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<front> Out of the darkness and into the light: bright field in <lb/>situ hybridisation for delineation of ERBB2 (HER2) <lb/>status in breast carcinoma <lb/> Aaron M Gruver, Ziad Peerwani, Raymond R Tubbs <lb/> ABSTRACT <lb/> Assessment of ERBB2 (HER2) status in breast <lb/>carcinomas has become critical in determining response <lb/>to the humanised monoclonal antibody trastuzumab. The <lb/>current joint College of American Pathologists and the <lb/>American Society of Clinical Oncology guidelines for the <lb/>evaluation of HER2 status in breast carcinoma involve <lb/>testing by immunohistochemistry and fluorescence in situ <lb/>hybridisation (FISH). However, neither of these modalities <lb/>is without limitations. Novel bright field in situ <lb/>hybridisation techniques continue to provide viable <lb/>alternatives to FISH testing. While these techniques are <lb/>not limited to evaluation of the HER2 gene, the extensive <lb/>number of studies comparing bright field in situ <lb/>techniques with other methods of assessing HER2 status <lb/>allow a robust evaluation of this approach. Analysis of the <lb/>literature demonstrates that, when used to assess HER2 <lb/> gene status, bright field in situ hybridisation demonstrates <lb/>excellent concordance with FISH results. The average <lb/>percentage agreement in an informal analysis of studies <lb/>comparing HER2 amplification by chromogenic in situ <lb/>hybridisation with FISH was 96% (SD 4%); k coefficients <lb/>ranged from 0.76 to 1.0. Although a much smaller <lb/>number of studies are available for review, similar levels <lb/>of concordance have been reported in studies comparing <lb/> HER2 amplification by methods employing metallography <lb/>(silver in situ hybridisation) with FISH. A summary of the <lb/>advancements in bright field in situ hybridisation, with <lb/>focus on those techniques with clinical applications of <lb/>interest to the practicing pathologist, is presented. <lb/></front>
<body> INTRODUCTION <lb/>Historical perspectives on in situ hybridisation <lb/> At the time of Watson and Crick's published <lb/>description of DNA structure in 1953, 1 Tjio and <lb/>Levan had yet to publish the first reliable determi-<lb/>nation of the normal human chromosome comple-<lb/>ment. 2 Much discovery was still needed before early <lb/>knowledge of DNA technology could be applied to <lb/>the field of cytogenetics. 3 Although the technique of <lb/>DNAeDNA hybridisation had been introduced in <lb/>1961, 4 it was not until 1969 that successful <lb/>attempts using radiographically labelled DNA and <lb/>RNA to identify chromosomal targets of cytological <lb/>preparations were made. 5 6 These early studies <lb/>relied upon a tritium-labelled RNA probe, derived <lb/>from mixtures of Xenopus 28S and 18S RNA, and <lb/>alkaline denaturation of extrachromosomal rDNA <lb/>from Xenopus oocytes. 5 Hybridised sequences were <lb/>detected by autoradiography. Although limited by <lb/>the resolution of the radiographic detection method <lb/>employed, Gall and Pardue were able to demonstrate <lb/>that RNA, and soon after DNA, can be hybridised <lb/>specifically to target sequences under conditions <lb/>that 'preserve the morphological integrity of the <lb/>nucleus'. 5 6 Furthermore, the ability of this in situ <lb/>technology to quantify relative amounts of target <lb/>sequence was suggested by the detection of a low <lb/>level gene amplification in premeiotic oogonia. 5 <lb/> Additional successes were soon reported in <lb/>employing autoradiographic detection of rRNA and <lb/>DNA hybrids in tissue sections and in cytological <lb/>specimens. 7 8 <lb/> Over the years, much improvement has been <lb/>made in the processes with which probes are <lb/>developed and labeled, including the introduction <lb/>of random primer labelling, nick translation reac-<lb/>tion and PCR-based labelling. 3 Revolutionary <lb/>discoveries were reported in 1982 by two groups <lb/>who performed hybridisation experiments with <lb/>probes labelled either fluorimetrically or cyto-<lb/>chemically, rather than with radioisotopes. 9 10 <lb/> These fluorescent labels provided many advantages <lb/>to the in situ hybridisation technique, including <lb/>improvements in the easy and safety of use, <lb/>increases in resolution, and the possibilities of <lb/>simultaneously identifying multiple targets within <lb/>the same nucleus. 11 This new technique, fluores-<lb/> cence in situ hybridisation (FISH), could be <lb/>accomplished using a probe labelled either directly <lb/>or indirectly with a fluorochrome, and the basic <lb/>principles of these labelling techniques have been <lb/>recently reviewed. 12 Briefly, direct labelling is the <lb/>process of incorporating fluorescently labelled <lb/>nucleotides into the nucleic acid probe; indirect <lb/>labelling often involves complexing the probe with <lb/>an intermediary hapten (eg, digoxigenin) that is <lb/>subsequently detected with a labelled antibody to <lb/>identify the target sequence of interest. <lb/>By 1985, another milestone in the in situ <lb/>hybridisation technique was achieved when Land-<lb/>egent et al demonstrated localisation of the human <lb/>thyroglobulin gene to a specific chromosome band <lb/>using a probe constructed from cosmid subclones of <lb/>the 3 9 region of the thyroglobulin gene. 13 By the <lb/>turn of the century, further refinement of the FISH <lb/>technique lead to routine localisation of DNA <lb/>targets as small as 10 kb and the ability to localise <lb/>segments as small as 1 kb. 11 Technical advance-<lb/>ments through the years have spawned a variety of <lb/>FISH technologies, 14 and many of these experi-<lb/>mental achievements are considered among the <lb/>most significant milestones in the field of cytoge-<lb/>netics and molecular pathology. 3 FISH has been <lb/>particularly successful for mapping single-copy and <lb/>repetitive DNA sequences using metaphase and <lb/></body>
<front>Department of Molecular <lb/>Pathology, Pathology and <lb/>Laboratory Medicine Institute, <lb/>Cleveland Clinic, Lerner College <lb/>of Medicine, Cleveland, Ohio, <lb/>USA <lb/> Correspondence to <lb/> Raymond R Tubbs, Department <lb/>of Molecular Pathology, <lb/>Pathology and Laboratory <lb/>Medicine Institute, Cleveland <lb/>Clinic, Lerner College of <lb/>Medicine, Cleveland, OH 44195, <lb/>USA; tubbsr@ccf.org <lb/>Accepted 23 November 2009 <lb/>This paper is freely available <lb/>online under the BMJ Journals <lb/>unlocked scheme, see http://jcp. <lb/>bmj.com/site/about/unlocked. <lb/>xhtml <lb/></front>
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<body> interphase nuclei, for detecting targeted chromosome trans-<lb/>locations, and for localising large repeat families to aid in chro-<lb/>mosome identification and karyotype analysis. The research <lb/>application of this technology is vast; clinically, FISH has proved <lb/>invaluable in the diagnosis, prognostication and pharmacoge-<lb/>nomic assessment of many diseases. <lb/>Despite the advantages of FISH, the technique is not without <lb/>drawbacks. Often cited limitations to the routine implementa-<lb/>tion of conventional FISH include the requirements of a dedi-<lb/>cated fluorescence imaging system and well-trained personnel <lb/>with specific expertise. Furthermore, FISH studies provide rela-<lb/>tively limited morphological assessment of overall histology, <lb/>reduced stability of the fluorescent detection signal(s), and <lb/>overall higher cost of testing. These limitations have prompted <lb/>new achievements in the arena of in situ hybridisation detec-<lb/>tion. The purpose of this review is to summarise the advance-<lb/>ments in bright field in situ hybridisation in use today with <lb/>a focus on those techniques with clinical applications of interest <lb/>to the practicing pathologist. <lb/> Clinical applications of bright field in situ hybridisation: the HER2 <lb/> story and beyond <lb/> The continuous evolution of our understanding of the molecular <lb/>pathogenesis of disease is perpetually altering our clinical decision <lb/>making and therapeutic strategies. These changes have placed <lb/>pressure upon clinical laboratories to provide adequate testing <lb/>platforms to provide insight into the status of the disease of an <lb/>individual patient. For many neoplastic processes, tissue micro-<lb/>scopic morphology is the foundation to a diagnosis being made, <lb/>and paraffin-embedded tissue provides an abundant source of <lb/>archived material for molecular testing. As the need for molecular <lb/>testing has increased, multiple techniques have been created or <lb/>incorporated into the clinical laboratory to provide these neces-<lb/>sary results. The various in situ hybridisation techniques meld <lb/>a focused genetic technique upon the histology slide platform. <lb/>Nonetheless, the amount of architectural information available <lb/>for review depends on the type of in situ hybridisation procedure <lb/>used. Bright field in situ hybridisation is particularly beneficial in <lb/>this regard, as the majority of the morphological detail present on <lb/>routine H&E-stained sections is preserved. Although in situ <lb/>hybridisation can be used to assess a myriad of different molecular <lb/>genetic aberrations, 15 investigation of the HER2 gene status in <lb/>breast carcinoma has been a major impetus for the development <lb/>of bright field in situ hybridisation techniques. <lb/>As one of the five leading causes of cancer deaths worldwide, <lb/>The World Health Organization recently projected that breast <lb/>cancer will cause 630 000 deaths in 2015. 16 This disease burden <lb/>necessitates efficient use of limited healthcare resources. The <lb/>ability to target specific genetic aberrations that are susceptible <lb/>to a specific therapy is becoming a best clinical practice for <lb/>treating a variety of diseases, particularly neoplasia. Discovery of <lb/>the role of HER2 in breast cancer, and subsequent discovery of <lb/>a viable corresponding gene-specific therapy, highlights the <lb/>central role of a specific genetic aberration in some breast cancers, <lb/>the ability to create therapeutics that target these specific aber-<lb/>rations, and the crucial necessity to identify the molecular <lb/>genetic status of an individual's breast cancer to personalise the <lb/>clinical management. The experience with HER2 in breast <lb/>carcinoma exemplifies the melding of a specific laboratory test <lb/>with a specific therapy, pharmacogenomics, and the important <lb/>role of in situ technology in clinical practice. <lb/> HER2 (ERBB2) is a proto-oncogene that encodes for a 185 <lb/>kDa protein that is a member of the ERB family of transmembrane <lb/>tyrosine kinase receptors. This receptor exists in a dimerisation-<lb/>ready conformation, and does not require ligand binding to form <lb/>functional dimers. Although it can form homodimers, it rarely <lb/>does. Rather, it preferentially forms heterodimers with the <lb/>remaining members of its family, particularly HER3. Depending <lb/>upon the heterodimer, various signalling pathways are activated. 17 <lb/> This results in HER2 playing a role in different cellular functions, <lb/>including the promotion of cell division and survival, while <lb/>inhibiting apoptosis. These various functions reflect its potential <lb/>to produce an oncogenic effect following HER2 gene amplification. <lb/>In 1987, Slamon et al published the first study identifying the <lb/>role of HER2 in a subset of breast cancers. 18 The authors <lb/>demonstrated that HER2 amplification by Southern blotting was <lb/>an independent variable linked to inferior overall survival and <lb/>progression-free survival in multivariate analysis. During this <lb/>same time period, Greene et al provided evidence that monoclonal <lb/>antibodies against the p185 product of HER2 inhibited HER2-<lb/> transformed cell lines implanted in nude mice. 19 These studies, <lb/>among others, laid the foundation for the development of <lb/>a targeted therapy in breast cancer: trastuzumab. <lb/>Trastuzumab, or Herceptin, is a humanised monoclonal anti-<lb/>body against the 185 kDa protein of HER2. Its effect upon HER2-<lb/> positive breast cancer is not limited to the immune response <lb/>upon antibody binding. Rather, its effects are diverse and include <lb/>the inhibition of dimerisation, induction of apoptosis, decreased <lb/>cellular proliferation, and the modulation of signal transduction <lb/>pathways. In 2001, the first phase III clinical trial of trastuzumab <lb/>was published by Slamon et al. 20 This prospective study exam-<lb/>ined the effect of trastuzumab on overall and progression-free <lb/>survival in a cohort of women with metastatic breast cancer. A <lb/>significant improvement in progression-free survival was <lb/>demonstrated when trastuzumab was added to the chemother-<lb/>apeutic protocols. Later studies, with a longer follow-up period, <lb/>confirmed a significant improvement in overall survival when <lb/>trastuzumab was added to the treatment of women with <lb/>metastatic breast cancer. In 2007, the HERA international <lb/>multicentre randomised trial reported on the use of trastuzumab <lb/>in patients with HER2-amplified early stage breast cancers. 21 A 2-<lb/>year follow-up of the study showed a significant improvement in <lb/>overall survival when trastuzumab was used in conjunction with <lb/>standard therapeutic regimens. Currently, trastuzumab is used in <lb/>the adjuvant setting for treatment of early stage breast cancer as <lb/>well as metastatic breast cancer. Although the role of trastu-<lb/>zumab as neoadjuvant therapy is still being investigated, 22 <lb/> preliminary studies have demonstrated a significantly better <lb/>pathological complete response in patients receiving neoadjuvant <lb/>trastuzumab in combination with other agents. 23e25 <lb/> Throughout these studies, the drug toxicity of trastuzumab <lb/>has been a concern, particularly the cardiac side effects. 26 These <lb/>ranged from mild left ventricular dysfunction to severe conges-<lb/>tive heart failure. The severity of side effects further emphasised <lb/>the clinical imperative to use trastuzumab only in the subset of <lb/>patients whose clinical benefit would outweigh the risk of <lb/>treatment side effects. <lb/>The clinical utility of trastuzumab, juxtaposed with the <lb/>potential for drug toxicity, mandates the use of this therapy in <lb/>the select group of patients who demonstrate HER2 amplifica-<lb/>tion. This creates a clinical laboratory imperative to provide <lb/>accurate and precise testing when assessing the HER2 status in <lb/>breast cancer patients. In 2007, the American Society for Clinical <lb/>Oncology and the College of American Pathologists (ASCO/ <lb/>CAP) published a joint guideline to standardise HER2 testing in <lb/>the USA. 27 28 The panel provided testing algorithms and test <lb/>interpretation guidelines. The concern of equivocal or false <lb/>positive results by immunohistochemistry is illustrated in the <lb/>
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interpretation guidelines. The panel redefined 2+ immunohis-<lb/>tochemical staining for the HER2 gene product as equivocal, <lb/>rather than positive. This change reflects two features previously <lb/>identified of this category of test results. First, a large portion of <lb/>cases that stain 2+ fail to show gene amplification by FISH. <lb/>Considering that FISH has a high concordance with Southern <lb/>blotting, it was decided that 2+ scoring was equivocal with <lb/>regards to HER2 status rather than positive. Second, up to 15% of <lb/>clinical cases assessed using immunohistochemistry (IHC) fall <lb/>within the equivocal category. Their recommendation requires <lb/>that cases that are equivocal by IHC be retested using a validated <lb/>assay for the HER2 gene status. Specific guidelines for interpre-<lb/>tation of bright field in situ hybridisation results were also <lb/>provided by the panel. <lb/>These pressures provide impetus to further develop laboratory <lb/>tests to fulfil this testing requirement. Although an armamen-<lb/>tarium of strategies to detect HER2 status in research and clinical <lb/>laboratories exists, including Southern blotting, PCR, IHC and <lb/>FISH, the limitations of each of these have been documented. 29 <lb/> More specifically, although FISH is a robust test, its complicated <lb/>procedure coupled with high technical expertise requirements <lb/>precludes its use except in laboratories equipped and staffed to <lb/>perform and interpret this highly complex testing. Over the last <lb/>decade, the development of bright field in situ hybridisation <lb/>techniques attempts to address the difficulties limiting wide-<lb/>spread FISH testing. <lb/>Although bright field in situ hybridisation has gained much <lb/>attention through the need to develop an accurate test of HER2 <lb/> status that can be performed efficiently and cost effectively in <lb/>many clinical laboratories, the application of this technology is <lb/>not confined to assessment of HER2 status in breast carcinomas. <lb/>Several other gene targets have been under investigation, and the <lb/>implications of testing for these have been reported. 30e34 <lb/> Recently, published studies have used chromogenic in situ <lb/>hybridisation as a means of assessing platelet-derived growth <lb/>factor receptor A in gliomas, 35 determining amplification of the <lb/>epidermal growth factor receptor (EGFR) gene in anal squamous <lb/>lesions, 36 and correlating the EGFR gene copy number with <lb/>therapy response in colorectal cancers. 37 Furthermore, the use of <lb/>silver-enhanced in situ hybridisation to evaluate EGFR status in <lb/>human glioblastomas has demonstrated strong concordance <lb/>with FISH and gene expression data. 38 The principles of various <lb/>bright field technologies in use today, along with their benefits <lb/>and limitations, are described below. <lb/> A NEW DAWN APPROACHES <lb/>Chromogenic in situ hybridisation <lb/> The basic principles of in situ hybridisation are straight forward <lb/>and can be simplified as: use of a DNA probe complementary to <lb/>a target sequence of interest followed by detection of the bound <lb/>probe. 39 Generation of the probe, method of labelling, condition <lb/>of hybridisation, and strategy for detection are all areas of nuance <lb/>that depend upon the type of in situ hybridisation technique <lb/>employed. 40 Chromogenic in situ hybridisation (CISH) was first <lb/> described by Tanner et al in 2000 as an alternative to FISH <lb/>detection of HER2 amplification in archival breast tissue. 41 In <lb/>that study, paraffin-embedded tissue sections were pretreated <lb/>and subsequently hybridised with a digoxigenin-labelled DNA <lb/>probe. The probe was detected by use of antidigoxigenin fluo-<lb/> rescein, followed by antifluorescein peroxidase and diamino-<lb/>benzidine. The basic principles of this CISH technique are <lb/>outlined in figure 1. <lb/>Although enzymatic DNA in situ hybridisation of CCNDI had <lb/>been previously described, the study by Tanner et al was the first <lb/> to examine the status of the HER2 gene in paraffin-embedded <lb/>tumour samples using a modified detection system with superior <lb/>sensitivity to the antidigoxigenin plus biotynlated-tyramine <lb/>amplification, with visualisation using diaminobenzidine and <lb/>hydrogen peroxide, already in use. 42 While improvements upon <lb/>the use of cosmid, P1, PAC and BAC clone probes for bright field <lb/> in situ hybridisation had been reported, 43 additional advances <lb/>were made in the CISH technique through pretreatment of tissue <lb/>sections by heating in a microwave followed by a short period of <lb/>enzyme digestion. The detection system used was an anti-<lb/>digoxigenin-fluorescein isothiocyanate antibody plus an anti-<lb/> fluorescein-isothiocyanate horseradish peroxidase conjugate. 41 <lb/> This original HER2 CISH procedure involved single-colour <lb/>detection of one probe, similar to the US Food and Drug <lb/>Administration (FDA) approved FISH testing for HER2 available <lb/>at the time. Comparison of CISH detection of HER2 to that of <lb/>FISH correlated well in the series of 157 breast cancers examined <lb/>(93.6% concordance). 41 <lb/> Since the study by Tanner et al, additional variations of CISH <lb/>technology have been evaluated. In general, the CISH technique <lb/>employs either antibodies or other proteins (eg, avidin) conju-<lb/>gated to an enzyme (eg, horseradish peroxidase) in order to <lb/>produce a chromogenic, rather than a fluorometric, reaction. <lb/>Unlike FISH, chromogenic in situ hybridisation performs best <lb/>when indirect labelling of the probe is used. 12 Examples of the <lb/>staining quality that is achievable through the CISH technique <lb/>are demonstrated in figure 2. Although a large variety of <lb/>commercial probes are available for testing by FISH, a relatively <lb/>limited number of probes are available for CISH. 12 However, <lb/>reliable protocols to generate probes for chromogenic and fluo-<lb/> rescence in situ hybridisation have been described. 44 <lb/> Our informal analysis comparing CISH with FISH for detection <lb/>of the HER2 gene in breast cancers demonstrates that the average <lb/>percentage agreement in the examined studies comparing HER2 <lb/> amplification by CISH and FISH is 96% (SD¼4%) (table 1). <lb/>Although not always performed, k coefficients ranged from 0.76 to <lb/> Figure 1 Conceptual schematic of single-colour chromogenic in situ <lb/>hybridisation demonstrating bright field detection of a digoxigenin-<lb/>labelled probe. The probe is recognised by an antidigoxigenin fluorescein <lb/>isothiocyanate primary antibody followed by detection with an anti-<lb/>fluorescein-isothiocyanate horseradish peroxidase (HRP). After addition <lb/>and oxidation of diaminobenzidine, a dark brown signal is deposited at the <lb/>target site. <lb/>
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1.0 with the exception of one study. 61 Of the studies reviewed, the <lb/> findings from several larger studies warrant additional attention. <lb/>In 2004, Isola et al reported a study of paired CISH/FISH <lb/>results from 192 breast cancers. 50 Similar to previously published <lb/>studies, the authors demonstrated excellent concordance <lb/>between CISH and FISH (93.8%), k coefficient 0.875. After <lb/>careful analysis of 12 cases that displayed discordance between <lb/>the two testing modalities, it was determined that 7 of the 12 <lb/>could have been resolved by having chromosome 17 information <lb/>when performing the CISH evaluation, and the other five <lb/> discrepant cases were due to histological features that were <lb/>difficult to identify in the sampled tumour. The authors <lb/>concluded that CISH is an accurate and feasible alternative to the <lb/>FDA-approved FISH test (Vysis PathVysion; Abbott Molecular, <lb/>Des Plaines, Illinois, USA). <lb/>In 2006, two groups independently published studies <lb/>comparing HER2 status determined by FISH and CISH. 57 58 The <lb/>two studies analysed 200 or more breast cancers. Hanna and <lb/>Kwok examined tumour samples by CISH and FISH in three <lb/>groups based upon HER2 status, as determined by IHC. Out of <lb/>groups with either 0/1+ or 3+ staining by IHC, concordance <lb/>between CISH and FISH was 97% and 98% respectively. Only 3 <lb/>of 119 cases demonstrated discordance by these two methods. <lb/>From the group with 2+ staining by IHC methodology, 9 of 135 <lb/>cases demonstrated discordance between CISH and FISH (93% <lb/>concordance). Discordant cases were of tumours displaying very <lb/>low or borderline amplification with FISH. Scoring of samples <lb/>with low-level FISH amplification is known to be difficult due in <lb/>part to the high level of interobserver variability with these <lb/>samples. 59 Overall, the authors conclude that evaluation of HER2 <lb/> by CISH may be a viable alternative to FISH analysis in the <lb/>testing algorithm. <lb/>Saez et al examined 200 cases of invasive breast cancer to <lb/>compare the status of HER2 as determined by CISH and FISH. 57 <lb/> The examined breast cancer cases were routinely examined by <lb/>IHC, and during a 4-year period 95 cases with 0/1+ staining, 43 <lb/>cases with 2+ staining, and 62 cases with 3+ staining, were <lb/>collected for the study. A tissue macroarray of these cases was <lb/>generated, and 174 of the cases were available for evaluation by <lb/>CISH and FISH. Overall, a concordance of 94.8% was found <lb/>between CISH and FISH. Of discordant cases, only one was <lb/>identified as amplified by FISH and not by CISH. Eight breast <lb/>cancers demonstrated amplification by CISH (two cases with low <lb/>level amplification), but no amplification by the FISH technique. <lb/>In 2007, van de Vijver et al published an international valida-<lb/>tion ring study involving five pathology laboratories who <lb/>undertook CISH assessment of HER2 in breast cancer cases. 59 A <lb/>total of 211 invasive breast carcinomas were analysed by CISH, <lb/>and the results compared with data generated by FISH testing. <lb/>Of the 76 cases with high levels of HER2 amplification (HER2/ <lb/>CEP17 ratio >4), 96% tested positive for amplification by CISH. <lb/>A concordance rate of 94% was achieved when testing 100 FISH-<lb/>negative cases. However, in cases with low-level HER2 amplifi-<lb/>cation by FISH (HER2/CEP17 ratio 2.0e4.0), only a 57% <lb/>concordance rate was achieved (20/35 CISH scores indicated <lb/>amplification). In addition to the difficultly in assessing low <lb/>amplification cases by FISH, part of this discordance was thought <lb/>to be due to assessment of only the HER2 locus by CISH, <lb/>without normalisation for the chromosome 17 copy number in <lb/>tumour samples. Although these cases pose difficulty for evalu-<lb/>ation by CISH alone, it was proposed that counting signals from <lb/>additional cells and using an additional CISH probe for chro-<lb/>mosome 17 on an additional slide would be helpful. Even though <lb/>it was estimated that the number of clinical breast cancers that <lb/>fall into the category of borderline amplification of HER2 by FISH <lb/>is 1e3%, 59 practical solutions to the level of discordance in the <lb/> Figure 2 Examples of HER2 detection using the chromogenic in situ <lb/>hybridisation technique in breast carcinoma. (A) Demonstration of non-<lb/>amplified HER2 producing 1e2 signals per nucleus. Examples of HER2 <lb/> amplification where the peroxidase signal exists as either a cluster of <lb/>gene copies (B) or as multiple individual gene copies (C). Original <lb/>magnification 3600. Reproduced from Tanner et al 41 with permission <lb/>from the American Society for Investigative Pathology. <lb/>
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van de Vijer study are needed. The overall conclusion of the study <lb/>by van de Vijer et al was that CISH and FISH have very high <lb/>concordance, and that CISH is a viable alternative to FISH for <lb/>assessment of HER2 in breast cancer cases. <lb/>The study by van de Vijer et al was not the first to identify that <lb/>assessment and interpretation of HER2 cases with very low level <lb/>of amplification (6e10 signals per cell) benefit from inclusion of <lb/>the chromosome 17 probe. Inclusion of the chromosome 17 probe <lb/>in such cases had proved to be robust and reproducible between <lb/>other laboratories. 64 A correlation of 100% was found between <lb/>CISH and FISH in one study in which samples scoring more than <lb/>two signals per nucleus were controlled using a chromosome 17 <lb/>CISH probe on adjacent tissue. 45 Using this approach, breast <lb/>cancers with aneusomy or polysomy of chromosome 17 can be <lb/>distinguished from genuine low-level HER2 amplification; <lb/>however, FISH was still deemed useful in some instances. 68 It has <lb/>been suggested that all breast carcinomas with a HER2 copy <lb/>number of 2e7 by FISH should also be analysed for chromosome <lb/>17. 69 A similar algorithmic use of a chromosome 17 probe by <lb/>CISH would require less time and resources than evaluating <lb/> HER2 and chromosome 17 in a routine fashion on all cases. <lb/>Recently, Gong et al published a multicentre study examining <lb/>the ability to detect HER2 gene status in breast cancer comparing <lb/>conventional scoring criteria with the new ASCO/CAP recom-<lb/>mendations. 66 The key difference between the ASCO/CAP <lb/>guidelines and USA FDA approved manufacturer scoring criteria <lb/>for HER2 amplification by CISH (Zymed SPOTLight HER2 <lb/>CISH; Invitrogen, Camarillo, California, USA) and FISH (Vysis <lb/>PathVysion) is that no equivocal category is used in the manu-<lb/>facturer's scoring criteria. The current ASCO/CAP guidelines for <lb/> HER2 detection by FISH have criteria for non-amplified (HER2/ <lb/>CEP17 ratio <1.8), equivocal (HER2/CEP17 ratio 1.8e2.2), and <lb/>amplified (HER2/CEP17 ratio >2.2). Similarly, the ASCO/CAP <lb/>guidelines for non-amplified, equivocal and amplified cases <lb/>detected by CISH are <4, 4e6, and >6, respectively. The authors <lb/>concluded that the concordance between CISH and FISH for <lb/>positive and negative cases of HER2 amplification was excellent <lb/>using the guidelines of the manufacturer and those of ASCO/ <lb/>CAP. Slightly higher concordance rates and reproducibility were <lb/>achieved at the two scoring sites using the ASCO/CAP guide-<lb/>lines. <lb/>Evaluation of the available published data leads to the <lb/>conclusion that bright field techniques, such as CISH, have <lb/>potential to be used as an alternative to FISH testing in the <lb/>assessment of HER2 status in breast cancers. In general, CISH is <lb/>thought to offer several advantages over the FISH technique <lb/>including: the ability to archive CISH prepared material indefi-<lb/>nitely, the use of a conventional bright field microscope to <lb/>interpret staining, the simultaneous assessment of morphology <lb/>and gene copy number in the same slide, and the identification of <lb/>tumour heterogeneity using low-level magnification. 12 In addi-<lb/>tion, CISH is CE marked and FDA approved. Many of these same <lb/>advantages are possible through use of another type of bright <lb/> field in situ hybridisation based on metallographic, rather than <lb/>chromogenic, probe detection. <lb/> Metallographic in situ hybridisation <lb/> Unlike CISH, enzyme metallographic in situ hybridisation <lb/>utilises an enzymatic reaction to facilitate the deposition of <lb/>metal directly from solution to identify the target site. In <lb/>addition to the advantages offered by chromogenic bright field in <lb/>situ hybridisation, metallographic in situ hybridisation provides <lb/>higher sensitivity and resolution for both amplified and non-<lb/>amplified genes. An excellent review of metallographic bright <lb/> field in situ hybridisation modalities was recently published 70 ; <lb/>discussion herein will be focused on describing the principles, <lb/>practicalities and relative utility of this technology. <lb/>Due to limitations of early Nanogold-silver enhancement <lb/>procedures that made them cumbersome for routine use, <lb/>a simplified gold-enhanced Nanogold-streptavidin method, <lb/>termed gold-facilitated in situ hybridisation (GOLDFISH) was <lb/>developed to assess HER2 gene status (figure 3). 71 This technique, <lb/>initially developed as a simplified way to qualitatively identify <lb/>confluent amplification signals in tissue sections rather than <lb/>a quantitative assessment of discreet dots, demonstrated much <lb/>initial promise. 72 The first generation gold-facilitated autome-<lb/>tallographic bright field in situ hybridisation displayed good <lb/>interobserver interpretative reproducibility in an examination of <lb/>a series of 66 breast carcinomas 73 ; however, the need to differ-<lb/>entiate cases with chromosome 17 aneusomy or polysomy from <lb/>those with low-level HER2 amplification necessitated use of <lb/>a quantitative interpretation method. 72 <lb/> It was subsequently discovered that horseradish peroxidase <lb/>can be used to selectively deposit metal from solution in the <lb/>absence of a particulate nucleating agent such as Nanogold. The <lb/>basic principles of this new technology, EnzMet, are presented in <lb/> figure 4A. As commercialised enzyme metallography is known as <lb/>silver in situ hybridisation (SISH). This advancement produced <lb/>discreet spots of metallic silver deposition, from the enzymatic <lb/>action of peroxidase on silver acetate in the presence of hydro-<lb/>quinone, at the target site, allowing a superior quantitative <lb/>assessment of gene copy number. 72 Results of the staining <lb/>achieved by this method are demonstrated in figure 4B,C. The <lb/>EnzMet Gene Pro assay, a form of SISH that incorporates <lb/>concomitant protein detection, has demonstrated excellent <lb/>interobserver reproducibility, 74 75 and several studies have now <lb/> Table 1 Comparison of HER2 status using CISH and FISH <lb/>methodologies <lb/> Reference <lb/>Sample <lb/>size <lb/>No. of <lb/>test sites <lb/>Concordance <lb/>(%)y <lb/> k <lb/> Coefficient* <lb/> Tanner et al 41 <lb/> 157 <lb/>1 <lb/>93.6 <lb/>0.81 <lb/>Zhao et al 45 <lb/> 62 <lb/>1 <lb/>100.0 <lb/>NS <lb/>Dandachi et al 46 <lb/> 38 <lb/>1 <lb/>100.0 <lb/>NS <lb/>Gupta et al 47 <lb/> 31 <lb/>2 <lb/>83.9 <lb/>NS <lb/>Park et al 48 <lb/> 188 <lb/>1 <lb/>94.1 <lb/>0.84 <lb/>Arnould et al 49 <lb/> 75 <lb/>8 <lb/>96.0 <lb/>0.97 <lb/>Isola et al 50 <lb/> 192 <lb/>2 <lb/>93.8 <lb/>0.88 <lb/>Hauser-Kronberger et al 51 <lb/> 38 <lb/>1 <lb/>100.0 <lb/>NS <lb/>Bhargava et al 52 <lb/> 102 <lb/>1 <lb/>100.0 <lb/>NS <lb/>Gong et al 53 <lb/> 80 <lb/>1 <lb/>95.0 <lb/>0.85e0.91 <lb/>Lin et al 54 <lb/> 25 <lb/>1 <lb/>92.0 <lb/>NS <lb/>Li-Ning-T et al 55 <lb/> 32 <lb/>1 <lb/>96.9 <lb/>NS <lb/>Loring et al 56 <lb/> 110 <lb/>1 <lb/>99.0 <lb/>NS <lb/>Saez et al 57 <lb/> 174 <lb/>1 <lb/>94.8 <lb/>0.86 <lb/>Hanna and Kwok 58 <lb/> 254 <lb/>1 <lb/>95.1 <lb/>0.91 <lb/>van de Vijver et al 59 <lb/> 211 <lb/>5 <lb/>88.6 <lb/>NS <lb/>Cayre et al 60 <lb/> 55 <lb/>1 <lb/>91.5 <lb/>0.76e0.88 <lb/>Sinczak-Kuta et al 61 <lb/> 55 <lb/>1 <lb/>NS <lb/>0.53 <lb/>Di Palma et al 62 <lb/> 161 <lb/>1 <lb/>100.0 <lb/>NS <lb/>Carbone et al 63 <lb/> 89 <lb/>5 <lb/>98.9 <lb/>NS <lb/>Di Palma et al 64 <lb/> 28 <lb/>7 <lb/>98.5 <lb/>0.91 <lb/>Pothos et al 65 <lb/> 88 <lb/>1 <lb/>100.0 <lb/>NS <lb/>Gong et al 66 <lb/> 226 <lb/>2 <lb/>98.8 <lb/>0.93e1.0 <lb/>Pedersen and Rasmussen 67 <lb/> 72 <lb/>1 <lb/>98.6 <lb/>0.97 <lb/> CISH, chromogenic in situ hybridisation; FISH, fluorescence in situ hybridisation; NS, not <lb/>specified. <lb/> *95% confidence level unless otherwise specified, coefficients rounded to two decimal <lb/>places. <lb/> yWeighted averages were calculated in some instances. <lb/>
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been published that compare HER2 gene status in breast <lb/>carcinomas as determined by SISH and FISH (table 2). <lb/>To date, the published studies examining HER2 gene copy <lb/>number by SISH have evaluated relatively small series of usually <lb/>less than 100 breast cancers each. In 2007, Dietel et al reviewed <lb/>a series of 99 invasive breast carcinomas by automated SISH and <lb/>FISH. 76 The results were analysed using the ASCO/CAP guide-<lb/>lines. Overall concordance was 96%. Discrepant cases were <lb/>usually attributable to the presence of intratumoral heteroge-<lb/>neity of HER2 amplification. The authors concluded that SISH <lb/>was as reliable as FISH in determining HER2 amplification. <lb/>One year later, Carbone et al published a multicentre study <lb/>that examined the staining and interpretative reproducibility of <lb/>the HER2 SISH assay (Ventana Medical Systems, Tucson, <lb/>Arizona, USA) from 89 breast carcinomas using multiple tech-<lb/>niques. 63 The reproducibility and efficacy of HER2 SISH staining <lb/>was excellent (median K w value 0.91). Overall concordance <lb/>between positive and negative SISH and FISH results was also <lb/>superb (93e100%). However, concordance between SISH and <lb/>FISH was lower (50%) for a group of eight cases in which the <lb/> HER2/CEP17 ratio was between 1.5 and 3.0. These results <lb/>suggest that the low-level or intermediate category of amplifi-<lb/>cation poses challenges for the SISH, as well as the CISH, method <lb/>of testing. This specific question has not been systematically <lb/>addressed for FISH to our knowledge. <lb/>In 2009, Sousha et al published an evaluation of HER2 ampli-<lb/> fication in 53 breast cancers by automated SISH and FISH. 77 In <lb/>94% of the cases examined, SISH and FISH results were identical <lb/>using scoring criteria provided by the manufacturer. Two of the <lb/>breast cancers were negative for HER2 amplification by SISH and <lb/>positive by FISH. Another breast cancer was scored negative by <lb/>FISH and positive for amplification by SISH. The authors agreed <lb/>with the conclusion reached by Dietel et al in stating that <lb/>automated SISH detection of HER2 in excised breast cancers <lb/>compares very favourably with FISH analysis. <lb/>A recent study examining 230 breast cancers with a rapid <lb/>SISH scoring technique determined a very high concordance <lb/>(99.6%) with FISH testing. 78 The authors employed a 'SISH <lb/> quick-score' when evaluating HER2 status by SISH. Similar to <lb/>the FDA-approved assessment of HER2 status with a single CISH <lb/>probe, the SISH quick-score relies upon the number of stain dots <lb/>present in tumour cell nuclei. With dots from an epithelial or <lb/> fibroblast cell used as a reference signal, the scorers evaluated <lb/> HER2 status as: non-amplification, aneusomy, polysomy, and <lb/>low-level or high-level amplification. The two evaluators in this <lb/>study were 100% concordant in their interpretation using the <lb/>SISH quick-score technique. In addition to confirming the ability <lb/>of automated SISH to accurately assess HER2 status in breast <lb/>cancers, the data of the study suggest that the use of SISH quick-<lb/>score is of additional utility in that it combines the resolution of <lb/>SISH the with straightforward interpretation style of the CISH <lb/>scoring method. <lb/>In sum, the studies available for review suggest that SISH is <lb/>a reliable substitution for FISH in the determination of HER2 <lb/> status in invasive breast carcinoma. Similar to FISH, SISH allows <lb/>enumeration of HER2 and chromosome 17 signals enabling <lb/>generation of a HER2/CEP17 ratio. The published ASCO/CAP <lb/>guidelines, including the equivocal range of HER2 gene amplifi-<lb/>cation (HER2/CEP17 ratio of 1.8e2.2), are also readily applied to <lb/>this bright field in situ hybridisation. The benefits of SISH <lb/>detection of HER2 include: very high sensitivity with high <lb/>resolution and signal separation, accurate quantitation of gene <lb/>amplification, excellent visualisation of tissue morphology, and <lb/>adaptability for automation. 70 SISH is currently CE marked but <lb/>has not yet been FDA approved. While assessment of HER2 using <lb/>currently available commercial technology opens up the benefits <lb/>of the CISH and SISH platforms to laboratories not able to <lb/>perform FISH, additional advances in bright field in situ <lb/>hybridisation technologies are currently under development. <lb/> Figure 3 (A) Schematic of gold-facilitated in situ hybridisation <lb/>(GOLDFISH) assay demonstrating recognition of the biotin-labelled probe <lb/>with a biotinylated tyramide followed by strepavidin-Nanogold (Nano-<lb/>probes). The Nanogold particulate nucleating agent facilitates auto-<lb/>metallographic deposition of gold from a solution of silver acetate in the <lb/>presence of hydroquinone at the target site. (B) Example of HER2 <lb/> detection using the GOLDFISH technique with demonstration of non-<lb/>amplified HER2 in infiltrating ductal carcinoma producing 1e2 signals per <lb/>nucleus. (C) Example of GOLDFISH technique in a breast carcinoma <lb/>containing amplified HER2 that is demonstrated by multiple large <lb/>confluent nuclear signals. Original magnification 3400. Reprinted from <lb/>Powel et al 70 with permission from Elsevier. <lb/>
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THE FUTURE IS BRIGHT <lb/> Dual-colour FISH is considered the 'gold standard' for in situ <lb/>assessment of gene copy number, in part because of the superior <lb/>spatial resolution offered by this technique and the FDA approval <lb/>status of Vysis PathVysion. 12 However, dual colour FISH has the <lb/>same disadvantages as single-colour FISH and the additional <lb/>limitation that probes producing more intense signal may lead to <lb/>the interpretation of biased ratios favouring the brighter probe. <lb/>Despite some limitations, the ability to directly assess both and <lb/>multiple targets in the same nucleus simultaneously is highly <lb/>desirable. Although studies evaluating multicolour detection <lb/>procedures for bright field microscopy using chromosome specific <lb/>probes had been reported in the past, 80e82 development of dual-<lb/>colour CISH using probes for HER2 and chromosome 17 was <lb/>reported more recently using single-colour detection of a digox-<lb/>igenin-labelled HER2 probe and a biotin labelled chromosome 17 <lb/>probe. 83 The results of dual-coloured CISH and FISH in that study <lb/>showed high concordance (91%, k coefficient 0.82), and the <lb/>contrast provided by the two colours allowed for immediate <lb/>distinction between HER2 amplification and chromosome 17 <lb/>aneuploidy. 84 Additional reports of dual-colour CISH for the <lb/>assessment of HER2 gene status found excellent concordance <lb/>when respectively compared with FISH results (98.6% and <lb/>94.6%). 67 85 Additional advancements in bright field in situ <lb/>hybridisation are aiming to provide assessment of both HER2 and <lb/>chromosome 17 through techniques to identify both targets either <lb/> Figure 4 (A) Schematic of enzyme metallography demonstrating <lb/>detection of the probe with a primary anti-hapten antibody followed by <lb/>a horseradish peroxidase (HRP)-labelled secondary antibody. Enzyme-<lb/>catalysed deposition of metallic silver from the silver acetate solution, in <lb/>the presence of hydroquinone, then occurs at the target site. (B) Example <lb/>of HER2 detection using the enzyme metallography EnzMet technique with <lb/>demonstration of non-amplified breast cancer; 1e2 signals are present in <lb/>each nucleus. (C) Example of breast carcinoma containing amplified HER2; <lb/> multiple distinct signals are present in each nucleus. Original magnification <lb/> 3400. Reprinted from Powel et al 70 with permission from Elsevier. <lb/> Table 2 Comparison of HER2 status using SISH and FISH <lb/>methodologies <lb/> Reference <lb/>Sample <lb/>size <lb/>No. of test <lb/>sites <lb/>Concordance <lb/>(%)y <lb/> k coefficient* <lb/> Sinczak-Kuta 61 <lb/> 63 <lb/>1 <lb/>NS <lb/>0.38 <lb/>Dietel et al 76 <lb/> 99 <lb/>5 <lb/>96.0 <lb/>0.75 <lb/>Carbone et al 63 <lb/> 89 <lb/>5 <lb/>98.9 <lb/>NS <lb/>Shousha et al 77 <lb/> 53 <lb/>1 <lb/>94.0 <lb/>NS <lb/>Collins et al 78 <lb/> 230 <lb/>2 <lb/>99.6 <lb/>NS <lb/>Bartlett et al 79 <lb/> 45 <lb/>7 <lb/>96.0 <lb/>NS <lb/> FISH, fluorescence in situ hybridisation; SISH, silver in situ hybridisation; NS, not specified. <lb/> *95% confidence level unless otherwise specified, coefficients rounded to two decimal places. <lb/> yWeighted averages were calculated in some instances. <lb/> Figure 5 Conceptual schematic demonstrating dual detection of HER2 <lb/> and chromosome 17 by bright field double in situ hybridisation. By this <lb/>technique, dual detection can be accomplished using individual single <lb/>haptens. The two probes are incompatible and two rounds of target DNA <lb/>denaturation, hybridisation and stringency washes are carried out <lb/>sequentially. DNP, dinitrophenol. <lb/>
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simultaneously or consecutively (figure 5). Examples of the <lb/>staining produced by such techniques are demonstrated in figure 6. <lb/>Recently, automated bright field double in situ hybridisation <lb/>(BDISH) applications have been described. 86 In the study by <lb/>Nitta et al, high consensus concordance was demonstrated <lb/>between FISH and BDISH methods. Future versions of this <lb/>approach will use simultaneous hybridisations with dual haptens <lb/>allowing dual colour detection (DISH) of HER2 and chromosome <lb/>17. Depending on the scoring criteria used (historical versus <lb/>ASCO/CAP) and whether FISH equivocal cases were included, <lb/>the concordance percentages ranged between 95.7% and 100% (k <lb/>coefficients 0.89e1.0). Since publication of that study, the utility <lb/>of this technology has become apparent, 87 and it has been <lb/>suggested that the BDISH automated technique might be used in <lb/>replacement of manual dual-colour FISH methods in the future. 86 <lb/> Alternatively, techniques combining IHC and BDISH methods <lb/>may become the new preferred method of HER2 assessment. 87 <lb/> In conclusion, the constant elucidation of the molecular <lb/>pathogenesis of disease requires that detailed genetic informa-<lb/>tion guide clinical decision making and therapeutic strategies. <lb/>This demand has placed pressure upon clinical laboratories to <lb/>provide testing platforms capable of accurately assessing <lb/>genomic signatures. In situ hybridisation techniques are, and <lb/>should continue to be, an important part of the pathologist's role <lb/>in the movement towards personalised medicine. 88 The bright <lb/> field in situ hybridisation techniques presented offer a glimpse at <lb/>where the state of diagnostics and pharmacogenomic testing is <lb/>headed in terms of accurately assessing the morphological status <lb/>and molecular status of a tumour cell simultaneously. <lb/> Figure 6 Examples of HER2 and <lb/>chromosome 17 detection in non-<lb/>amplified (A) and amplified (B) breast <lb/>carcinomas using the dual-colour dual-<lb/>hapten approach. Single HER2 gene (C) <lb/>and chromosome 17 polysomy (D) are <lb/>demonstrated using bright field double <lb/>in situ hybridisation. Magnification <lb/> 3100. Reproduced from Nitta et al 86 <lb/> with author permission. <lb/> Take-home messages <lb/> < Bright field in situ hybridisation is a molecular technique that <lb/>enables visualisation of cellular target DNA using chromogenic <lb/>(eg, chromogenic in situ hybridisation) or enzyme metallo-<lb/>graphic (eg, silver in situ hybridisation) methods of detection <lb/>with conventional light microscopy. <lb/> < Benefits to bright field in situ hybridisation include: the ability <lb/>to archive prepared material indefinitely, the use of a conven-<lb/>tional bright field microscope to interpret staining, the <lb/>simultaneous assessment of morphology and gene copy <lb/>number on the same slide, and the identification of tumour <lb/>heterogeneity using low-level magnification. <lb/> < An informal analysis of the literature demonstrates excellent <lb/>concordance among published comparisons of bright field in <lb/>situ hybridisation and fluorescence in situ hybridisation <lb/>assessment of ERBB2 (HER2) gene status in breast carcinoma. <lb/> < Current American Society for Clinical Oncology and College of <lb/>American Pathologists interpretation guidelines for FISH <lb/>detection of ERBB2 (HER2) gene status in breast carcinoma <lb/>are readily applied to bright field in situ hybridisation techniques. <lb/> < Effective advocacy, use and interpretation of bright field in situ <lb/>hybridisation can be an important part of the role of the <lb/>pathologist in the movement towards personalised medicine. <lb/> Interactive multiple choice questions <lb/> This JCP review has an accompanying set of multiple choice <lb/>questions (MCQs). To access the questions, click on BMJ <lb/>Learning: Take this module on BMJ Learning from the content <lb/>box at the top right and bottom left of the online article. For more <lb/>information please go to: http://jcp.bmj.com/education Please <lb/>note: the MCQs are hosted on BMJ Learningdthe best available <lb/>learning website for medical professionals from the BMJ Group. <lb/>If prompted, subscribers must sign into JCP with their journal's <lb/>username and password. All users must also complete a one-<lb/>time registration on BMJ Learning and subsequently log in (with <lb/>a BMJ Learning username and password) on every visit. <lb/>
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<div type="acknowledgement"> Acknowledgements The authors thank Dr Jorma Isola, Dr Hiroaki Nitta, the <lb/>American Society for Investigative Pathology, and Elsevier, for providing access to <lb/>examples of bright field in situ hybridisation techniques. <lb/></div>
<div type="annex">Funding This work was supported by federal grants to Nanoprobes and The Cleveland <lb/>Clinic (NIH 1R43CA84875-01, NIH/NCI 1R41CA83618-01, NIH/NCI 1R41CA83618-02, <lb/>NIH 1R43GM64257-01, NIH/NIGMS 1R43GM0628250-01 and NIH 1R43 <lb/>CA111182-01) and an Industry sponsored grant from Ventana Medical Systems <lb/>to RRT. <lb/></div>
<div type="annex">Competing interests The senior author of the manuscript receives research support <lb/>and honoraria for speaking on behalf of Ventana Medical Systems. <lb/></div>
<div type="annex">Provenance and peer review Commissioned; externally peer reviewed. <lb/></div>
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<note place="headnote"> Review <lb/></note>
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