© Springer International Publishing Switzerland 2016
Debra G.B. Leonard (ed.)Molecular Pathology in Clinical Practice10.1007/978-3-319-19674-9_3333. Breast Cancer
(1)
Genomic Health Inc., 301 Penobscot Drive, Redwood City, CA 94063, USA
Abstract
Breast cancer treatment has evolved over the past 50 years, often as a direct consequence of molecular testing advances. In fact, molecular testing of predictive markers in breast cancer, including hormone receptors and HER2, is the model for personalized cancer treatment. Clinical practice guidelines specify that every primary invasive breast cancer and putative recurrence be tested for ER, PR, and HER2 expression to identify those cancers likely to respond to corresponding targeted treatments. The newest tests for breast cancer management are the tissue-based prognostic and/or predictive molecular assays, which identify patients with biologically indolent breast cancer who will not benefit from cytotoxic chemotherapy and those with intrinsically aggressive disease who may benefit. This chapter reviews clinically standard predictive marker and Oncotype DX® testing, as well as several emerging molecular testing systems.
Keywords
Breast cancerPredictive markersEstrogen receptorHER2ImmunohistochemistryIn situ hybridizationPrognostic molecular assaysIntrinsic subtypesIntroduction
Breast carcinoma is the most common malignancy in women in the USA and occurs in two major forms: sporadic and hereditary. Sporadic breast cancer is the topic of this chapter; hereditary breast cancer, caused by mutations in BRCA1 and BRCA2 and other germline DNA mutation syndromes, is discussed in Chap. 22. Molecular assays for detecting circulating tumor cells are discussed in Chap. 39.
Molecular Pathogenesis of Breast Cancer
The cause of common, sporadic breast cancer and the reasons for its progression are unknown. What is amply clear is that no simple, single alteration is responsible for sporadic breast cancer. Putative oncogenic processes include impaired cell-number regulation, mutated oncogenes and tumor suppressor genes, dysfunctional epigenetic controls, and deranged intercellular interaction [1]. In addition, emerging evidence supports a theory that transforming epithelia communicate aberrantly with their surrounding mammary stroma, such that the entire tissue region is likely involved in the oncogenic process. Collectively, these processes determine the behavior of a specific breast cancer.
Two dominant models of mammary carcinogenesis are (1) the stochastic clonal evolution model and (2) the cancer stem cell model (both reviewed in Ref. 2). The stochastic clonal evolution model proposes that random mutational events occur in any mammary cell; uncontrolled cell division results when a cell accumulates a sufficient number and type of mutations to gain a selective growth advantage. In this model, the expanding aberrant clone acquires additional DNA alterations that enable it to invade adjacent stroma and spread to distant sites via lymphatics and blood vessels. The clone “evolves” to an invasive and metastatic phenotype.
The cancer stem cell hypothesis derives from the theoretical normal breast parenchymal development pathway. A breast stem cell divides asymmetrically to produce a daughter stem cell (self-renewal) and a common progenitor cell, which can divide and differentiate into either a myoepithelial cell or a luminal progenitor cell. The latter is the parent of two more-differentiated lineages: luminal and ductal epithelia [2]. The cancer stem cell hypothesis posits that the initiating carcinogenic event occurs in one of the small number of mammary stem cells or their uncommitted progenitors but never in differentiated luminal or myoepithelial cells. Following initiation, clonal evolution may progress to the fully malignant phenotype. An appealing variation of the cancer stem cell hypothesis has cancer initiation restricted to the second generation common progenitor cell, usually a common luminal progenitor. If correct, this would explain why global gene expression profiling parses human breast cancers into four to five major molecular classes (intrinsic subtypes), which include luminal A and B groups [3–5].
Anatomic Classification of Breast Cancer
The vast majority of breast cancers arise in the hormonally sensitive, physiologically active, terminal duct lobular units; only a small percentage arise in the larger ducts. The two morphologic subtypes of breast cancer, ductal and lobular, are named for the histologic structures from which they appear to arise, the terminal ducts and the lobules, respectively. Both ductal and lobular carcinomas occur in invasive and in situ forms; this chapter focuses on invasive carcinoma.
Invasive ductal carcinoma (IDC) is the most common form of invasive breast cancer, accounting for approximately 85 % of cases. Several lines of evidence indicate that IDC directly evolves from ductal carcinoma in situ (DCIS). Invasive lobular carcinoma (ILC) accounts for fewer cases of invasive breast cancer (approximately 15 %). Like IDC, ILC appears to evolve from a morphologic precursor lesion, lobular carcinoma in situ (LCIS). At the genomic level, IDC is far more heterogeneous than ILC. While the two subtypes share some recurrent molecular alterations, they differ at many more loci. Invasive ductal and lobular carcinomas are managed clinically in the same way.
Overview of the Clinical Management of Breast Cancer
Breast cancer treatment has evolved over the past 50 years, often as a direct consequence of molecular testing advances. Currently, potentially curable, early invasive breast cancer (Stages I, II, and III) is treated both as local-regional and systemic disease. The goal of local-regional treatment is to eradicate cancer from the breast and regional lymph nodes, whereas systemic treatment seeks to eliminate occult microscopic deposits of cancer at remote sites in the body.
Treatment selection rests on two sets of clinicopathologic factors: (1) anatomic staging [6] to estimate prognosis, which is the likelihood that a person will survive the disease independent of systemic treatment, and (2) results of predictive marker tests, which estimate the likelihood a breast cancer will respond to specific targeted treatments. Only two targeted treatments are in current common use for breast cancer: antiestrogens (e.g., tamoxifen, aromatase inhibitors) and trastuzumab (Herceptin™; Genentech, South San Francisco, CA), a monoclonal antibody targeting the human epidermal growth factor receptor-2 protein (simplified to HER2 in this chapter). Currently, tests for these predictive markers are used to determine the use of these targeted therapies for breast cancer: estrogen receptor (ER) levels and progesterone receptor (PR) levels for the antiestrogens and HER2 overexpression for trastuzumab.
Molecular testing of prognostic and predictive markers in breast cancer has a long, successful history and is a paradigm for personalized cancer treatment. Clinical practice guidelines specify that every primary invasive breast cancer and putative recurrence be tested for expression of ER, PR, and HER2 [7, 8]. These are called predictive markers because they predict the likelihood a cancer will respond to antiestrogen and anti-HER2 treatment. The next sections describe the standard and emerging molecular tests for newly diagnosed breast cancer, including ER and PR tests, HER2 tests, molecular profiling for intrinsic breast cancer subtypes, and the commercially marketed prognostic and/or predictive tests for breast cancer management.
Hormone Receptors in Breast Cancer
Molecular Basis for Targeting Hormone Receptor Expression in Breast Cancer
Like normal mammary glandular tissue, most invasive breast cancers (75–80 %) express ER and/or PR [9]. These receptors, which bind endogenous estrogen and progesterone, are ligand-inducible transcription factors that bind to regulatory DNA sequences associated with target genes, activating a variety of cellular events that give cancer cells a survival advantage [10, 11]. Aside from this direct DNA interface, ER also interacts with cytoplasmic proteins that have an indirect action on gene transcription [12]. Interfering with this key hormone-receptor interaction is a mainstay of ER-positive breast cancer treatment.
Estrogen receptor protein exists in two isoforms, ERα and ERβ, which are encoded by two highly homologous genes, ESR1 and ESR2, respectively. ERα (ESR1) is the clinically important isoform of ER in breast cancer. Similarly, there are two PR isoforms (A and B), which are products of the same gene. PR isoform A is a truncated form of B [12]. Estrogen receptor-ligand complexes activate the transcription of PR; thus, nearly all ER-positive breast cancers also express PR. The ER test result dominates clinical decision-making. Antiestrogen treatment is recommended for all ER-positive (>1 % cells) cancers. The small fraction (approximately 3 %) of ER-negative, PR-positive cancers also are treated with antiestrogens because they are presumed to have partially intact ER transcriptional activity [13].
Three therapeutic approaches are used to nullify estrogen’s ER-mediated effects on breast cancer: (1) remove the ovaries to reduce endogenous estrogen, (2) pharmacologically inactivate ER, and (3) medicate to prevent inactive forms of endogenous estrogen from converting to active molecules. Surgical oophorectomy, the oldest endocrine treatment for breast cancer, still has a role in some circumstances [14]. Inactivating ER directly is accomplished by treatment with tamoxifen (Nolvadex), one of the most commonly used targeted therapies. Tamoxifen is an oral medication that binds stably to the ER to form an unwieldy complex that is sterically incapable of binding to DNA to activate gene transcription. Lastly, aromatase inhibitors (AIs), such as anastrozole (Arimidex) and exemestane (aromasin), bind to the aromatase enzyme to prevent adrenal androgens, the main source of endogenous estrogen after menopause, from being converted to estrogen. AIs are most useful in postmenopausal patients, reducing circulating estradiol to near zero levels [13, 68].
Clinical Utility of ER/PR Expression Testing
ER/PR testing is critical because the ER content of a breast cancer is the strongest predictor of antiestrogen treatment efficacy, regardless of how the estrogen-reduced state is achieved (oophorectomy, tamoxifen, or AI). Breast cancers that entirely lack ER do not respond to antiestrogens and are omitted from the therapeutic plan [15]. All patients (women and men) with ER-positive invasive breast cancer based on ER/PR test results are offered antiestrogen treatment, unless contraindicated by specific comorbid conditions [9, 16]. The decision whether to use antiestrogen therapy is highly significant because adjuvant tamoxifen reduces mortality by at least 33 % at 15 years [15, 17].
Available Assays for Hormone Receptor Content Testing
Currently, immunohistochemistry (IHC) of formalin-fixed, paraffin-embedded (FFPE) tissue sections is the standard method for ER/PR testing. IHC testing for ER/PR expression has many advantages: ER/PR in invasive cancer cells can be assessed specifically and normal tissue expression ignored; results are not confounded by endogenous hormone levels; no requirement for fresh or frozen tissue, so that IHC can be applied retrospectively and to small (≤1.0 cm) invasive cancers, which are now commonplace; and lastly, IHC staining kits and instrumentation are widely available in nonacademic centers, where most breast cancers are diagnosed and treated. Any proposed new testing platform must retain or improve on these advantages to supplant IHC hormone receptor testing.
In late 2008, The American Societ of Clinical Oncology (ASCO) partnered with the College of American Pathologists (CAP) to convene a panel to determine best practices for ER/PR testing in breast cancer. The standardization effort was driven by academic oncologists who discovered significant discordances between local and central predictive marker testing results for patients entered in clinical trials. The panel analyzed numerous published studies and clinical trials’ results correlating the ER/PR content to outcomes of antiestrogen treatment [8, 18]. The resulting 2010 ASCO/CAP Guideline Recommendations for IHC Testing of ER/PR in Breast Cancer were widely adopted and remain the standard practice [8].
The guideline recommends using IHC of FFPE tissue sections to test all primary invasive breast cancers and putative recurrences for ER/PR content, whenever tissue is available. The guideline defines how to control and document preanalytic, analytic, and postanalytic variables to ensure analytic and clinical validity and clinical utility [8].
Preanalytic Standardization
The 2010 ASCO/CAP Guideline Recommendations for IHC testing of ER/PR in Breast Cancer specify how to handle FFPE tissues that are likely to be tested (see summary below). The overall goal is to standardize three key preanalytic variables: tissue handling, fixation type, and fixation duration [8, 18]. Preanalytic factors are most easily controlled in the core biopsy setting which, therefore, is the preferred tissue for testing.
Documenting cold ischemia time requires cooperation between persons performing the breast cancer biopsies and resections with pathology personnel, which can be a challenge. Adding designated spaces for this biopsy or resection timing data to the surgical pathology requisition form or in the electronic ordering process can aid clinicians in providing the data. Reporting templates help the pathologist to document the preanalytic factors in the report.
Summary of ASCO/CAP Guideline Recommendations for Preanalytic Variables for Hormone Receptor and HER2 Immunohistochemistry:
Minimize cold ischemia time (time from excision to initiation of fixation) to 1 h or less.
Use 10 % neutral buffered formalin (NBF) as the standard fixative.
If nonstandard fixatives or those containing decalcifying agents are used, add a disclaimer to the report.
Do not use microwave-type processors for ER/PR staining, because results have not been clinically validated.
Fix the cancer in NBF for at least 6 h, but no more than 72 h, before paraffin embedding.
Analytic Standardization
Antibodies and controls are the key analytic variables for ER/PR testing. The 2010 ASCO/CAP Guideline Recommendations for IHC Testing of ER/PR in Breast Cancer advise using specific ER antibody clones (1D5, 6F11, SP1, and 1D5 + ER.2.123), which have slightly different staining profiles but have been sufficiently validated to provide comparable results. Test kits with validated scoring schemes that are approved or cleared by the US Food and Drug Administration (FDA) are considered optimal. Laboratory developed tests (LDTs) are acceptable, if stringently validated [8].
The interpreting pathologist must verify that the internal and external controls stain appropriately. Internal controls consist of immunoreactive benign mammary epithelia in the same section or in another equivalently processed section from the same specimen [8]. Where appropriate internal controls are lacking, an apparently negative ER or PR stain is considered uninterpretable. Breast biopsies are rarely problematic, but lung and bone, which are common sites of metastasis, may be more difficult to interpret. In this context, the pathologist should add a disclaimer that a negative staining result cannot be confirmed as accurate in tissues that do not normally express ER/PR (no internal control). Also, bone from pathologic fractures due to metastatic cancer typically must be decalcified. With decalcification, an apparently negative ER/PR stain must be interpreted cautiously and a disclaimer added to the report for a negative result, noting that decalcification can adversely affect antigen preservation in tissue and result in a false-negative stain. Finally, it is essential for a laboratory initiating IHC for ER/PR to validate the assays; the CAP provides guidelines for validation [19].
Postanalytic Standardization
The 2010 ASCO/CAP Guideline Recommendations for IHC Testing of ER/PR in Breast Cancer allow the pathologist to choose a scoring system (Allred system, H-score, etc.) but recommend that the report indicates both the percentage of stained invasive carcinoma cell nuclei and the stain intensity (see below). Some FDA-approved or FDA-cleared staining systems mandate the use of a specific scoring system to ensure clinical validity. For example, the FDA 510(k)-cleared DakoCytomation ER/PR pharmDx™ (Dako Corp, Carpinteria, CA) staining kit requires pathologists to report stain results using the Allred scoring scheme [20]. Semiquantitative visual estimates of nuclear staining are sufficient for guiding clinical decisions. Computer-assisted imaging is more expensive and does not add value to testing, given that antiestrogen treatment is considered for any level of staining ≥1 %.
Interpreting ER/PR stains (adapted from Ref. 8):
ER- or PR-positive cancer is one in which ≥1 % of invasive carcinoma cell nuclei are immunoreactive.
ER- or PR-negative cancer is one in which <1 % of invasive carcinoma cell nuclei are immunoreactive.
ER and/or PR status is not interpretable when no invasive carcinoma cell nuclei are immunoreactive and appropriately stained controls are lacking.
Laboratory and Regulatory Issues
The Clinical Laboratory Improvement Amendments (CLIA) of 1988 and the derived CLIA regulations provide the analytic validity standards for predictive factor assays such as ER, PR, and HER2 tests. Laboratories performing these high complexity tests must be surveyed semiannually, with defined criteria and actions required when performance is deficient [21]. Semiannual proficiency testing for ER/PR analysis is now a mandatory part of the CAP Laboratory Accreditation Program (LAP) (http://www.cap.org/web/home/lab/accreditation/laboratory-accreditation-program. Accessed 3/10/2015). Competence is assessed by periodic review of test performance against peers and failure mandates remediation.
Future Directions
Transitioning routine ER/PR expression testing to a molecular platform, for example, a reverse transcription-polymerase chain reaction (RT-PCR) analysis using paraffin tissues, would not be straightforward. IHC has many advantages, including wide availability, and a strong evidence base that allows ER/PR results to be used to triage cancers for additional molecular testing, as discussed later.
The 2010 ASCO/CAP Guideline Recommendations for IHC Testing of ER/PR in Breast Cancer require new ER/PR tests be validated clinically and operationally [8]. Although ER/PR expression profiling results are provided with the Oncotype DX® Recurrence Score (Genomic Health™, Redwood City, CA), these may serve to confirm IHC stain results but have not been validated for clinical decision-making [8, 21]. It is likely that paraffin-IHC will remain the platform of choice for some time.
HER2 in Breast Cancer
Molecular Basis for Targeting HER2 Expression in Breast Cancer
The other important predictive marker in breast cancer is HER2, a cell-surface membrane glycoprotein involved in cell proliferation control. HER2 gene amplification, leading to protein overexpression, is found in 15–20 % of invasive breast cancers [21]. Early investigations used Southern blot analysis to identify HER2 gene amplification in fresh or frozen samples of breast cancer and showed that patients with HER2-amplified breast cancers had higher recurrence and death rates than those with HER2-normal cancers [22, 23]. Defining HER2 as a possible prognostic marker drove the first laboratory testing efforts using monoclonal antibodies for IHC staining [24]. Later, as HER2 became far more important as a drug target, the goals of testing shifted to predicting therapeutic response to drugs targeting the HER2 protein.
The first anti-HER2-targeted treatment, trastuzumab (Herceptin™; Genentech, South San Francisco, CA), was developed in the mid-1990s and is now the standard treatment for HER2-overexpressing breast cancers. Trastuzumab is a recombinant humanized monoclonal antibody that specifically binds to the extracellular juxtamembrane domain of HER2 and inactivates its intracellular tyrosine kinase function by several possible mechanisms, resulting in reduced growth and reduced survival of HER2-dependent cancers [25]. Trastuzumab is most effective when combined with chemotherapy agents active against breast cancer, such as taxanes, doxorubicin, and cyclophosphamide, with trastuzumab monotherapy not being the standard care at this time (http://www.nccn.org/professionals/physician_gls/pdf/breast.pdf. Accessed 03/19/2015).
Following the landmark Genentech-sponsored trials that demonstrated the efficacy of trastuzumab in HER2-overexpressing metastatic breast cancer, trastuzumab was marketed with a companion diagnostic IHC assay, HercepTest™ (Dako Corp, Carpinteria, CA). In 1998, the FDA approved the HercepTest™ designed to identify HER2 overexpression in invasive breast cancer to select patients for trastuzumab treatment [26]. The FDA advocates the importance of companion diagnostic testing for all emerging targeted cancer treatments.
Other anti-HER2 agents (pertuzumab, lapatinib, and ado-trastuzumab emtansine [T-DM1]) are in the clinical trial pipeline. Testing for HER2 overexpression/amplification is also used to select patients for these newer agents [21].
Clinical Utility of HER2 Expression Testing
Following trastuzumab’s introduction, HER2 joined ER/PR expression as a standard predictive marker in breast cancer management. The ASCO Tumor Marker Guidelines Panel added routine HER2 expression testing of all invasive breast cancers to its 2001 recommendations [7]. Because DCIS is never treated with chemotherapy and trastuzumab, DCIS should not be tested for HER2 amplification.
The goal of HER2 expression testing is to identify patients who are likely to benefit from trastuzumab treatment, i.e., those with breast cancers that overexpress HER2 protein and/or have HER2 gene amplification by in situ hybridization (ISH). Trastuzumab can be lifesaving for patients with HER2-overexpressing breast cancer but requires a costly (approximately $100,000), yearlong course of intravenous therapy that is not risk-free. As is true for ER/PR expression testing, accurate, reliable, and reproducible testing is essential to direct anti-HER2 treatment to those who can benefit and spare those who will not.
Whether trastuzumab benefits patients with HER2-negative or HER2-equivocal cancers has been controversial, although the weight of evidence from early exploratory trials suggested little, if any, effect [21, 25]. A prospective randomized clinical trial (NSABP B-47; NCT01275677) will answer this question definitively, but current clinical practice is to treat only HER2-positive invasive breast cancer with trastuzumab [21, 27].
Available Assays for HER2 Overexpression/HER2 Amplification Testing
Clinical laboratories can use IHC to test for HER2 overexpression or ISH to detect HER2 gene amplification. Gene amplification is the preponderant mechanism for HER2 overexpression in breast cancer. IHC and ISH share several advantages over Southern blot analysis: (1) the signals are interpreted in the context of the histopathology on tissue sections, permitting specific scoring in morphologically confirmed invasive cancer cells; (2) standard FFPE tissues are acceptable specimens, whereas Southern blot analysis requires fresh or frozen tissue to obtain intact DNA, not fragmented by the fixation and embedding processes; and (3) small cancer specimens, such as core needle biopsies, are acceptable for IHC and ISH, whereas larger specimens are needed for Southern blot analysis, which requires a large amount of DNA [21, 26]. ISH testing, especially fluorescence ISH (FISH), has the disadvantages of being more labor-intensive and costly than IHC; because of expense, ISH is a second-line, confirmatory test in most clinical settings. Newer bright-field ISH platforms mitigate some of these disadvantages and may have a future in testing.
The optimal procedures for HER2 IHC testing, scoring, and reporting of results have been long debated. Some phase III clinical trials of trastuzumab found significant discordances between local and central laboratory results for HER2, highlighting the need to standardize HER2 testing [27]. Accurate testing for HER2 expression in the clinical trial context is critically important, because treating cancers with false-positive HER2 overexpression results can confound interpretation of treatment efficacy [21, 27]. Obviously, accurate testing in the patient care context is no less essential.
The ASCO and the CAP came together in 2006 to address HER2 testing inaccuracy (both false-positive and false-negative results) and develop guidelines for testing and interpretation. They convened an expert panel to (1) determine the optimal testing algorithm for HER2 testing, and (2) develop strategies to ensure optimal performance, interpretation, and reporting of results across US laboratories. The first ASCO/CAP HER2 Guideline Recommendations were published in 2007 [28]. An updated guideline in 2013 was informed by new clinical trial data and stakeholder suggestions and addresses newer testing platforms, such as FDA-cleared bright-field ISH assays ([21, 29]; http://www.asco.org/guidelines/her2; http://www.cap.org. Accessed 03/10/2015). Molecular pathologists can expect periodic guideline updates as investigators publish new clinical trial data for anti-HER2 agents.
Testing, Interpreting, and Reporting HER2 Test Results
The 2013 ASCO/CAP HER2 Guidelines recommend using IHC or ISH (FISH or bright-field ISH) on FFPE tissue sections to test all primary and recurrent invasive breast cancers for HER2 overexpression. Increasingly, oncologists biopsy suspected breast cancer recurrences to obtain tissue for accurate HER2 and ER/PR results to guide the treatment of metastatic disease.
The goal of the ASCO/CAP HER2 Guidelines is the same as for ER/PR testing: to standardize preanalytic, analytic, and postanalytic variables to ensure analytic and clinical validity and clinical utility [21, 28]. If available, a core biopsy sample of the primary cancer is used for the first HER2 test. If clearly positive, no further testing of the resected primary cancer is recommended. If the result is negative, no further testing is recommended, unless there are concerns about the core biopsy tissue handling, histopathologic discordance, or tumor heterogeneity, in which case testing can be repeated on the resection specimen [21].
Preanalytic Standardization
Analytic Standardization
Antibodies and controls are the key analytic variables for IHC HER2 testing. ASCO/CAP, as well as the maker of trastuzumab and pertuzumab (Genentech, South San Francisco, CA), recommends using FDA-approved or FDA-cleared assays for HER2 testing. The FDA-approved HercepTest™ (Dako Corp, Carpinteria, CA) has validated scoring schemes and is considered optimal. As is true for ER/PR expression testing, LDTs are acceptable, if stringently validated [21].
Ideally, a new HER2 assay should be validated using well-annotated breast cancer specimens from prospective therapeutic trials of anti-HER2 therapy. This task is difficult to accomplish because such specimens are relatively rare and limited. As a substitute, ASCO/CAP will endorse HER2 assays that show high-level concordance with other established HER2 tests, as long as concordance studies use data sets with a representative distribution of HER2 overexpression/HER2 amplification states.
The interpreting pathologist must verify that the controls stain appropriately. Benign mammary epithelia, which have normal HER2 gene copy number and do not overexpress HER2 protein, function as negative controls. External controls are extremely important as positive controls and include known HER2-overexpressing and HER2-nonoverexpressing invasive breast carcinomas, which must be run concurrently. The HercepTest™ kit (Dako Corp, Carpinteria, CA) is an IHC test kit that includes three FFPE cell line controls with different HER2 copy numbers that stain negative (score 0), negative (score 1+), and positive (score 3+), respectively. Where appropriately stained controls are lacking, or nonstandard conditions have occurred, an apparently negative HER2 stain must be considered uninterpretable and be accompanied by a disclaimer noting the problem [21, 28]. HER2 staining results are interpreted as positive (3+), equivocal (2+), or negative (score 0 or 1+), as summarized below.
Interpreting IHC for HER2 overexpression status (adapted from Ref. 21):
1.
IHC-positive (3+) carcinoma shows:
Circumferential complete, intense membrane staining in >10 % of contiguous and homogeneous cancer cells
2.
IHC-equivocal (2+) carcinoma shows:
Complete, circumferential intense membrane staining in ≤10 % of cancer cells
Circumferential membrane staining that is incomplete and/or weak/moderate intensity in >10 % of contiguous and homogeneous cancer cells
3.
IHC-negative (1+) carcinoma shows:
Incomplete, faint/barely perceptible membrane staining in >10 % of cancer cells
4.
IHC-negative (0) carcinoma shows:
No staining
Incomplete, faint/barely perceptible membrane staining in ≤10 % of cancer cells
The 2013 ASCO/CAP HER2 Guidelines indicate that ISH for HER2 gene amplification may be single-probe (HER2 alone) or dual-probe (HER2 and chromosome 17 centromere, CEP17). The first FDA-approved, dual-probe FISH test for HER2 is the PathVysion HER2 DNA Probe Kit (Abbott Molecular, Des Plaines, Illinois) and is used by most laboratories in the USA. Dual-probe FISH has been the standard method of detecting and semiquantitating HER2 gene amplification in breast cancer since the late 1990s but has some disadvantages: suboptimal morphologic detail, which can make identification of carcinoma cells difficult; signals that fade quickly, not allowing for review; a 2–3-day turnaround time (TAT); and significant cost due to the need for a fluorescence imaging system and specially trained personnel. These drawbacks have stimulated development of more user-friendly bright-field approaches.
Bright-field ISH offers better microscopic detail, permanent signals that allow later review, less hands-on technician time, easier identification of tumor heterogeneity using low-power magnification, and lower cost (conventional light microscope) [30–32]. An important question has been whether bright-field ISH is as sensitive as FISH. While 15–20 % of invasive breast cancers have HER2 amplification, among FISH-positive cases, nearly half (47 %) have only borderline or low levels of HER2 amplification [33].
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