specimens
American Journal of Clinical Pathology 2015; 144(5): 686-703
Daniela Furrer1,2,3, François Sanschagrin1,2,4,5, Simon Jacob1,2,4,5, Caroline Diorio1,2,3,4
1Centre de Recherche sur la cancer de l’Université Laval, Quebec City, QC G1V 0A6, Canada; 2Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Hôpital
du Saint-Sacrement, 1050 chemin Ste-Foy, Quebec City, QC G1S 4L8, Canada; 3Département
de médecine sociale et préventive, Faculté de Médecine, Quebec City, QC G1V 0A6, Canada;
4Centre des Maladies du Sein Deschênes-Fabia, Hôpital du St-Sacrement, 1050 chemin Ste-
Foy, Quebec City, QC G1S 4L8, Canada; 5Département de biologie moléculaire, de biochimie
médicale et de pathologie, Faculté de Médecine, Quebec City, QC G1V 0A6, Canada
Corresponding author: Caroline Diorio, Axe Oncologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Hôpital du Saint-Sacrement, 1050 chemin Ste-Foy, Quebec City, QC G1S 4L8, Canada
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Résumé
Le facteur 2 de croissance épidermique humain (HER2) est un marqueur prédictif du cancer du sein. L’évaluation fiable du statut HER2 est essentielle afin de déterminer l’éligibilité des patientes atteintes d’un cancer du sein au traitement anti-HER2. Plusieurs méthodes existent pour l’évaluation d’HER2.
Dans cette revue, les principaux avantages et inconvénients des techniques qui ont été développées pour évaluer HER2 dans les spécimens de cancer du sein seront discutées. Puisque chaque technique a ses propres avantages et inconvénients, à date aucun consensus n’a été atteint concernant la meilleure technique d’évaluation.
L’accent doit être mis sur la standardisation des procédures, l’exécution d’un contrôle de qualité et l’évaluation de la performance des méthodes déjà existantes. Le développement de nouvelles méthodes robustes et fiables doit également être encouragé. De plus, des grands essais cliniques sont nécessaires afin d’identifier la technique qui mieux prédit la réponse des patientes.
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Abstract
Background. 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 breast cancer patients 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).
Design. In this review we will 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.
Conclusion. Currently, emphasis must be put on standardization of procedures, internal and external quality control assessment, and competency evaluation of already existing methods in order 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 in order to identify the technique that the most reliable predict positive response to anti-HER2 drugs.
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Introduction
Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor belonging to the family of epidermal growth factor receptors (EGFR) (1). The protein is encoded by the HER2 (ERBB2) gene, which is located on the long arm of chromosome 17 (17q12-21.32) (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 breast cancer 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) is a recombinant humanized monoclonal antibody that specifically targets the extracellular domain of the HER2 protein (7). Trastuzumab improves the outcomes of HER2- positive breast cancer patients in both the metastatic (8, 9) and adjuvant settings (10, 11). The 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 2006. Lapatinib (Tykerb®/Tyverb®, GlaxoSmithKline) is a small molecule inhibitor of the intracellular tyrosine kinase domain of both HER2 and EGFR receptors (12). In 2007 Lapatinib has been approved by FDA as combination therapy with capecitabine for the treatment of patients with HER2-positive advanced breast cancer who have 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 at 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 last few decades for the determination of HER2 status in breast
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cancer specimens. The principle of each method will be 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 2.1., whereas each technique and its advantages and disadvantages are thoroughly depicted in the next section.
Principles of the methods of analysis, advantages and
disadvantages of each described technique
Southern blot
The Southern blot technique has been used to determine HER2 gene amplification in breast cancer samples (16-22). 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-labelled HER2 single stranded DNA (ssDNA) probe. Labelled HER2 ssDNA probe will hybridize with the HER2 ssDNA sequence on the basis of strand annealing between complementary ssDNA molecules. The labelled HER2 sequence is then visualized by autoradiography (18-20, 22). Autoradiograms are then scanned with a densitometer (23).
The HER2 gene copy number is then compared to that of a control gene (16), to control DNA extracted from blood (18) or to DNA extracted from normal breast tissues (19). Tumors that showed a more than twofold increase in copy number compared to control unamplified DNA are considered amplified (16).
Advantages. Since DNA is very stable, it is considerably less degraded in tissues compared
to protein and mRNA (24).
Disadvantages. Although very reliable, this technique is not applicable in routine diagnostic
settings, since it is time-consuming and requires large amount of DNA (25, 26). In addition, this technique does not allow the morphologic preservation of tissue and therefore the evaluation of histological features of tumor. Since non-amplified non-neoplastic cells present in tumor cannot be isolated from cancer cells, results obtained potentially underestimate the real HER2 gene amplification of the sample through a dilution effect (21).
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Northern blot
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. After extraction from homogenized tissue sample, total cellular RNA or 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-labelled complementary probe (27). The labelled HER2 sequence is then visualized by autoradiography. The relative optical density (OD) of bands is measured by densitometry scanning. Tumors are then divided into 4 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); 4+ (> 1.5 OD units) (21).
Advantages. 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).
Disadvantages. 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 of being a labor-intensive technique, does not allow an exclusive evaluation of HER2 status in cancer cells, as the morphology of tissues is destroyed during the homogenization of tissue samples (21).
Enzyme-linked immunosorbent assay (ELISA)
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 serum concentration of HER2 ECD breast cancer patients (33-37). HER2 ECD is detected using 2 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 labelled with horseradish peroxidase (HRP).
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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 labelled with chemiluminescent compounds (i.e., 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) and one manual ELISA assay (Siemens Healthcare Diagnostics) have been approved by the FDA in 2000. Another automated platform (ADVIA CentaurTM, Siemens Healthcare Diagnostics) has also received FDA approval in 2003 (39).
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 cut off was derived from the sera of 242 healthy women (36)) in HER2-positive breast cancer patients may be associated with the progression of primary tumors to metastatic breast cancer (39). In both metastatic and early breast cancer patients, ECD levels might reflect the HER2 full-length protein expression, as elevated HER2 ECD levels in serum (≥15 ng/ml) have been correlated with higher scores at the HER2 IHC (34, 35, 40, 41). In metastatic breast cancer patients, high HER2 ECD serum concentrations (≥15 ng/ml) have been also associated with resistance to endocrine therapy and chemotherapy (42) and in both metastatic and early breast cancer patients 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 metastatic breast cancer patients, 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 on which cut off value was considered (for a recent review see (47). Based on these conflicting data, the clinical use of the ELISA assay for the determination of HER2 ECD in patient’s serum has not yet been widely implemented (47, 48).
Advantages. ELISA is a quick and simple assay in addition of 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).
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Disadvantages. Results obtained by ELISA might not be reliable if the serum samples are from patients receiving trastuzumab treatment, since trastuzumab still present in patient’s serum might compete with the two antibodies used in the assay (49).
Western blot
Western blot has been used to evaluate HER2 protein expression in frozen and FFPE breast cancer tissue samples (21, 50, 51). Following protein extraction from tissues, sample proteins are separated by size using SDS-polyacrylamide gel electrophoresis. Separated proteins are then transferred to a nitrocellulose or a polyvinylidene fluoride (PVDF) membrane. Membrane is firstly 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 is 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).
Advantages. High sensitivity (as little as 0.1 ng of protein can be detected in a sample)
represents the major advantages of the Western blot technique (51).
Disadvantages. 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 (PCR)-based assays
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 single-stranded DNA template. Oligonucleotide primers, i.e. 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.
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Polymerase chain reaction (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-labelled sequence-specific primers. Fluorescence signal produced during the amplification process is detected using 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 ≥ 2.0 is regarded as HER2 amplification (25, 55, 58-60, 62).
RT-PCR, often denoted as real time RT-PCR or quantitative reverse transcriptase PCR (qRT- 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 ΔΔCT 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 tissues samples. Several cut off values have been used in the literature to define HER2 overexpression determined by RT-PCR (24, 25, 54, 56-58, 61). Importantly, the Oncotype DxTM (Genomic
Health, Redwood City, CA) assay is based on RT-PCR-technology to analyse the expression of 21 genes involved in breast cancer biology, including HER2, estrogen receptor (ER) and progesterone receptor (PR). The test, performed on mRNA extracted from FFPE tumor tissues, is used to predict the likelihood of breast cancer recurrence in early-stage, node- negative, ER-positive breast cancer patients, in addition to predict their chemotherapy benefit (65).
Advantages. Real-time PCR allows a rapid and quantitative analysis of gene amplification (60,
66). Being real-time PCR a DNA-based technique, variations in tissue fixation and processing have little impact on the results. Moreover, since only small quantities of DNA fragments are
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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). Real time RT-PCR presents several advantages, including a large dynamic range and an accurate quantification (67).
Disadvantages. 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 non-amplified stroma cells or non-invasive 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 histological subtype might considerably influence the efficacy of this assay. The impact of surrounding stromal and non- malignant 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 reverse-transcriptase PCR (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 (MLPA)
Multiplex ligation-dependent probe amplification (MLPA) is a recent 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 3 probes for the HER2 gene, 11 probes for chromosome 17, and 25 control probes located chromosomes other than chromosome 17 (26). Each MLPA probe consists of two halves. One half is
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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 probe-specific length and a universal primer sequence. Since the two probe halves recognize adjacent DNA target