM, Allred DC, Mohsin S, Chamness G, ElledgeRM. Survival of patients with metastatic breast carcinoma:importance of prognostic markers of the primary tumor. Cancer2003;97:545-53.

[33] Zeillinger R, Kury F, Czerwenka K, et al. HER-2 amplification,steroid receptors and epidermal growth factor receptor in primarybreast cancer. Oncogene 1989;4:109-14.

[34] Bacus SS, Zelnick CR, Plowman G, Yarden Y. Expression of theerbB-2 family of growth factor receptors and their ligands in breast

cancers. Implication for tumor biology and clinical behavior. Am JClin Pathol 1994;102:S13-24.

[36] PegramMD, Finn RS, Arzoo K, Beryt M, Pietras RJ, Slamon DJ. Theeffect of HER-2/neu overexpression on chemotherapeutic drugsensitivity in human breast and ovarian cancer cells. Oncogene1997;15:537-47.

[37] Fornier M, Risio M, Van Poznak C, Seidman A. HER2testing and correlation with efficacy of trastuzumab therapy.Oncology (Huntingt). 2002;16:1340-8, 1351-52; discussion,1352, 1355-58

[38] Konecny G, SlamonDJ. HER2 testing and correlation with efficacy oftrastuzumab therapy. Oncology (Huntingt). 2002;16:1576, 1578

[39] Pegram MD, Slamon DJ. Combination therapy with trastuzumab(Herceptin) and cisplatin for chemoresistant metastatic breast cancer:evidence for receptor-enhanced chemosensitivity. Semin Oncol 1999;26(4 Suppl 12):89-95.

[40] Fornier MN, Seidman AD, Schwartz MK, et al. Serum HER2extracellular domain in metastatic breast cancer patients treated withweekly trastuzumab and paclitaxel: association with HER2 status byimmunohistochemistry and fluorescence in situ hybridization andwith response rate. Ann Oncol 2005;16:234-9.

[41] Feldman AM, Lorell BH, Reis SE. Trastuzumab in the treatment ofmetastatic breast cancer: anticancer therapy versus cardiotoxicity.Circulation 2000;102:272-4.

[42] Nissenblatt MJ, Karp GI. Bleeding risk with trastuzumab (Herceptin)treatment. JAMA 1999;282:2299-301.

[43] Chen Y, Dong J, Lu Y, McGee JO. Quantitative detection ofamplification of proto-oncogenes in breast cancer. Chin Med J (Eng)1995;108:849-54.

[44] Donovan-Peluso M, Contento AM, Tobon H, Ripepi B, Locker J.Oncogene amplification in breast cancer. Am J Pathol 1991;138:835-45.

[45] Valeron PF, Chirino R, Fernandez L, et al. Validation of a differentialPCR and an ELISA procedure in studying HER-2/neu status in breastcancer. Int J Cancer 1996;65:129-33.

[46] van de Vijver MJ, Mooi WJ, Wisman P, Peterse JL, Nusse R.Immunohistochemical detection of the neu protein in tissue sectionsof human breast tumors with amplified neu DNA. Oncogene 1988;2:175-8.

[47] Molina R, Ciocca DR, Tandon AK, et al. Expression of HER-2/neuoncoprotein in human breast cancer: a comparison of immunohisto-chemical and western blot techniques. Anticancer Res 1992;12:1965-71.

[48] Press MF, Pike MC, Chazin VR, et al. Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerizedimage analysis and association of overexpression with increased riskof recurrent disease. Cancer Res 1993;53:4960-70.

[49] Birner P, Oberhuber G, Stani J, et al. Austrian Breast & ColorectalCancer Study Group. Evaluation of the United States Food andDrug Administration–approved scoring and test system of HER-2protein expression in breast cancer. Clin Cancer Res 2001;7:1669-75.

[50] O'Malley FP, Parkes R, Latta E, et al. Comparison of HER2/neustatus assessed by quantitative polymerase chain reaction andimmunohistochemistry. Am J Clin Pathol 2001;115:504-11.

[51] Pauletti G, Godolphin W, Press MF, Slamon DJ. Detection andquantitation of HER-2/neu gene amplification in human breast cancerarchival material using fluorescence in situ hybridization. Oncogene1996;13:63-72.

[52] Fernandez JL, Goyanes V, Lopez-Fernandez C, Buno I, Gosalvez J.Quantification of C-ERB-B2 gene amplification in breast cancer cellsusing fluorescence in situ hybridization and digital image analysis.Cancer Genet Cytogenet 1996;86:18-21.

[53] Press MF, Bernstein L, Thomas PA, et al. HER-2/neu geneamplification characterized by fluorescence in situ hybridization:poor prognosis in node-negative breast carcinomas. J Clin Oncol1997;15:2894-904.

http://www.cancer.org/docroot/CRI/content/CRI_2_4_1X_What_are_the_key_statistics_for_breast_cancer_5.asp?sitearea




[54] Xing WR, Gilchrist KW, Harris CP, Samson W, Meisner LF. FISHdetection of HER-2/neu oncogene amplification in early onset breastcancer. Breast Cancer Res Treat 1996;39:203-12.

[55] Gonzalez-Angulo AM, Hortobagyi GN, Esteva FJ. Adjuvant therapywith trastuzumab for HER-2/neu–positive breast cancer. Oncologist2006;11:857-67.

[56] Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvantchemotherapy for operable HER2-positive breast cancer. N Engl JMed 2005;353:1673-84.

[57] Joensuu H, Kellokumpu-Lehtinen PL, Bono P, et al. Adjuvantdocetaxel or vinorelbine with or without trastuzumab for breastcancer. N Engl J Med 2006;354:809-20.

[58] Coudert BP, Arnould L, Moreau L, et al. Pre-operative systemic (neo-adjuvant) therapy with trastuzumab and docetaxel for HER2-overexpressing stage II or III breast cancer: results of a multicenterphase II trial. Ann Oncol 2006;17:409-14.

[59] Limentani SA, BrufskyAM, Erban JK, JahanzebM, Lewis D. Phase IIstudy of neoadjuvant docetaxel, vinorelbine, and trastuzumabfollowed by surgery and adjuvant doxorubicin plus cyclopho-sphamide in women with human epidermal growth factor receptor2–overexpressing locally advanced breast cancer. J Clin Oncol 2007;25:1232-8.

[60] Gouvea AP, Milanezi F, Olson SJ, Leitao D, Schmitt FC, Gobbi H.Selecting antibodies to detect HER2 overexpression by immunohis-tochemistry in invasive mammary carcinomas. Appl Immunohisto-chem Mol Morphol 2006;14:103-8.

[61] Baak JP, Chin D, van Diest PJ, Ortiz R, Matze-Cok P, Bacus SS.Comparative long-term prognostic value of quantitative HER-2/neuprotein expression, DNA ploidy, and morphometric and clinicalfeatures in paraffin-embedded invasive breast cancer. Lab Invest1991;64:215-23.

[62] Press MF, Hung G, Godolphin W, Slamon DJ. Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error inimmunohistochemical studies of oncogene expression. Cancer Res1994;54:2771-7.

[63] Johnson RC, Ricci Jr A, Cartun RW, Ackroyd R, Tsongalis GJ.p185HER2 overexpression in human breast cancer using molecularand immunohistochemical methods. Cancer Invest 2000;18:336-42.

[64] Wang S, Saboorian MH, Frenkel E, Hynan L, Gokaslan ST, AshfaqR. Laboratory assessment of the status of Her-2/neu protein andoncogene in breast cancer specimens: comparison of immunohis-tochemistry assay with fluorescence in situ hybridisation assays. JClin Pathol 2000;53:374-81.

[65] Tubbs RR, Pettay JD, Roche PC, Stoler MH, Jenkins RB, GroganTM. Discrepancies in clinical laboratory testing of eligibility fortrastuzumab therapy: apparent immunohistochemical false-positivesdo not get the message. J Clin Oncol 2001;19:2714-21.

[66] Seidman AD, Fornier MN, Esteva FJ, et al. Weekly trastuzumab andpaclitaxel therapy for metastatic breast cancer with analysis ofefficacy by HER2 immunophenotype and gene amplification. J ClinOncol 2001;19:2587-95.

[67] Tedesco KL, Thor AD, Johnson DH, et al. Docetaxel combined withtrastuzumab is an active regimen in HER-2 3+ overexpressing andfluorescent in situ hybridization–positive metastatic breast cancer: amulti-institutional phase II trial. J Clin Oncol 2004;22:1071-7.

[68] Wolff AC, Hammond ME, Schwartz JN, et al. American Society ofClinical Oncology; College of American Pathologists. AmericanSociety of Clinical Oncology/College of American Pathologistsguideline recommendations for human epidermal growth factorreceptor 2 testing in breast cancer. J Clin Oncol 2007;25:118-45.

[69] Press MF, Slamon DJ, Flom KJ, Park J, Zhou JY, Bernstein L.Evaluation of HER-2/neu gene amplification and overexpression:comparison of frequently used assay methods in a molecularlycharacterized cohort of breast cancer specimens. J Clin Oncol 2002;20:3095-105.

[70] Lal P, Salazar PA, Hudis CA, Ladanyi M, Chen B. HER-2 testingin breast cancer using immunohistochemical analysis and

fluorescence in situ hybridization: a single-institution experienceof 2,279 cases and comparison of dual-color and single-colorscoring. Am J Clin Pathol 2004;121:631-6 [Erratum in: Am JClin Pathol, 2004;6:58.

[71] Ma Y, Lespagnard L, Durbecq V, et al. Polysomy 17 in HER-2/neustatus elaboration in breast cancer: effect on daily practice. ClinCancer Res 2005;11:4393-9.

[72] Persons DL, Tubbs RR, Cooley LD, et al. HER-2 fluorescence in situhybridization: results from the survey program of the College ofAmerican Pathologists. Arch Pathol Lab Med 2006;130:325-31.

[73] Dal Lago L, Durbecq V, Desmedt C, et al. Correction for chro-mosome-17 is critical for the determination of true Her-2/neu geneamplification status in breast cancer. Mol Cancer Ther 2006;5:2572-9.

[74] Vera-Roman JM, Rubio-Martinez LA. Comparative assays for theHER-2/neu oncogene status in breast cancer. Arch Pathol Lab Med2004;128:627-33.

[75] Tanner M, Gancberg D, Di Leo A, et al. Chromogenic in situhybridization: a practical alternative for fluorescence in situhybridization to detect HER-2/neu oncogene amplification in archivalbreast cancer samples. Am J Pathol 2000;157:1467-72.

[76] Dandachi N, Dietze O, Hauser-Kronberger C. Chromogenic in situhybridization: a novel approach to a practical and sensitive methodfor the detection of HER2 oncogene in archival human breastcarcinoma. Lab Invest 2002;82:1007-14.

[77] Gupta D, Middleton LP, Whitaker MJ, Abrams J. Comparison offluorescence and chromogenic in situ hybridization for detection ofHER-2/neu oncogene in breast cancer. Am J Clin Pathol 2003;119:381-7.

[78] Isola J, Tanner M, Forsyth A, Cooke TG, Watters AD, Bartlett JM.Interlaboratory comparison of HER-2 oncogene amplification asdetected by chromogenic and fluorescence in situ hybridization. ClinCancer Res 2004;10:4793-8.

[79] Peiro G, Mayr D, Hillemanns P, Lohrs U, Diebold J. Analysis ofHER-2/neu amplification in endometrial carcinoma by chromogenicin situ hybridization. Correlation with fluorescence in situ hybridiza-tion, HER-2/neu, p53 and Ki-67 protein expression, and outcome.Mod Pathol 2004;17:227-87.

[80] Gong Y, Gilcrease M, Sneige N. Reliability of chromogenic in situhybridization for detecting HER-2 gene status in breast cancer:comparison with fluorescence in situ hybridization and assessment ofinterobserver reproducibility. Mod Pathol 2005;18:1015-21.

[81] Loring P, Cummins R, O'Grady A, Kay EW.HER2 positivity in breastcarcinoma: a comparison of chromogenic in situ hybridization withfluorescence in situ hybridization in tissue microarrays, with targetedevaluation of intratumoral heterogeneity by in situ hybridization. ApplImmunohistochem Mol Morphol 2005;13:194-200.

[82] Bhargava R, Lal P, Chen B. Chromogenic in situ hybridization for thedetection of HER-2/neu gene amplification in breast cancer with anemphasis on tumors with borderline and low-level amplification:does it measure up to fluorescence in situ hybridization? Am J ClinPathol 2005;123:237-43.

[83] Nitta H, Christensen K, Grogan TM. Automated colormetrictyramide signal amplification in situ hybridization (TISH) detectionof single HER-2/neu oncogene copies. Acta Histochem Cytochem2002;35:224.

[84] Laakso M, Tanner M, Isola J. Dual-colour chromogenic in situhybridization for testing of HER-2 oncogene amplification in archivalbreast tumours. J Pathol 2006;210:3-9.

[85] Faulk WP, Taylor GM. An immunocolloid method for the electronmicroscope. Immunochemistry 1971;8:1081-3.

[86] Handley DA. The development and application of colloidal gold as amicroscopic probe; methods for synthesis of colloidal gold. In: HayatMA, editor. Colloidal gold: principles, methods and applications. SanDiego, CA: Academic Press; 1989. p. 1-33.

[87] Wu M, Davidson N. Transmission electron microscopic method forgene mapping on polytene chromosomes by in situ hybridization.Proc Natl Acad USA Sci 1981;78:7059-63.


[88] Hutchison NJ, Langer-Safer PR, Ward DC, Hamkalo BA. In situhybridization at the electron microscope level: hybrid detection byautoradiography and colloidal gold. J Cell Biol 1982;95:609-18.

[89] Ferguson DJ, Burns J, Harrison D, Jonasson JA, McGee JO.Chromosomal localization of genes by scanning electron microscopyusing in situ hybridization with biotinylated probes: Y chromosomerepetitive sequences. J Histochem 1986;18:266-70.

[90] Danscher G. Localization of gold in biological tissue. A photo-chemical method for light and electronmicroscopy. Histochemistry1981;71:81-8.

[91] Danscher G, Norgaard JO. Light microscopic visualization ofcolloidal gold on resin-embedded tissue. J Histochem Cytochem1983;31:1394-8.

[92] Hacker GW, Springall DR, Van Noorden S, Bishop AE, Grimelius L,Polak JM. The immunogold-silver staining method. A powerful toolin histopathology. Virchows Arch A Pathol Anat Histopathol 1985;406:449-61.

[93] Hacker GW, Graf AH, Hauser-Kronberger C, et al. Application ofsilver acetate autometallography and gold-silver staining methods forin situ DNA hybridization. Chin Med J (Engl) 1993;106:83-92.

[94] Hainfeld JF, Powell RD. New frontiers in gold labeling. J HistochemCytochem 2000;48:471-80.

[95] Hainfeld JF. STEM analysis of Janssen Auroprobe One. In: BaileyGW, editor. Proc. XII Int. Cong. Elec. Microsc. San Francisco, CA:San Francisco Press; 1990. p. 954-5.

[96] Behnke O, Ammitzboll T, Jessen H, et al. Non-specific binding ofprotein-stabilized gold sols as a source of error in immunocytochem-istry. Eur J Cell Biol 1986;41:326-38.

[97] Kramarcy NR, Sealock R. Commercial preparations of colloidalgold–antibody complexes frequently contain free active antibody. JHistochem Cytochem 1990;39:37-9.

[98] Sun XJ, Tolbert LP, Hildebrand JG. Using laser scanning confocalmicroscopy as a guide for electron microscopic study: a simplemethod for correlation of light and electron microscopy. J HistochemCytochem 1995;43:329-35.

[99] Hainfeld JF, Furuya FR. Silver enhancement of Nanogold andUndecagold. In: Hayat MA, editor. Immunogold-silver staining:principles, methods and applications. Boca Raton, FL: CRC Press;1995. p. 71-96.

[100] Hacker GW, Zehbe I, Hainfeld J, et al. High-performance Nanogoldin situ hybridization and in situ PCR. Cell Vision 1996;3:209-14.

[101] Zehbe I, Hacker GW, Su H, Hauser-Kronberger C, Hainfeld JF,Tubbs R. Sensitive in situ hybridization with catalyzed reporterdeposition, streptavidin-Nanogold, and silver acetate autometallo-graphy: detection of single-copy human papillomavirus. Am J Pathol1997;150:1553-61.

[102] Hacker GW, Hauser-Kronberger C, Zehbe I, et al. In situ localizationof DNA and RNA sequences: super-sensitive in situ hybridizationusing streptavidin-Nanogold-silver staining: minireview, protocolsand possible applications. Cell Vision 1997;4:54-65.

[103] Hainfeld JF, Powell RD. Silver- and gold-based autometallography ofNanogold. In: Hacker GW, Gu J, editors. Gold and silver staining:techniques in molecular morphology. Boca Raton: CRC Press; 2002.p. 29-46.

[104] Owen GR, Meredith DO, Ap Gwynn I, Richards RG. Enhancement ofimmunogold-labelled focal adhesion sites in fibroblasts cultured onmetal substrates: problems and solutions. Cell Biol Int 2001;25:1251-9.

[105] Cheung AL, Graf AH, Hauser-Kronberger C, Dietze O, Tubbs RR,Hacker GW. Detection of human papillomavirus in cervicalcarcinoma: comparison of peroxidase, Nanogold, and catalyzedreporter deposition (CARD)–Nanogold in situ hybridization. ModPathol 1999;12:689-96.

[106] Powell RD, Joshi VN, Halsey CMR, et al. Combined Cy3/Nanogoldconjugates for immunocytochemistry and in situ hybridization. In:Bailey GW, JeromeWG, McKernan S, Mansfield JF, Price RL, editors.Microsc Microanal, 5 (Suppl. 2: Proceedings); 1999. p. 478-9.

[107] Tubbs R, Pettay J, Skacel M, et al. Gold-facilitated in situhybridization: a bright-field autometallographic alternative to

fluorescence in situ hybridization for detection of Her-2/neu geneamplification. Am J Pathol 2002;160:1589-95.

[108] Tubbs R, Skacel M, Pettay J, et al. Interobserver interpretativereproducibility of GOLDFISH, a first generation gold-facilitatedautometallographic bright field in situ hybridization assay for HER-2/neu amplification in invasive mammary carcinoma. Am J Surg Pathol2002;26:908-13.

[109] Hicks D, Ruddy T, Downs-Kelly E, et al. Quantitative image analysisof GOLDFISH, (a first generation gold-facilitated autometallographicbright field in-situ hybridization assay) for HER2 gene amplificationin invasive breast cancer. 26th San Antonio Breast CancerSymposium; 2003; poster #611.

[110] Hainfeld JF, Eisen RN, Tubbs RR, Powell RD. Enzymaticmetallography: a simple new staining method. In: Lyman CE,Albrecht RM, Carter CB, Dravid VP, Herman B, Schatten H, editors.Microsc Microanal, 8 (Suppl. 2: Proceedings); 2002. p. 916 CD.

[111] Hainfeld JF, Powell RD, Hacker GW. Nanoparticle molecular labels.In: Mirkin CA, Niemeyer CM, editors. Nanobiotechnology. Ger-many: Wiley-VCH, Weinheim; 2004. p. 353-86. [ch. 23].

[112] Tubbs R, Pettay J, Hicks D, et al. Novel bright field molecularmorphology methods for detection of HER2 gene amplification. JMol Histol 2004;35:589-94.

[113] Tubbs RR, Pettay J, Skacel M, et al. Gold and silver-facilitatedmetallographic in situ hybridization procedures for detection ofHER2 gene amplification. In: Hacker GW, Tubbs RR, editors.Molecular morphology in human tissues. Boca Raton, FL: CRCPress; 2005. p. 101-6. [ch. 5].

[114] Tubbs R, Pettay J, Powell R, et al. Enzyme metallography (EnzMet):a novel second generation bright field assay for detection of HER2gene amplification in breast carcinoma. J Mol Diagn (AMP 2003Meeting Abstracts) 2003;5:279.

[115] Bonin S, Petrera F, Rosai J, Stanta G. DNA and RNA obtained fromBouin's fixed tissues. J Clin Pathol 2005;58:313-6.

[116] Sato T, Abe K, Kurose A, Uesugi N, Todoroki T, Sasaki K.Amplification of the c-erbB-2 gene detected by FISH in gastriccancers. Pathol Int 1997;47:179-82.

[117] Ishikawa T, Kobayashi M, Mai M, Suzuki T, Ooi A. Amplificationof the c-erbB-2 (HER-2/neu) gene in gastric cancer cells. Detectionby fluorescence in situ hybridization. Am J Pathol 1997;151:761-8.

[118] Tubbs R, Pettay J, Powell R, et al. High-resolution immunophenotypingof subcellular compartments in tissue microarrays by enzymemetallography. Appl Immunohistochem Mol Morphol 2005;13:371-5.

[119] Tubbs RR, Pettay J, Roche P, et al. Concomitant oncoproteindetection with fluorescence in situ hybridization (CODFISH): afluorescence-based assay enabling simultaneous visualization of geneamplification and encoded protein expression. J Mol Diagn 2000;2:78-83.

[120] Downs-Kelly E, Pettay J, Hicks D, et al. Analytical validation andinterobserver reproducibility of EnzMet GenePro: a second-genera-tion bright-field metallography assay for concomitant detection ofHER2 gene status and protein expression in invasive carcinoma of thebreast. Am J Surg Pathol 2005;29:1505-11.

[121] Skacel M, Skilton B, Pettay JD, Tubbs RR. Tissue microarrays: apowerful tool for high-throughput analysis of clinical specimens: areview of the method with validation data. Appl ImmunohistochemMol Morphol 2002;10:1-6.

[122] Myers J. Automated slide stainers for SS, IHC, and ISH: A review ofcurrent technologies and commercially available systems. MLO MedLab Obs 2004;36:28-30.

[123] Rimsza LM, Vela EE, Frutiger YM, et al. Rapid automated combinedin situ hybridization and immunohistochemistry for sensitivedetection of cytomegalovirus in paraffin-embedded tissue biopsies.Am J Clin Pathol 1996;106:544-8.

[124] Lloyd RV. Practical markers used in the diagnosis of neuroendocrinetumors. Endocr Pathol 2003;14:293-301.

[125] Lloyd RV. Advances in pituitary pathology: use of novel techniques.Front Horm Res 2004;32:146-74.

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