Advantages and Disadvantages of Technologies for HER2 Testing in Breast Cancer Specimens

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1 and of Technologies for HER2 Testing in Breast Cancer Specimens Daniela Furrer, MSc, 1-3 François Sanschagrin, PhD, 4,5 Simon Jacob, MD, 4,5 and Caroline Diorio, PhD 1-4 From the 1 Centre de recherche sur le cancer de l Université Laval, Quebec City, Canada; 2 Axe Oncologie, Centre de recherche du CHU de Québec, Hôpital du St-Sacrement, Quebec City, Canada; 3 Département de médecine sociale et préventive, Faculté de Médecine, Quebec City, Canada; 4 Centre des Maladies du Sein Deschênes-Fabia, Hôpital du St-Sacrement, Quebec City, Canada; and 5 Département de biologie moléculaire, de biochimie médicale et de pathologie, Faculté de Médecine, Quebec City, Canada. Key Words: Breast cancer; HER2 status; Trastuzumab; Lapatinib; HER2 inhibitors Am J Clin Pathol November 2015;144: ABSTRACT Objectives: Human epidermal growth factor receptor 2 (HER2) plays a central role as a prognostic and predictive marker in breast cancer specimens. Reliable HER2 evaluation is central to determine the eligibility of patients with breast cancer to targeted anti-her2 therapies such as trastuzumab and lapatinib. Presently, several methods exist for the determination of HER2 status at different levels (protein, RNA, and DNA level). Methods: In this review, we discuss the main advantages and disadvantages of the techniques developed so far for the evaluation of HER2 status in breast cancer specimens. Results: Each technique has its own advantages and disadvantages. It is therefore not surprising that no consensus has been reached so far on which technique is the best for the determination of HER2 status. Conclusions: Currently, emphasis must be put on standardization of procedures, internal and external quality control assessment, and competency evaluation of already existing methods to ensure accurate, reliable, and clinically meaningful test results. Development of new robust and accurate diagnostic assays should also be encouraged. In addition, large clinical trials are warranted to identify the technique that most reliably predicts a positive response to anti-her2 drugs. Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor belonging to the family of epidermal growth factor receptors (EGFRs). 1 The protein is encoded by the HER2 (ERBB2) gene, which is located on the long arm of chromosome 17 (17q ). 2 HER2 is an essential mediator of cell proliferation and differentiation in the developing embryo and in adult tissues. 3 Its inappropriate activation, however, is associated with the development of several malignancies, including breast, ovarian, gastric, colorectal, pancreatic, and endometrial cancers. 4 In human breast cancer, HER2 gene amplification and receptor overexpression, which occur in 15% to 20% of patients, are important prognostic markers for poor prognosis, including a more aggressive disease and a shorter survival. 5 Moreover, HER2-positive status is considered a predictive marker of response to HER2-targeted drugs, including trastuzumab and lapatinib. 6 Trastuzumab (Herceptin; Roche, Basel, Switzerland) is a recombinant humanized monoclonal antibody that specifically targets the extracellular domain of the HER2 protein. 7 Trastuzumab improves the outcomes of patients with HER2-positive breast cancer in both the metastatic 8,9 and adjuvant settings. 10,11 The US Food and Drug Administration (FDA) approved trastuzumab for the treatment of HER2-positive metastatic breast cancer in 1998 and as adjuvant treatment for HER2-positive early stage breast cancer in Lapatinib (Tykerb/Tyverb; GlaxoSmithKline, Philadelphia, PA) is a small-molecule inhibitor of the intracellular tyrosine kinase domain of both HER2 and EGFR receptors. 12 In 2007, lapatinib had been approved by the FDA as combination therapy with capecitabine for the treatment of patients with HER2-positive advanced breast cancer who 686 Am J Clin Pathol 2015;144:

2 had progressed on trastuzumab-based regimens. 13 Given its prognostic, predictive, and therefore therapeutic implications, an accurate evaluation of HER2 status is crucial for identification of patients who would most likely benefit from targeted anti-her2 therapies. Several techniques have been developed for the evaluation of HER2 status in breast cancer specimens in clinical practice, at the protein level, DNA level, and RNA level. Currently, there are several FDA-approved methods to evaluate HER2 status, including immunohistochemical (IHC) determination of HER2 protein expression or assessment of HER2 gene amplification using in situ hybridization (ISH), most commonly fluorescent ISH (FISH). 14,15 However, since each technique has its own advantages and disadvantages, there is still no consensus on which method is superior for assessing the HER2 status in breast cancer specimens. This review provides an overview of the techniques that have been developed and tested over the past few decades for the determination of HER2 status in breast cancer specimens. The principle of each method is briefly presented. The central objective of the present review article, however, is to highlight the main advantages and disadvantages of each described technique. Main characteristics of the presented techniques are summarized in Table 1, whereas each technique and its advantages and disadvantages are thoroughly depicted in the next section. Principles of the Methods of Analysis,, and of Each Described Technique Southern Blot The Southern blot technique has been used to determine HER2 gene amplification in breast cancer samples Following DNA extraction from breast cancer frozen tissues, DNA is digested through a restriction enzyme. Digested DNA fragments are then separated by gel electrophoresis on agarose gel. Following DNA denaturation, DNA specimens are transferred from the gel to a membrane and hybridized with a radioactive-labeled HER2 single-stranded DNA (ssdna) probe. The labeled HER2 ssdna probe will hybridize with the HER2 ssdna sequence on the basis of strand annealing between complementary ssdna molecules. The labeled HER2 sequence is then visualized by autoradiography ,22 Autoradiograms are then scanned with a densitometer. 23 The HER2 gene copy number is then compared with that of a control gene, 16 control DNA extracted from blood, 18 or DNA extracted from normal breast tissues. 19 Tumors that showed a more than twofold increase in copy Table 1 Main Characteristics of the Described Techniques Method of Analysis Target Specimen Type Southern blot DNA Frozen tissue No Northern blot mrna Frozen tissue No ELISA Protein Blood sample Yes Western blot Protein Frozen tissue and FFPE tissue No PCR-based assays number compared with control unamplified DNA are considered amplified. 16 Since DNA is very stable, it is considerably less degraded in tissues compared with protein and mrna. 24 Although very reliable, this technique is not applicable in routine diagnostic settings, since it is time-consuming and requires a large amount of DNA. 25,26 In addition, this technique does not allow the morphologic preservation of tissue and therefore the evaluation of histologic features of tumor. Since nonamplified nonneoplastic cells present in tumor cannot be isolated from cancer cells, results obtained potentially underestimate the real HER2 gene amplification of the sample through a dilutional effect. 21 Northern Blot qpcr: DNA; RT-PCR: mrna Frozen tissue and FFPE tissue FDA Approval MLPA DNA FFPE tissue No IHC Protein FFPE tissue Yes FISH DNA FFPE tissue Yes CISH DNA FFPE tissue Yes SISH DNA FFPE tissue No GOLDFISH DNA FFPE tissue No BDISH DNA FFPE tissue Yes HER2-gene protein Protein and FFPE tissue No assay DNA mrna in situ mrna FFPE tissue No hybridization IQFISH DNA FFPE tissue Yes Automated HER2 FISH assay DNA FFPE tissue No BDISH, bright-field double in situ hybridization; CISH, chromogenic in situ hybridization; ELISA, enzyme-linked immunosorbent assay; FDA, US Food and Drug Administration; FFPE, formalin-fixed, paraffin-embedded; FISH, fluorescence in situ hybridization; GOLDFISH, gold-facilitated autometallographic in situ hybridization; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; IQFISH, instant-quality fluorescence in situ hybridization; MLPA, multiplex ligation-dependent probe amplification; mrna, messenger RNA; PCR, polymerase chain reaction; qpcr, quantitative PCR; RT-PCR, reverse transcription PCR; SISH, silver-enhanced in situ hybridization. The Northern blot assay allows the detection of HER2 RNA in frozen breast cancer specimens. 21 This method is very similar to the Southern blot, with the exception that RNA molecules are detected instead of DNA sequences. No Am J Clin Pathol 2015;144:

3 Furrer et al / Pros and Cons of HER2 Diagnostic Tests After extraction from homogenized tissue sample, total cellular RNA or messenger RNA (mrna) is separated by size via electrophoresis in an agarose gel and transferred to a nitrocellulose membrane. The HER2 RNA is then visualized via hybridization with an isotopic-labeled complementary probe. 27 The labeled HER2 sequence is then visualized by autoradiography. The relative optical density (OD) of bands is measured by densitometry scanning. Tumors are then divided into four RNA expression categories: 1+ (0.1 to 0.5 OD units), 2+ (>0.5 to 1.0 OD units), 3+ (>1.0 to 1.5 OD units), and 4+ (>1.5 OD units). 21 Northern blot reagents are not too expensive, which allows the running of many gels at low cost. Moreover, quantity and quality of RNA can be verified after gel electrophoresis. 28 One of main disadvantages of the Northern blot technique is that RNA molecules are often degraded in tissues. Indeed, even a slight degradation of RNA can compromise the quality of data and therefore the ability to quantify gene expression. Similar to Southern blot, the Northern blot technique, in addition to being a labor-intensive technique, does not allow an exclusive evaluation of HER2 status in cancer cells, since the morphology of tissues is destroyed during the homogenization of tissue samples. 21 Enzyme-Linked Immunosorbent Assay HER2 protein is composed of a cytoplasmic domain with tyrosine kinase activity, a transmembrane domain, and an extracellular domain (ECD). 29 The HER2 ECD can be cleaved from the full-length HER2 receptor present on the breast cancer cell membrane by matrix metalloproteases 30 and released into the serum. 31 Enzyme-linked immunosorbent assay (ELISA) allows the detection and quantification of proteins in fluids or cell lysates. 32 In breast cancer, manual and automated ELISA assays have been used for the determination of HER2 ECD serum concentration HER2 ECD is detected using two monoclonal antibodies recognizing two distinct epitopes of the antigen. In the manual assay, HER2 ECD is immobilized using a 96-well plate coated with the first monoclonal antibody. The immobilized protein is then incubated with the second monoclonal antibody, which is labeled with horseradish peroxidase (HRP). After application of the HRP substrate, detection is accomplished by assessing the colored end product with spectrophotometry, which correlates to the HER2 ECD concentration in sample. 37 In the automated assay, HER2 ECD is visualized through direct chemiluminescent technology, using antibodies that are labeled with chemiluminescent compounds (ie, acridinium ester, fluorescein). 38 The measured chemiluminescence is directly proportional to the HER2 ECD concentration in sample. 35 Among commercially available ELISA assays, one automated (Immuno-1; Siemens Healthcare Diagnostics, Tarrytown, NY) and one manual ELISA assay (Siemens Healthcare Diagnostics) were approved by the FDA in Another automated platform (ADVIA Centaur; Siemens Healthcare Diagnostics) also received FDA approval in Some studies suggest that HER2 ECD could be used as a biomarker for the monitoring of the disease course and the patient s response to therapy. Circulating levels of HER2 ECD greater than 15 ng/ml (this reference cutoff was derived from the serum samples of 242 healthy women 36 ) in patients with HER2-positive breast cancer may be associated with the progression of primary tumors to metastatic breast cancer. 39 In patients with metastatic and early breast cancer, ECD levels might reflect the HER2 full-length protein expression, since elevated HER2 ECD levels in serum ( 15 ng/ml) have been correlated with higher scores at the HER2 IHC. 34,35,40,41 In patients with metastatic breast cancer, high HER2 ECD serum concentrations ( 15 ng/ml) have also been associated with resistance to endocrine therapy and chemotherapy 42 and, in patients with both metastatic and early breast cancer, with worse survival. 43,44 In metastatic patients treated with trastuzumab, decreased HER2 ECD serum levels (>20%) were predictive of response to treatment. 45 Another study conducted on patients with metastatic breast cancer, however, did not observe a clear association between the changes in ECD levels and response to trastuzumab therapy. 46 In general, results regarding the associations between ECD circulating levels and prognostic and predictive factors are very variable, depending on which assay was used or which cutoff value was considered (for a recent review, see Lam et al 47 ). Based on these conflicting data, the clinical use of the ELISA assay for the determination of HER2 ECD in the patient s serum has not yet been widely implemented. 47,48 ELISA is a quick and simple assay in addition to being a less invasive (only blood samples are needed) and quantitative test. 32,47 Moreover, since HER2 ECD can be measured directly in serum, ELISA can be used to monitor the dynamic changes of HER2 status over the course of the disease progression or following treatment. 47 Results obtained by ELISA might not be reliable if the serum samples are from patients receiving trastuzumab treatment, since trastuzumab still present in the patient s serum might compete with the two antibodies used in the assay Am J Clin Pathol 2015;144:

4 Western Blot Western blot has been used to evaluate HER2 protein expression in frozen and formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue samples. 21,50,51 Following protein extraction from tissues, sample proteins are separated by size using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Separated proteins are then transferred to a nitrocellulose or a polyvinylidene fluoride membrane. Membrane is first incubated with a primary antibody directed against HER2, followed by the incubation with a secondary antibody (radioactive or HRP labeled) raised against the primary antibody host species. Radioactive-labeled bands are visualized by autoradiography. 52 HER2 expression levels in individual tumors are then determined by densitometric scanning and expressed as HER2 units based on a laboratory standard. 51 More commonly, nowadays signal from HRP-labeled target can be detected through enhanced chemiluminescence. 50 High sensitivity (as little as 0.1 ng protein can be detected in a sample) represents the major advantages of the Western blot technique. 51 Since proteins are less stable than DNA, they are less well preserved in tissues than DNA. 24 The Western blot technique presents the same disadvantage as the other blotting techniques namely, that it is a time-consuming method, and the morphology of tissue samples is not preserved. 21,51,53 Polymerase Chain Reaction Based Assays Polymerase chain reaction (PCR) is a method for the detection of DNA samples through the exponential amplification of target DNA sequences. At first, double-stranded DNA is denatured into a single-stranded DNA template. Oligonucleotide primers (ie, short single-stranded sequences that match the DNA sequence at each end of the region to be amplified) are then annealed to the single-stranded DNA template. In the following step, DNA polymerase synthetizes a new DNA strand by adding deoxynucleotide triphosphates complementary to the bases of the single-stranded DNA template. As DNA doubles during each PCR cycle, this results in exponential accumulation of the targeted DNA fragment. The products of a PCR reaction are usually analyzed by agarose gel electrophoresis, which allows the detection of the presence (but not the quantification) of the target sequence and the length of the fragment. PCR-based assays can evaluate changes in both HER2 gene copy number and expression. Quantitative PCR (qpcr) and reverse transcription PCR (RT-PCR) have been used to evaluate HER2 gene amplification and HER2 expression, respectively, in breast cancer specimens in both FFPE and frozen tissue. 24,25,54-63 qpcr, also called real-time PCR, is a form of PCR that is used for the DNA quantification in samples. DNA amplification is monitored while the reaction proceeds through the implementation of either DNA-binding dyes or fluorescently labeled sequence-specific primers. Fluorescence signal produced during the amplification process is detected using a thermal cycler equipped with a detector to monitor the emitted fluorescence. As fluorescence signal increases with a growing amount of PCR products, qpcr allows quantifying the amount of DNA formed after each cycle. HER2 gene and the reference gene are simultaneously quantified. A ratio between HER2 and the reference gene of 2.0 or more is regarded as HER2 amplification. 25,55,58-60,62 RT-PCR, often denoted as real-time RT-PCR or quantitative reverse transcriptase PCR, allows the quantification of mrna in biological samples. Following RNA extraction from tissue samples, extracted RNA is reverse transcribed into complementary DNA (cdna). cdna is then measured by qpcr. The relative fold change in gene expression is usually calculated using the comparative ΔΔC T method. 64 At first, the relative quantitation of HER2 gene expression is calculated comparing the target gene expression with that of one or several housekeeping genes. The relative HER2 gene expression measured in samples is then normalized to a calibrator obtained by mixing RNA from several normal breast tissue samples. Several cutoff values have been used in the literature to define HER2 overexpression determined by RT- PCR. 24,25,54,56-58,61 Importantly, the Oncotype Dx (Genomic Health, Redwood City, CA) assay is based on RT-PCR technology to analyze the expression of 21 genes involved in breast cancer biology, including HER2, estrogen receptor (ER), and progesterone receptor. The test, performed on mrna extracted from FFPE tumor tissues, is used to predict the likelihood of breast cancer recurrence in patients with early-stage, node-negative, ER-positive breast cancer, in addition to their chemotherapy benefit. 65 Real-time PCR allows a rapid and quantitative analysis of gene amplification. 60,66 Since real-time PCR is used to quantify DNA in samples, variations in tissue fixation and processing have little impact on the results. Moreover, since only small quantities of DNA fragments are required, real-time PCR represents a suitable technique for the evaluation of HER2 gene amplification from DNA isolated from FFPE tissues. In addition, real-time PCR is an easy, quick, and inexpensive technique that yields reliable results even in cases with low-level amplification. 55 Moreover, since real-time PCR is a quantitative method, it is less sensitive to interobserver variability. 62 Am J Clin Pathol 2015;144:

5 Furrer et al / Pros and Cons of HER2 Diagnostic Tests Real time RT-PCR presents several advantages, including a large dynamic range and an accurate quantification. 67 Although the PCR technique is an easy and reproducible technique, PCR technology has not yet been approved as a diagnostic tool for the evaluation of HER2 amplification. The main reason is that PCR results are often associated with false-negative results due to the dilution of amplified tumor cells with surrounding nonamplified stroma cells or noninvasive breast lesions. 59,63,66 However, this effect can at least in part be resolved through laser-assisted microdissection (LAM), which allows the isolation of tumor cells from archival FFPE tissues. 59 Some authors suggest that the tumor histologic subtype might considerably influence the efficacy of this assay. The impact of surrounding stromal and nonmalignant cells on test result might be in fact significantly bigger in tumors in which tumor cells are scattered throughout the stroma, such as diffuse lobular tumors. 55 LAM might be therefore particularly important in this subgroup of breast carcinomas. Moreover, since mrna integrity can be damaged by several factors, including tissue fixation and processing and storage time, 24 the evaluation of HER2 status at the mrna level by RT-PCR using FFPE can be problematic. 60 Therefore, the widespread RNA fragmentation observed in archival FFPE tissues (but not in frozen tissues) 24 may limit the use of RT-PCR for the evaluation of HER2 status for clinical purposes. 57,63 Moreover, similar to qpcr, RT-PCR fails to detect equivocal cases at IHC/FISH and produces false-negative results. 57 Multiplex Ligation-Dependent Probe Amplification Multiplex ligation-dependent probe amplification (MLPA) is a recently developed PCR method used to detect copy number variation, such as gene amplifications and gene deletions. 68 After DNA denaturation and fragmentation, genomic DNA is hybridized with sequence-specific MLPA probes. The MLPA kit commercially available for the analysis of the HER2 gene in FFPE tissue (P004 HER-2 kit; MRC Holland, Amsterdam, the Netherlands) contains three probes for the HER2 gene, 11 probes for chromosome 17, and 25 control probes located on chromosomes other than chromosome Each MLPA probe consists of two halves. Half is composed of a target-specific sequence flanked by a universal primer sequence. The other half consists of a target-specific sequence, a sequence showing a probespecific length, and a universal primer sequence. Since the two probe halves recognize adjacent DNA target sequences, they are joined through the action of a ligase. This produces a complete probe flanked by universal primer binding sites that can be amplified by PCR, whereas unbound probe halves cannot be amplified. 68 Since each complete probe is characterized by a unique length, the resulting PCR products can be separated and identified by capillary electrophoresis. Since the amounts of probe are proportional to the target copy number, copy number variations such as deletions and amplifications of target sequence can be recognized by the relative peak heights. 68 HER2 copy number is determined by comparing the mean ratio of the HER2 probe peaks with the reference probes located in stable regions of the genome. Mean values smaller than 1.5 indicate nonamplified cases, mean values between 1.5 and 2.0 indicate low-level amplified cases, and mean values bigger than 2.0 designate HER2- amplified cases. 26 The MLPA assay is a fast, accurate, and inexpensive technique. Moreover, only small quantities of DNA extracted from paraffin-embedded materials are required. Fragmentation of DNA does not have an impact on the reliability of results. In addition, this method yields quantitative results. 26 Some probe signals may be affected by factors such as sample purity and small changes in experimental conditions. Therefore, copy number changes detected by MLPA should always be confirmed by other methods. Furthermore, tissue morphology is not conserved using this assay, and tumor heterogeneity can be missed. 69 Moreover, the MLPA assay requires special equipment, including a thermocycler and a capillary sequencer. 48 IHC IHC assays involve the detection of specific antigens in FFPE tissues using specific antibodies. Immunohistochemical analysis is a commonly used method for the evaluation of HER2 protein overexpression in FFPE breast cancer specimens. 14,15 After antigen retrieval, tissue sections are incubated with a primary antibody directed against the HER2 protein. A visualization reagent (secondary antibody directed against the primary antibody and dextran polymer conjugated with HRP) is subsequently applied. Antigen is visualized by adding the enzyme-specific substrate 3,3 -diaminobenzidine tetrahydrochloride (DAB). Through the action of the enzyme, DAB is converted to a brownish reaction product, resulting in membranous staining. 70,71 So far, several commercially available diagnostic tests for the determination of HER2 protein expression have been approved by the FDA for the identification of patients who might benefit from targeted anti-her2 therapies: the HercepTest kit (DAKO, Glostrup, Denmark), the Pathway kit (clone 4B5; Ventana Medical Systems, Tucson, AZ), the InSite HER2/neu kit (clone CB11; BioGenex Laboratories, 690 Am J Clin Pathol 2015;144:

6 San Ramon, CA), and the Bond Oracle HER2 IHC System (Leica Biosystems, Newcastle, UK). Membranous staining is scored on a semiquantitative scale. According to the American Society of Clinical Oncology (ASCO), the College of American Pathologists (CAP), and Canadian recommendations for HER2 testing in breast cancer published in 2007, HER2 expression is scored as 0 (no staining), 1+ (weak or incomplete membrane staining in any percentage of tumor cells), 2+ (strong, complete membrane staining in 30% of tumor cells or weak/moderate heterogeneous complete membrane staining in 10% of tumor cells), or 3+ (strong, complete, homogeneous membrane staining in >30% of tumor cells). 15 In 2013, the ASCO/CAP updated the guidelines to clarify the recommendations published in According to the newest ASCO/CAP guidelines, HER2 expression is scored as 0 (no staining or weak/ incomplete membrane staining in 10% of tumor cells), 1+ (weak, incomplete membrane staining in >10% of tumor cells), 2+ (strong, complete membrane staining in 10% of tumor cells or weak/moderate and/or incomplete membrane staining in >10% of tumor cells), or 3+ (strong, complete, homogeneous membrane staining in >10% of tumor cells). 14 For both the 2007 and 2013 ASCO/CAP guidelines, HER2 immunohistochemical status is considered negative if the immunohistochemical score is 0 or 1+, equivocal if the score is 2+, and positive if the score is 3+. Patients with a positive HER2 status at the IHC are eligible for targeted anti-her2 therapy. The IHC 2+ group is regarded as borderline, and confirmatory testing using a gene amplification test (an ISH assay) should be performed. Patients with a tumor equivocal at the IHC but HER2 amplified at the gene amplification test are also eligible for therapy with HER2 inhibitors, whereas patients with a tumor that scores 0 or 1+ at the IHC and that is not HER2 amplified are not eligible. 14,15 IHC is easy to perform and relatively inexpensive. 72 Since staining results can be viewed using a conventional bright-field microscope, protein overexpression can be evaluated in the context of tissue morphology. 73 Furthermore, since staining is permanent, slides can be stored. Another advantage is that IHC detects the overexpression of the HER2 protein, which represents the direct target of trastuzumab and other anti-her2 therapies. The ability to accurately determine HER2 protein expression status by IHC can be significantly affected by numerous factors, 22,74,75 including warm/cold ischemic time, 76 delay and duration of fixation, and fixative used. 77,78 The method for antigen retrieval and the training of the staff also play an important role. 14,15,79 Furthermore, since commercially available antibodies display different specificity and sensitivity, HER2 overexpression rates vary considerably depending on the antibody used An important factor regards the interpretation of results. Since the interpretation is based on semiquantitative scoring, immunohistochemical analysis is susceptible to considerable interobserver variability and therefore to substantial discrepancies in HER2 IHC results, 86 especially for cases scoring Variability in staining results, however, has remarkably decreased since the introduction of the highly standardized HercepTest. 48 Moreover, staining variability can be further reduced through the use of fully automated staining systems such as Ventana BenchMark (for the PATHWAY HER-2/ neu assay, clone 4B5). 88 To reduce variability in scoring results, ASCO/CAP recommendations have been published in 2007 and updated in 2013 for the interpretation of HER2 overexpression. 14,15 Moreover, subjectivity in the interpretation of membrane staining can be reduced through the implementation of quantitative image analysis, including the automated cellular imaging system (ACIS; ChromaVision Medical Systems, San Juan Capistrano, CA) 89,90 and HER2-CONNECT (Visiopharm A/S, Hoersholm, Denmark). 91 ASCO/CAP guidelines recommend the use of quantitative image analysis, especially for cases showing a weak HER2 membrane staining (IHC score 1+ and 2+). 15 FISH FISH is a cytogenetic technique that uses fluorescentlabeled probes to detect specific DNA sequences in tissue samples. 92 In breast cancer specimens, the FISH assay is used to quantify the HER2 gene copy number within tumor cell nuclei. 92 FISH can be performed either as a single-color assay (HER2 probe only) or dual-color assay (using differentially labeled HER2 and chromosome 17 centromere probes simultaneously). 93 Following enzymatic digestion of tissues, fluorescentlabeled probes are applied to tissue sections. After DNA denaturation, probes hybridize with their targets in tissue. The HER2 probe, labeled with an orange fluorophore, targets the HER2 gene locus on chromosome 17. The green fluorophore labeled chromosome 17 centromere probe targets the a satellite DNA sequence located at the centromeric region of chromosome 17. Nuclei are counterstained with 4,6 -diamino-2-phenylindole (DAPI). Fluorescent hybridization signals can be visualized using a fluorescence microscope equipped with 100 oil immersion objectives and appropriate filters (Spectrum Orange for locus-specific probe HER2, Spectrum Green for centromeric probe 17, and the UV filter for the DAPI nuclear counterstain). 94,95 Three FISH assay kits have been approved by the FDA so far for the evaluation of HER2 gene amplification in Am J Clin Pathol 2015;144:

7 Furrer et al / Pros and Cons of HER2 Diagnostic Tests breast cancer specimens: the single-probe INFORM HER2 FISH DNA kit (Ventana Medical Systems; currently discontinued), the dual-probe PathVysion HER-2 DNA probe kit (Abbott Molecular, Des Plaines, IL), and the dual-probe HER2 FISH PharmDx kit (DAKO). According to the ASCO/CAP and Canadian recommendations for HER2 testing in breast cancer published in 2007, a mean HER2 gene copy number of less than 4 signals/ nucleus or a HER2 gene/chromosome 17 copy number ratio (HER2/CEP17) of less than 1.8 is considered FISH negative (nonamplified), a mean HER2 gene copy number of 4 or more and 6 or less signals/nucleus or a HER2/CEP17 ratio between 1.8 and 2.2 is considered FISH equivocal, and a mean HER2 gene copy number of more than 6 signals/nucleus or a HER2/CEP17 ratio of 2.2 or more is considered FISH positive (amplified). 15 According to the updated ASCO/CAP guidelines published in 2013, a mean HER2 gene copy number of less than 4 signals/nucleus or a HER2/CEP17 ratio of less than 2.0 with a mean HER2 copy number of less than 4 signals/nucleus is considered FISH negative (nonamplified), a mean HER2 gene copy number of 4 or more and 6 or less signals/nucleus or a HER2/CEP17 ratio of less than 2.0 with a mean HER2 copy number of 4 or more and less than 6 signals/nucleus is considered FISH equivocal, and a mean HER2 gene copy number of 6 or more signals/nucleus or a HER2/CEP17 ratio of less than 2.0 with a mean HER2 copy number of 6 or more signals/nucleus or a HER2/CEP17 ratio of 2.0 or more is considered FISH positive (amplified). 14 A minimum of 20 tumor cell nuclei are counted in at least two invasive tumor areas. For equivocal FISH samples, results are confirmed by counting 20 additional cells or repeating the FISH test. 14,15 Similar to IHC, FISH is performed on FFPE tissues. 92 The FISH assay yields results that are considered more objective and quantitative than immunohistochemical scoring. 92,96 Although still a matter of debate, several investigators consider FISH as being more accurate and reliable than IHC in the determination of HER2 status in breast cancer specimens. 81,86,97-99 Moreover, since DNA is more stable than protein, preanalytical factors have less impact on assay results compared with IHC. 32 Another advantage is represented by the presence of internal controls, consisting of nonamplified signals in nonneoplastic cells (ductal epithelial cells and stromal cells) adjacent to the tumor. 93 The dual-probe system evaluates HER2 gene amplification as a ratio of the total HER2 signals to those of chromosome 17. The inclusion of the chromosome 17 probe enables the recognition of chromosome 17 polysomy and therefore allows the distinction between pseudoamplification due to polysomy from true HER2 gene amplification. 100 Although some studies report that chromosome 17 polysomy is a rare event and therefore rarely affects HER2 results, and although clinical relevance of chromosome 17 polysomy in breast cancer is not well established yet, 63,104,105 some authors argue that inclusion of the CEP17 probe is of central relevance for the accurate determination of HER2 amplification, particularly in cases with low-level amplification In fact, chromosome 17 polysomy is reported as a frequent reason for discordance of results obtained with mono-color and dual-color FISH. 109,110 Although FISH has been established as a very robust method to evaluate HER2 gene amplification, this technique presents several disadvantages. FISH is nine times more time-consuming (36 hours vs 4 hours) 71 and almost three times more expensive compared with immunohistochemical analysis ($140 vs $50). 111 Moreover, FISH testing requires costly equipment for signal detection and recognition. Expensive fluorescence microscopes equipped with highmagnification oil immersion objectives and multiband filters are indispensable, making it difficult to integrate the FISH assay in every routine diagnostic laboratory. 85 Furthermore, as fluorescence signals fade within a few weeks, hybridization results should ideally be recorded with an expensive CCD camera. 85,112 Since morphologic aspects of the tissues are difficult to evaluate under fluorescence, distinguishing invasive breast cancer from breast carcinoma in situ, where HER2 gene amplification or protein overexpression has different clinical significance, is rather complicated and timeconsuming. 113 Therefore, the FISH assay can be interpreted only by well-trained personnel. 113,114 Some authors suggest that since FISH analysis is performed at high magnification, intratumoral heterogeneity can be missed. 115,116 Although the FISH assay is considered a more objective and quantitative technique than IHC, reproducibility of FISH results depends on recognition of the invasive component. 117 Moreover, discrepancies among observers occur mainly in samples showing low-level amplification. 106 Another important aspect is that fluorescence signal counting is time consuming. To overcome this problem, image analysis software for the automated evaluation of fluorescent signals has been developed. Several studies have demonstrated an excellent agreement between HER2/CEP17 ratios calculated through manual counting and those resulting from automated image analysis software Some image analysis software has been approved by the FDA for the automated evaluation of HER2 gene amplification, including the Metafer (MetaSystems, Altussheim, Germany) and the Ariol HER2/neu FISH (Applied Imaging, San Jose, CA). In addition to reducing the time required for the analysis of fluorescent signals, this software allows 692 Am J Clin Pathol 2015;144:

8 the storing of captured images and therefore the archiving of cases for future studies. 118 Bright-Field ISH Methods To overcome FISH limitations, alternative in situ methods, such as chromogenic ISH (CISH), silver-enhanced ISH (SISH), gold-facilitated autometallographic ISH (GOLD- FISH), and bright-field double ISH (BDISH), have been developed to detect HER2 amplification. These recent techniques combine practical advantages of immunohistochemical analysis with the reproducibility of the FISH method. 121 Similar to FISH, these alternative ISH methods allow the quantification of HER2 and CEP17 signals and therefore the identification of HER2 gene amplification. However, since visualization is achieved using other reactions than fluorescence-labeled probe, signals can be analyzed using a standard bright-field microscope, allowing the simultaneous analysis of gene copy numbers and morphologic features of tissue. 121 Signals can be therefore exclusively analyzed in an invasive compartment, excluding normal tissue elements and noninvasive carcinoma. Similar to immunohistochemical analysis and FISH, BDISH assays are performed on FFPE tissue sections. 121 In the following subsections, we will briefly describe each single BDISH technique, as well the distinctive advantages and disadvantages of each assay. CISH CISH allows the visualization of target genes in tissue sections using peroxidase enzyme-labeled probes. 121 Similar to FISH, CISH can be performed as either a single-color (HER2 probe only) or a dual-color (HER2 and chromosome 17 probes) assay. The single-color CISH assay (SPOT-Light HER2 CISH kit; Life Technologies, Carlsbad, CA) and the dual-color CISH assay (HER2 CISH PharmDx kit; DAKO) were approved by the FDA in 2008 and 2011, respectively, for the evaluation of HER2 gene amplification in breast cancer specimens, either as a primary test to evaluate HER2 status or to retest equivocal immunohistochemical results (IHC score 2+). 14 With the monochromatic CISH, only the absolute HER2 copy number is scored. 73,122 Following heat-induced epitope retrieval and enzymatic digestion, a digoxigenin-labeled HER2 DNA probe is applied to the slide. After DNA denaturation and hybridization, the hybridized HER2 probe is visualized by sequential incubation with mouse antidigoxigenin and polymerized HRP-goat antimouse, followed by development with DAB as chromogen. HER2 gene copies are then recognizable as intranuclear brown chromogenic reaction product signals. Slides are counterstained with hematoxylin. 96,116, Evaluation of chromosome 17 can be performed on a consecutive slide, using the same protocol as for the HER2 probe, with the exception that a biotinlabeled chromosome 17 centromeric (CEP17) probe will used instead of the digoxigenin-labeled HER2 DNA probe. The CEP17 probe is visualized through incubation with HRP-conjugated antibiotin antibodies and DAB. 96,124 HER2 signals are recognizable either as large peroxidase-positive intranuclear signal clusters or as numerous individual peroxidase-positive small signals. 123 Cases with low-level amplification show six to 10 signals per nucleus in more than 50% of cancer cells, whereas high-level amplified cases are characterized by a mean HER2 copy number of more than 10 or by large gene copy clusters in more than 50% of tumor cell nuclei. 73,106,123,125 Similar to the dual-color FISH, the dual-color CISH assay allows the simultaneous visualization of the HER2 probe and the CEP17 probe on the same slide. 126,127 The dual-color CISH assay is performed using sequential HRP and alkaline phosphatase (AP) enzymatic reactions to detect digoxigenin-labeled HER2 probes and biotin or dinitrophenyl-labeled chromosome 17 probes. 128,129 HRP-conjugated antibodies are then visualized using a green chromogen (HER2 signals) and AP-conjugated antibodies using a red chromogen (CEP17 signals). Alternatively, AP-conjugated antibodies and HRPconjugated antibodies can be used to detect Texas red labeled HER2 DNA probe and fluorescein isothiocyanate (FITC) labeled CEP17 probe, respectively. AP-conjugated anti Texas red antibodies are then developed using a red chromogen (HER2 signals), whereas the HRP-conjugated anti-fitc antibodies are developed using a blue chromogen (CEP17 signals). 126,127,130 Red fluorescence signals produced by Texas red are therefore converted to red chromogenic HER2 signals, and green fluorescence signals produced by FITC are converted to blue chromogenic CEP17 signals. Slides are then counterstained with hematoxylin. Results obtained by dual-color CISH are reported as dualcolor FISH. 15,113,126, Interpretation of results is performed using equipment already available in routine histopathology laboratories, such as a conventional bright-field microscope. 123 Moreover, since results can be visualized using a standard bright-field microscope, CISH analysis allows the simultaneous evaluation of tissue morphology and copy number alterations, reducing the risk of analyzing the nonmalignant compartment. 126,131 Similar to FISH, the CISH assay has the advantage of the built-in internal control. 93 Another advantage over FISH is that tumor heterogeneity is promptly recognizable, even at 20 magnification. 132 Furthermore, chromogenic signals are permanent. 73 Slides can therefore be stored and used for reevaluation or retrospective Am J Clin Pathol 2015;144:

9 Furrer et al / Pros and Cons of HER2 Diagnostic Tests studies. 126,127 Comparatively to FISH, the CISH assay is less expensive ($98/slide vs $183/slide) 48 and quicker (248 vs 314 minutes). 96 Since the CISH assay allows an easier identification of the invasive component compared with FISH, analysis of CISH signals is consequently less timeconsuming than FISH. 96,126 The inclusion of a CEP17 probe in the dual-color assay allows the calculation of the HER2/ CEP17 ratio, enabling exclusion of chromosome 17 polysomy. 124 In addition, the dual-color assay can be performed on a programmable automated slide stainer, improving the assay efficiency and reducing the risk of errors. 128,129 The main disadvantage of mono-color CISH is that the use of monochromatic signals does not allow the determination of the HER2/CEP17 ratio and therefore the detection of chromosome 17 polysomy, making it necessary to hybridize the control probe (chromosome 17) on an adjacent section. This additional staining is time-consuming and cost ineffective. 126 The evaluation of HER2 gene copy number can be difficult in areas presenting high-level amplification, since overlapping dots lead to formation of signal clusters. 126 Although relatively infrequent, technical problems, including over- or underfixation of tissue samples, overor underdigestion of tissue sections, and high background debris, can lead to erroneous results or loss of signal. 73,112,122 SISH SISH is an automated enzyme metallography procedure. 133 Enzymatic metallography is a recently developed method for ISH, in which an enzyme reaction is used to selectively deposit metal from solution to produce a black, sharply defined staining. 133 In the specific case of SISH, HRP-conjugated probes are used to deposit metallic silver at the reaction site. 133 All steps of the automated assay, including deparaffinization and counterstaining, are performed on the Ventana BenchMark XT automated slide stainer HER2 and chromosome 17 analysis is performed on sequential slides After heat-induced epitope retrieval and proteolytic pretreatment, slides are incubated with a dinitrophenol (DNP) labeled HER2 probe and a DNP-labeled CEP17 probe, respectively. Following DNA denaturation and hybridization, both HER2 and CEP17 probes are visualized by application of a rabbit anti-dnp antibody followed by an HRP-labeled goat anti-rabbit antibody. 134,135,137,138 As mentioned above, probes are visualized through the process of enzyme metallography. This process is driven by the sequential addition of silver acetate, hydroquinone, and hydrogen peroxide. Silver ions are reduced to metallic silver ions through the action of the reducing agent, hydroquinone. This reaction is stimulated by hydrogen peroxide, which is a substrate for HRP. The silver precipitation is deposited in the nucleus, and HER2 or CEP17 signals are recognizable as black dots within cell nuclei. 135,137,139 Similar to FISH, HER2 gene amplification status obtained by SISH is reported as a HER2/CEP17 ratio, according to the ASCO/CAP guidelines. 15,115, ,140,141 SISH is a fully automated bright-field ISH assay. In addition, since SISH protocol is automated, this assay is six times faster to perform than FISH (6 hours vs 36 hours). 135 Moreover, staining results obtained with SISH show a higher signal resolution compared with those obtained with DAB-based methods (IHC, CISH). 133 Discrete black dots and signal clusters are easier to evaluate compared with other bright-field ISH techniques. 142,143 Furthermore, image analysis software (Ventana Image Analysis System; Ventana Medical Systems) for the automated quantification of HER2 gene and CEP17 copy number in sequential sections has been developed. 121 To correct for chromosome 17 aneusomy, the hybridization of a further section is required for separate evaluation of CEP17 copy number. 142 Moreover, the SISH assay is a relatively new technique and requires a rather expensive automated slide stainer that may not be available in many routine pathology laboratories. 128 GOLDFISH GOLDFISH is a recently developed method that combines the use of streptavidin-labeled gold nanoparticles with gold-based autometallography. Autometallography is the process through which gold nanoparticles are enlarged and therefore visualized through bright-field microscopy by selective deposition of gold ions onto their surface. 144,145 GOLDFISH has been tested for the determination of HER2 gene amplification in breast cancer specimens. 145,146 The GOLDFISH assay is based on the tyramide signal amplification principle. 147 At first, tissue sections are hybridized with biotin-labeled probe. The probe signal is amplified through the addition of streptavidin-peroxidase and biotinylated tyramine. Tissue sections are then incubated with Streptavidin-Nanogold (Nanoprobes, Yaphank, NY), 1.4-nm diameter gold particles covalently conjugated with streptavidin, and GoldEnhance (Nanoprobes), a reagent that catalyzes the deposition of gold ions in solution around the gold nanoparticles, thereby increasing their sizes. 145 This produces a dense black, permanent signal visible with a conventional bright-field microscope Am J Clin Pathol 2015;144:

10 Cells are considered nonamplified if up to two small nonconfluent black signals are identified within nuclei of invasive carcinoma. Amplified cases are characterized by large clusters of confluent black signals, whereas cases showing low-level amplification are characterized by four to eight separately distinguishable black small signals. 145 Since amplification cases are immediately recognized by the presence of massive confluent deposit in cell nuclei, signal counting in amplified cases is not required, allowing a quicker examination of amplified cases. 146 The signals obtained with the GOLDFISH technique are less discrete compared with those obtained by SISH. 142 BDISH BDISH or dual-color in situ hybridization (dual ISH) is a fully automated bright-field ISH assay for the simultaneous detection of HER2 and CEP17 signals on the same FFPE breast cancer tissue section. 142,148 This technique combines the detection of HER2 gene copies through the deposition of metallic silver particles at the reaction site, as in the monocolor SISH assay, with the visualization of CEP17 copies with fast red, similar to the CISH procedure. 142,149 The whole assay is performed with the FDA-approved dual-color ISH assay (INFORM HER2 Dual ISH DNA probe cocktail assay; Ventana Medical Systems) using the BenchMark XT automated slide stainer (Ventana Medical Systems). 142,148,149 Following antigen retrieval, DNPlabeled HER2 probe is applied to the slide. HER2 probe is visualized with a rabbit anti-dnp antibody followed by an HRP-labeled goat anti-rabbit antibody. HRP enzyme is then developed with a silver precipitate. HER2 signals are visualized as discrete black spots in the nuclei. 142,148,149 Sequentially, DNP-labeled CEP17 probe is applied to the slide. CEP17 probe is visualized by the incubation with a rabbit anti-dnp antibody, followed by an AP-labeled goat anti-rabbit antibody. AP enzyme is finally developed by fast red reagent. The CEP17 signals are visualized as red spots in the nuclei. CEP17 red spots are slightly larger than the HER2 black spots. Slides are then counterstained with hematoxylin. 142,148 Similar to FISH and SISH, HER2 gene amplification status determined by BDISH is reported as HER2/CEP17 ratios, as recommended by the ASCO/CAP guidelines. 15 The BDISH assay allows the simultaneous visualization of both HER2 and CEP17 targets on the same slide. This is very relevant, especially for cases displaying chromosome 17 aneusomy or intratumoral heterogeneity. 142 Furthermore, since the assay procedure is completely automated, reproducibility of results is increased, since risk of human errors is diminished. 142,143 Moreover, in analogy to the SISH assay, HER2 signals are visualized as discrete black dots that are particularly easy to count. 142,143 As the HER2 signals and CEP17 signals differ in color and size, both signals can be distinguished from each other, even though they colocalize within cell nuclei. 142 Moreover, since the assay is completely automated, results are available within 6 hours. 143 Similar to other bright-field ISH techniques, HER2 amplification status can be examined with a conventional bright-field microscope in the context of tissue morphology. Moreover, slides can be permanently archived. 142 The BDISH assay presents the same disadvantages as CISH and SISH. HER2 Gene-Protein Assay These recently developed assays allow the simultaneous evaluation of HER2 protein expression and HER2 gene amplification on single FFPE breast cancer tissue sections. Several combinations have been described so far in the literature. Downs-Kelly and collaborators 150 combined IHC with mono-color SISH (EnzMet GenePro, Ventana Medical Systems). HER2 gene amplification assessment was performed using an HRP-based silver deposition method, followed by HER2 immunohistochemical staining using AP and fast red substrate. They observed an excellent correlation between the results obtained with the combination gene-protein assay and those obtained by IHC alone. Ni and collaborators 151 combined HER2 immunohistochemical staining with mono-color CISH. Similar to Downs- Kelly et al, 150 HER2 immunohistochemical staining was achieved using an AP-based fast red system. Subsequently, the CISH assay was performed using a digoxigenin-labeled HER2 probe, which was visualized via an antimouse antidigoxigenin antibody and an antimouse HRP polymer conjugate, followed by incubation with DAB. The combined protocol was performed partially on an automated slide stainer. Reisenbichler and collaborators 152 tested a HER2 geneprotein assay consisting of the sequential performance of mono-color CISH followed by HER2 immunohistochemical staining. The digoxigenin-labeled HER2 probe was visualized via an antimouse antidigoxigenin antibody and an antimouse HRP polymer conjugate, followed by incubation with DAB. HER2 protein expression was evaluated with an HRP-based DAB system. They observed 100% agreement between the gene amplification results obtained when CISH was performed alone compared with the sequential protocol. Compared with separately performed immunohistochemical staining, Am J Clin Pathol 2015;144: