Krishnamurti, Uma MD, PhD; Silverman, Jan F. MD
The human epidermal growth factor receptor 2 (HER2) is 1 of the 4 membrane receptor tyrosine kinases (RTKs). HER2 oncogene is a member of the human epidermal growth factor receptor family located on chromosome 17q12.1 It was first identified as a novel gene from rat neuroblastomas that transformed NIH 3T3 cells.2 King et al3 reported that DNA from human breast carcinoma had amplification of this gene. The sequence of the neu oncogene was homologous to the erb-B oncogene and its 185 kD phosphoprotein was antigenetically related to the epidermal growth factor receptor.4 The other members in the epidermal growth factor receptor family are: HER1 (EGFR), HER3 (erbB3), and HER4 (erbB4). All the 4 RTKs are transmembrane single subunit glycoproteins that have an extracellular ligand–binding domain, a transmembrane domain, and an intracellular tyrosine kinase catalytic domain. On ligand activation, the receptors dimerize forming homodimers or heterodimers. This is followed by transphosphorylation which activates several intracellular signaling pathways such as Ras/mitogen-activated protein kinase pathway, the phosphatidylinositol 3 kinase (PI3K)/Akt pathway, the Janus kinase/signal transducer and activator of transcription pathway, and the phospholipase C pathway, which ultimately affects cell proliferation, survival, motility, and adhesion.5 There is no known ligand for HER2 receptors to form homodimers. It can form heterodimers with other HER family members. The conformation of the extracellular region of HER2 without bound ligand is similar to the activated conformation of RTK with bound ligands.6
BIOLOGICAL SIGNIFICANCE OF HER2 IN BREAST CANCER
The overall frequency of HER2 overexpression/positivity in breast cancer in all of the earlier studies was around 22%, with a range of 9% to 74%.7 In current practice, HER2 positive rates are <20%, with most investigators currently reporting that the true-positive rate is in the range of 15% to 20%.7 The HER2-positive rate may be higher when metastatic lesions are tested, and tertiary hospitals and cancer centers report slightly higher rates than community hospitals and national reference laboratories.7,8 Breast cancers can have up to 25 to 50 copies of the HER2 gene and up to a 40- to 100-fold increase in HER2 protein expression.9,10HER2 gene amplification is responsible for HER2 overexpression in 90% of breast carcinomas.11
Prognostic Value of HER2
The value of HER2 as a prognostic factor is controversial. In a great majority of cases, abnormalities in HER2 expression at the gene or protein level have been associated with an adverse prognosis in both lymph node–negative and lymph node–positive breast cancer.7 Of >100 studies that looked at close to 40,000 patients, 88% of the studies determined that either HER2 gene amplification or HER2 protein overexpression predicted breast cancer outcome on either univariate or multivariate analysis.7 In 68 of the 93 studies (73%) that utilized multivariate analysis of outcome data, the adverse prognostic significance of HER2 gene, message, or protein overexpression was independent of all other prognostic variables. In only 13 of the studies (12%) there was no correlation between HER2 status and clinical outcome identified.7
In earlier studies, HER2 overexpression was demonstrated to be an independent adverse prognostic factor in node-positive patients,11–13 and the prognostic role of HER2 in node-negative patients was unclear. The majority of available data support the view that HER2 overexpression is associated with a poorer prognosis in node-positive and node-negative breast cancer.14–16
However, all these studies were performed before trials demonstrating the benefit of adjuvant trastuzumab therapy when added to other chemotherapeutic agents. Thus, although the weight of data suggests that a positive HER2 is a negative prognostic factor, its significance in clinical practice is doubtful as the outcomes are heavily influenced by therapy. The ASCO expert panel on tumor markers in breast cancer did not recommend the use of HER2 for determining prognosis.
Predictive Value of HER2
Therapy targeted against HER2 includes the monoclonal antibody trastuzumab (Herceptin), and newer drugs such as small molecule tyrosine kinase inhibitor lapatinib, as well as others such as pertuzumab and ertumaxomab.17 The initial HER2-targeting antibody was murine, and was directed against the extracellular domain of the receptor.18 Subsequently a humanized version was created by inserting the antigen-binding residues of the murine antibody into a cloned human immunoglobulin G (IgG) framework.19 Use of trastuzumab was FDA approved in 1998 for the treatment of metastatic disease. Exact mechanisms through which trastuzumab exert its effects are not completely clear, but is believed to include antibody-dependent cellular cytotoxicity, disruption of downstream signaling pathway, inhibition of cell cycle progression, and antiangiogenic effects.20 After extensive clinical testing it was first used in phase II studies as a single agent, and then in a landmark phase III trial it was used along with chemotherapy in a metastatic setting.21 Most response rates in clinical trials were approximately 35% with response rates varying from 12% to 68%.17,20 Responses were higher in patients who were 3+ by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) positive with negligible responses in patients who were 2+ by IHC or FISH negative. The median duration of response was prolonged by 2 to 4 months by the addition of trastuzumab therapy. Even in patients progressing on trastuzumab there was improvement in progression-free survival by 2 to 3 months.20 After the improvement in outcomes by use of trastuzumab in a metastatic setting it was tested as an adjunct to chemotherapy in the adjuvant setting. Interim outcomes were reported in 2005 from 4 large multicenter randomized trials: NSABP B-31, NCCTG N9831, HERA, and BCIRG 006.20 Predominantly node-positive patients and some patients who were node negative with tumors measuring at least 1 cm were included in these trials. Patients were randomized to 1 to 2 years of therapy. Interim analysis after only 1 year showed a statistical improvement in disease-free survival with a hazard ratio of 0.54.20 On median follow-up of about 2 years there was a significant 34% reduction in risk of death. Currently 1 year of adjuvant trastuzumab is the standard of care for HER2-positive tumors. The cost of 1 year of trastuzumab therapy ranges from 70,000 to 110,000 dollars.20
Trastuzumab toxicity: In the landmark phase III trial, trastuzumab plus anthracyclines dramatically increased the rate of cardiac dysfunction from 8% in the anthracycline-only group to 27% in the anthracycline plus trastuzumab group.21 Concurrent use of anthracyclines and trastuzumab is not advised apart from the neoadjuvant setting. After 5 years of follow-up, asymptomatic cardiac dysfunction is reported in 5% to 15% of patients and symptomatic cardiac dysfunction in 2% to 4%. There was a 4% incidence of class III/IV cardiac failure or cardiac death in patients who received trastuzumab in the B31 trial. Cardiac monitoring is imperative in patients on trastuzumab and patients should have left ventricular function monitored every 3 to 4 months. Besides cardiac toxicity, trastuzumab is well tolerated with only about 25% patients having first-time infusion reactions which generally did not recur in subsequent infusions.20
Despite its success in the treatment of early and advanced HER2-positive breast cancer, a proportion of patients who receive trastuzumab, and nearly all who receive it in a metastatic setting progress and relapse. Some of the proposed mechanisms of resistance are: inhibition of interaction of trastuzumab with its target by heterodimerization with other HER2 family members, or increased expression of membrane glycoprotein MUC4,22 or shedding of the extracellular domain leaving behind the truncated form of the receptor (p95) which retains kinase activity but cannot bind to trastuzumab.23
Non–HER2-targeted Therapy in HER2-positive Tumors
Tamoxifen: Overall disease-free survival and overall survival in patients receiving tamoxifen with or without chemotherapy is significantly lower in the hormone receptor–positive/HER2-positive group compared with the receptor-positive/HER2-negative group.24
Most studies show benefit of anthracycline-based chemotherapy in HER2-positive tumors,25,26 and also show that response rates for taxanes was significantly greater in HER2-positive tumors than in HER2-negative tumors.27,28
HER2 Status and Molecular Classification of Breast Cancers
Breast cancers are molecularly classified into 4 main subtypes based on gene expression profiling: Luminal A subtype are ER positive and/or PR positive and HER2 negative (not amplified) with low Ki-67 (<14%); Luminal B subtype are ER positive and/or PR positive and HER2 positive or if HER2 negative have high Ki-67 (>14%). These have a higher histologic grade than luminal A; HER2-amplified subtype are ER/PR negative, HER2 amplified, and more likely high grade, and Triple-negative subtype are ER, PR, and HER2 negative. A subset of the triple-negative breast cancers are basal like and these express cytokeration 5/6 and/or epidermal growth factor receptor.29,30 Luminal A and B subtypes respond to endocrine therapy, but the response in the luminal B is lower and luminal B shows greater response to chemotherapy. The HER2-amplified subtype responds to trastuzumab and anthracycline-based chemotherapy. The basal like show no response to endocrine or trastuzumab therapy, but seem to be sensitive to platinum-based chemotherapy, paclitaxel, and PARP inhibitors. The pathologic complete response rates in the luminal A and B subtypes is low (7% and 0%, respectively). It is much higher in the HER2-amplified and basal-like groups (45%).31
HER2 Expression and Histopathology of Breast Cancer
HER2 gene amplification occurs at a significantly lower rate in classic invasive lobular carcinoma (10%) compared with invasive ductal carcinoma, and is found almost exclusively in the pleomorphic variant of invasive lobular carcinoma which is linked to an adverse outcome.32 HER2 positivity is extremely rare in tubular carcinoma,33 and extremely low in mucinous (colloid) breast cancers, but occasionally associated with aggressive disease.34 Medullary breast carcinoma is HER2 negative,35 and so are most cases of inflammatory breast cancer.36 HER2 overexpression and HER2 amplification have been consistently noted in both mammary and extramammary Paget disease.37 Breast sarcomas and phyllodes tumors are HER2 negative.38HER2 amplification and HER2 overexpression have been associated with adverse outcome in some studies of male breast carcinoma,39 but not in others.40 HER2 positivity seems to be lower in male breast cancer than in female breast cancer.40,41 The incidence of HER2 positivity in ductal carcinoma in situ (DCIS) (24-38%) is higher than that for invasive breast cancer and has been associated with extensive, higher grades and DCIS with comedo-type necrosis.42 However, routine testing for HER2 status in DCIS is not widely performed. Hereditary breast cancer including cases that have either BRCA1 or BRCA2 germline mutations have shown a lower HER2 positivity rate.43 In addition, HER2 gene amplification and protein overexpression have been associated consistently with high tumor grade, high cell proliferation rate, and greater negativity for estrogen and progesterone receptors.44
HER2 Expression in Primary Versus Metastatic Breast Cancer
The majority of studies that have compared the HER2 status in paired primary and metastatic tumor tissues have found an overwhelming consistency in the patient’s status.45 However, recent studies have reported 20% to 30% discordance rates between the HER2 status of primary and metastatic lesions and suggest that there are changes in HER2 expression between primary and metastatic disease. This is particularly true after intervening HER2-directed therapy, but also occurs in the absence of such treatment.46,47
TESTING FOR HER2 IN BREAST CANCER
The 2007 ASCO guidelines recognized that HER2 is an important prognostic, predictive, and therapeutic marker in invasive breast cancer. Therefore, HER2 should be evaluated in every primary breast cancer either at the time of diagnosis or recurrence to guide therapy. It is also crucial to standardize testing techniques to accurately assess HER2 status. Currently HER2 testing is carried out by several methods: IHC, ELISA analysis of serum or tumor cytosol, and the Western blot test for HER2 protein overexpression. FISH, chromogenic in situ hybridization (CISH), silver in situ hybridization (SISH), Southern blot, and PCR are used to evaluate HER2 gene amplification.
Of all these tests, IHC and FISH are the most frequently used tests to evaluate the HER2 status in breast cancer48 and the majority (about 80%) of HER2 testing begins with screening by IHC.7
Immunohistochemistry for HER2
The assessment of HER2 status is quantitative rather than qualitative, because HER2 is expressed in all breast epithelial cells. To provide a meaningful interpretation of a HER2 immunostain, it was necessary to show a relationship between the number of HER2 receptors on a cell’s surface and the distribution and intensity of the immunostain. Using cell lines, it was possible to establish a standardized IHC procedure and scoring system in which cells containing <20,000 receptors show no staining (0), cells containing approximately 100,000 receptors show partial membrane staining with <10% of the cells showing complete membrane staining (1+), cells containing approximately 500,000 receptors show light to moderate complete membrane staining in >10% of the cells (2+), and cells containing approximately 2,300,000 receptors show strong, complete membrane staining in >10% of the cells (3+).7
Advantages of IHC testing are its wide availability, relatively low cost, easy preservation of stained slides, and use of routine microscopy. However, HER2 interpretation can be problematic. Comparisons of HER2 overexpression as measured by IHC at local laboratories versus HER2 amplification by FISH tested at a reference laboratory revealed low concordance rates (66% to 87%), whereas concordance rates between local and reference FISH results were higher (87% to 92%).48 However, the ability to accurately determine HER2 protein expression status by IHC can be significantly impacted by preanalytic, analytic, and postanalytic issues. The issues that affect reporting of the HER2 status by IHC are: the duration and type of tissue fixation, type of antibody (polyclonal vs. monoclonal), intensity of antigen retrieval, lack of a positive internal control signal, variability in system control samples, and last but not the least the difficulties in applying a semiquantitative subjective slide-scoring system.49–51 Studies have shown that when a standardized IHC assay is performed on specimens that are carefully fixed, processed, and embedded, there is good to excellent correlation between gene copy status and protein expression levels.49,52 It is on this basis that the ASCO/CAP guidelines were updated in 2007. The updated guidelines detailed the sources of HER2 testing variation, recommendations for testing and interpretation of both IHC and FISH, sample exclusion criteria to perform and interpret a HER2 IHC and FISH assay, IHC and FISH interpretation criteria, and the reporting elements for IHC and FISH.53 We present below a few key elements of the ASCO guidelines.
For HER2 testing by IHC, 30 anti-HER2 antibodies (7 polyclonal and 21 monoclonal) have been used in IHC assays. The 2 FDA-approved IHC-based tests for testing for HER2 overexpression are the Dako Hercep test which uses a polyclonal antibody (A085) and the Pathway (Ventana) which uses a monoclonal antibody (4B5). According to the recently issued 2013 ASCO/CAP guidelines,54 the scoring method for HER2 IHC is semiquantitative and is based on 4 classes (0/1+, 2+, 3+) which is as follows: score 0 (negative): no staining is observed in invasive tumor cells; score 1+ (negative): weak, incomplete, membrane staining in any proportion of invasive tumor cells, or weak, complete membrane staining in <10% of invasive tumor cells; score 2+ (equivocal): circumferential membrane staining that is incomplete and/or weak/moderate and in >10% of the invasive tumor cells; or complete and circumferential membrane staining that is intense and in ≤10% of the invasive tumor cells; score 3+ (positive): circumferential membrane staining that is complete and intense in a homogenous and contiguous population and present in >10% of invasive tumor cells that is readily appreciated using a low-power objective. By IHC, only a score of 3+ is reported as positive for HER2 amplification. All 2+ equivocal cases have to undergo subsequent testing by FISH (Figs. 1, 2). In the latest 2013 guidelines, the cutoff for HER2 3+ (positive) staining has changed to complete intense membrane staining in >10% invasive tumor cells from complete membrane staining in >30% invasive tumor cells in the 2007 guidelines. In 2007, the cutoff for HER2 3+ (positive) staining was changed from the prior cutoff of complete membrane staining in >10% invasive tumor cells to complete membrane staining in >30% invasive tumor cells.
There should be laboratory documentation of the following: proof of initial testing validation in which positive and negative HER2 categories are 95% concordant with alternative or same validated method for HER2, ongoing quality assurance procedures, participation in external proficiency testing, and current accreditation by a valid accrediting agency. Specimen fixation for <6 hours or >72 hours is not recommended. The test is rejected or repeated if controls are not as expected, if artifacts involve most of the sample, or if the sample has strong membrane staining of normal breast ducts. Slide scoring can be improved by avoiding overinterpretation of specimen edges, retraction artifacts, underfixation or overfixation artifacts, cytoplasmic tumor cell staining, and membranous tumor cell staining that lacks a complete circumferential staining pattern. The use of a quantitative image analysis system can substantially reduce slide scoring variability among pathologists, especially in the 2+ cases.55,56 In the United Kingdom, it has been recommended that HER2 IHC testing be restricted to laboratories performing an annual minimum of 250 IHC tests.57
Fluorescence In Situ Hybridization
FISH is a more reliable, sensitive, and accurate procedure and is less affected by preanalytic and analytic variables than IHC. However, it requires special equipment, special-trained personnel, and is more tedious, expensive, and time consuming. Three types of FISH assays are FDA approved: PathVysion by Abbott (Abbott Laboratories, Abbott Park, IL) and the Dako PharmDx FISH test (Dako Corporation) that use dual-probes, one for HER2 and one for the centromere (CEP17), and the Ventana Inform (Ventana Medical Systems) that uses a single-probe only for HER2.
For FISH, according to the 2013 ASCO/CAP guidelines,54 amplification for HER2 is: Dual-probe HER2/CEP17 ratio ≥2.0 with an average HER2 copy number ≥4.0 signals/cell or dual-probe HER2/CEP17 ratio ≥2.0 with an average HER2 copy number <4.0 signals/cell or dual-probe HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥6.0 signals/cell or single-probe average HER2 copy number ≥6 copies/cell; ISH equivocal is: Dual-probe HER2/CEP17 ratio <2.0 with an average HER2 copy number ≥4.0 and <6.0 signals/cell or single-probe ISH average HER2 copy number ≥4.0 and <6 signals/cell and negative for HER2 amplification is: Dual-probe HER2/CEP17 ratio <2.0 with an average HER2 copy number <4.0 signals/cell or average HER2 gene copy number of <4.0 signals/cell for single-probe test systems (Fig. 3).
These guidelines are different from the 2007 guidelines for testing systems using a dual probe. Previously HER2/CEP17 ratio >2.2 was required for an ISH-positive result. A case is defined as having centromere 17 copy number increase or chromosome 17 “polysomy” if there is ≥3 CEP17 copies/cell. These cases may or may not have HER2 amplification.
Newer Modalities of HER2 Testing
Chromogenic In Situ Hybridization
FDA approved the SPOT-Light HER2 CISH assay (Invitrogen, CA) in 2008. CISH combines the advantages of IHC (lower cost, routine microscopy, preservation of morphologic features, permanent signals which will not fade with slide storage, and need for less expertise) with those of FISH (more reliable). CISH uses a single HER2 probe. There is a very high concordance rate of 97% to 99% between CISH and FISH.58–60
Silver In Situ Hybridization
SISH is based upon the deposition of silver at the target site after an enzymatic reaction. It uses both HER2 and CEP17 probes that are hybridized on separate slides. SISH also shows a high concordance rate with FISH.61,62 Bright-field double in situ hybridization is a newer automated technique where dual probes targeting HER2 and the centromere are used and there is simultaneous evaluation of morphologic features by a bright-field microscope. This has also been demonstrated to have high concordance rates with FISH.63 PCR and RT-PCR are other methods to assess HER2 status. RT-PCR is used to assess HER2 m-RNA levels in the 21-gene Oncotype DX assay and the 70-gene Mammaprint assay. Dimerization assays that quantify number of HER2 homodimers have predicted potential resistance to trastuzumab. This in combination with HER2 receptor number has been commercialized (HERmarkTM; Monogram Biosciences, CA).7
Next Generation Sequencing
In the last decade of the targeted therapy, there has been a great interest in evaluating the cancer cell gene sequence to predict response to therapy. There is continuous demand for more rapid and low-cost sequencing in place of the traditional Sanger sequencing which is slow and expensive. There are recent thorough in-depth sequencing technologies referred to as next-generation sequencing (NGS) or massively parallel sequencing that have been developed to meet the demand of the massive genetic information required for targeted anticancer therapy. Some of the commercial NGS platforms are: Illumina, 454 pyrosequencing, Helicos heliscope, Supported oligonucleotide ligation and detection (SOLid), and the Ion torrent sequencing.64 Some platforms are better for wide genome scans, and others, sequence targeted genomic regions. NGS identifies base substitutions, deletions, and insertions across entire exons of hundreds of cancer-related genes. It can also identify gene copy number changes such as HER2 amplification. Lipson and colleagues tested 35 formalin-fixed paraffin-embedded invasive breast carcinomas previously tested by FISH for HER2 status. They found that HER2 status determined by NGS demonstrated 97% accuracy relative to the HER2 status determined by FISH.65 In addition, NGS can identify HER2 mutations in cancers lacking HER2 amplification. Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification, of which the majority of the mutations were activating mutations.66 The 25 patients with HER2 mutation-positive breast cancer were identified from a total of 1499 patients, suggesting an overall HER2 mutation rate of approximately 1.6%. The mutations were found to be clustered in 2 major areas, with 20% (5/25) mutations in the extracellular domain and 68% (17/25) mutations in the kinase domain. The HER2 mutation frequency in relapsed or metastatic breast cancers is currently unknown and could be >1.6%. Neratinib, an irreversible HER2 inhibitor, was found to be a very potent inhibitor for all of the HER2 mutations, whereas lapatinib was less potent. The effect of trastuzumab and pertuzumab could not be tested in entirety in the Matrigel-based model that was used for testing response to anti-HER2 drugs.66
Although several newer modalities exist to evaluate the HER2 status in breast cancer, they are investigational and their efficacy looking at response to HER2-targeted therapies has not been validated in a prospective clinical trial.7,67
Testing by IHC and FISH are the recommended methods by ASCO/CAP.
EFFECT OF CENTROMERE 17 COPY NUMBER ALTERATION
Besides HER2 amplification, a subset of breast cancers have increased copy numbers of the chromosome 17 centromere, commonly referred to as chromosome 17 polysomy. Cases with chromosome 17 polysomy may or may not be associated with HER2 gene amplification. By ASCO/CAP 2007 guidelines, a case is defined as having centromere 17 copy number increase or chromosome 17 “polysomy” without HER2 amplification if there is ≥3 CEP17 copies/cell with a HER2/CEP17 ratio <1.8. In the literature, the reported incidence of chromosome 17 polysomy in breast carcinoma ranges from 5% to 50%.68–70 However, recent studies such as array-based comparative hybridization studies, extended FISH, and multiplex ligation-dependent probe amplification analyses have demonstrated that true polysomy of chromosome 17 is a very rare event in breast cancer and that increased copy numbers of CEP17 as demonstrated by CEP17 FISH or CISH are often due to CEP17 duplication or amplification or due to gains or amplification in the pericentromeric region of chromosome 17.71–74 Although HER2 overexpression is an established adverse prognostic factor in breast cancer, there is a paucity of literature on the correlation of increased centromere 17 numbers/chromosome 17 polysomy with important prognostic and predictive pathologic factors in invasive breast carcinoma. There are conflicting results with some studies showing greater adverse parameters in polysomic cases, whereas others do not.69,70,75–78 Currently, patients with polysomy 17 without HER2 amplification are treated similar to patients who show neither amplification or polysomy. Although the use of CEP17 to correct HER2 copy numbers might not be ideal and the use of HER2:CEP17 ratio to define HER2 positivity has been questioned, it is also believed that in some cases this might provide misleading HER2 gene status results.73,74,79 If the absolute number of HER2 copies are >6/cell nucleus, there is no plausible explanation why extra CEP17 copies would block the effect of extra HER2 copies and there is no reason to assume that patients with >6 HER2 copies/cell, but with HER2/CEP17 ratio <1.8 should not respond to trastuzumab. It has been proposed that in cases tested by dual-color FISH, clinical decision regarding therapy should be made and trials testing efficacy of anti-HER2 agents should select patients not only on the basis of HER2/CEP17 ratios, but also on absolute HER2 gene copy numbers. In addition, although most patients with polysomy 17 without HER2 amplification have a 2+ equivocal expression of HER2 protein on IHC, some patients have a 3+ positive score on IHC.69,76 Hofmann and colleagues reported 4 patients with polysomy 17 without HER2 amplification and 3+ HER2 IHC expression, with 2 of these 4 patients responding to treatment with trastuzumab. The authors in that study concluded that chromosome 17 polysomy seemed to be a major cause of clinical responses to trastuzumab in FISH-negative cases and they propose that patients who are HER2 IHC 3+ positive but FISH-negative should be first retested by IHC, and patients with 3+ positive IHC results should be considered for trastuzumab therapy.80 However, the updated 2013 ASCO/CAP guidelines54 will help to accurately classify patients with increased chromosome 17 copy numbers. It will also be interesting to see the results of the ongoing NSABP B47 study that is testing the efficacy of anti-HER2 therapy on node-negative HER2 low patients, as many polysomic patients will be included in this group.
The HER2 oncogene located on chromosome 17 codes for HER2 which is a RTK. The HER2 protein is overexpressed in a subset of breast cancers and HER2 gene amplification is the most common pathway by which the protein is overexpressed. Although HER2-positive breast cancers are more aggressive, targeted therapy has improved the prognosis in these patients. The pathologist has a crucial role in evaluating for overexpression of HER2 in breast carcinoma and therefore directs disease treatment. Adherence to the guidelines established by ASCO/CAP will help in accurately classifying patients for therapy.
1. Popescu NC, King CR, Kraus MH .Localization of the human erbB-2 gene on normal and rearranged chromosomes 17 to bands q12-21.32.Genomics. 1989; 4:362–366.
2. Shih C, Padhy LC, Murray M, et al .Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts.Nature. 1981; 290:261–264.
3. King CR, Kraus MH, Aaronson SA .Amplification of a novel v-erbB-related gene in a human mammary carcinoma.Science. 1985; 229:974–976.
4. Akiyama T, Sudo C, Ogawara H, et al .The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity.Science. 1986; 232:1644–1646.
5. Moasser MM .The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis.Oncogene. 2007; 26:6469–6487.
6. Cho HS, Mason K, Ramyar KX, et al .Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.Nature. 2003; 421:756–760.
7. Ross JS, Slodkowska EA, Symmans WF, et al .The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine.Oncologist. 2009; 14:320–368.
8. Wolff AC, Hammond ME, Schwartz JN, et al .American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer.J Clin Oncol. 2007; 25:118–145.
9. Gutierrez C, Schiff R .HER2: biology, detection, and clinical implications.Arch Pathol Lab Med. 2011; 135:55–62.
10. Kallioniemi OP, Kallioniemi A, Kurisu W, et al .ERBB2 amplification in breast cancer analyzed by fluorescence in situ hybridization.Proc Natl Acad Sci U S A. 1992; 89:5321–5325.
11. Slamon DJ, Clark GM, Wong SG, et al .Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science. 1987; 235:177–182.
12. Borg A, Tandon AK, Sigurdsson H, et al .HER-2/neu amplification predicts poor survival in node-positive breast cancer.Cancer Res. 1990; 50:4332–4337.
13. Hartmann LC, Ingle JN, Wold LE, et al .Prognostic value of c-erbB2 overexpression in axillary lymph node positive breast cancer. Results from a randomized adjuvant treatment protocol.Cancer. 1994; 74:2956–2963.
14. Press MF, Pike MC, Chazin VR, et al .Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease.Cancer Res. 1993; 53:4960–4970.
15. Ro JS, el-Naggar A, Ro JY, et al .c-erbB-2 amplification in node-negative human breast cancer.Cancer Res. 1989; 49:6941–6944.
16. Andrulis IL, Bull SB, Blackstein ME, et al .neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group.J Clin Oncol. 1998; 16:1340–1349.
17. Shah S, Chen B .Testing for HER2 in breast cancer: a continuing evolution.Pathol Res Int. 2011; 2011:1–16.
18. Fendly BM, Winget M, Hudziak RM, et al .Characterization of murine monoclonal antibodies reactive to either the human epidermal growth factor receptor or HER2/neu gene product.Cancer Res. 1990; 50:1550–1558.
19. Carter P, Presta L, Gorman CM, et al .Humanization of an anti-p185HER2 antibody for human cancer therapy.Proc Natl Acad Sci U S A. 1992; 89:4285–4289.
20. Murphy CG, Modi S .HER2 breast cancer therapies: a review.Biologics. 2009; 3:289–301.
21. Slamon DJ, Leyland-Jones B, Shak S, et al .Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.N Engl J Med. 2001; 344:783–792.
22. Nahta R, Esteva FJ .HER2 therapy: molecular mechanisms of trastuzumab resistance.Breast Cancer Res. 2006; 8:215–222.
23. Scaltriti M, Rojo F, Ocana A, et al .Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer.J Natl Cancer Inst. 2007; 99:628–638.
24. Carlomagno C, Perrone F, Gallo C, et al .c-erb B2 overexpression decreases the benefit of adjuvant tamoxifen in early-stage breast cancer without axillary lymph node metastases.J Clin Oncol. 1996; 14:2702–2708.
25. Muss HB, Thor AD, Berry DA, et al .c-erbB-2 expression and response to adjuvant therapy in women with node-positive early breast cancer.N Engl J Med. 1994; 330:1260–1266.
26. Thor AD, Berry DA, Budman DR, et al .erbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer.J Natl Cancer Inst. 1998; 90:1346–1360.
27. Harris LN, Broadwater G, Lin NU, et al .Molecular subtypes of breast cancer in relation to paclitaxel response and outcomes in women with metastatic disease: results from CALGB 9342.Breast Cancer Res. 2006; 8:R66–R77.
28. Hayes DF, Thor AD, Dressler LG, et al .HER2 and response to paclitaxel in node-positive breast cancer.N Engl J Med. 2007; 357:1496–1506.
29. Badve S, Dabbs DJ, Schnitt SJ, et al .Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists.Mod Pathol. 2011; 24:157–167.
30. Schnitt SJ .Classification and prognosis of invasive breast cancer: from morphology to molecular taxonomy.Mod Pathol. 2010; 23:suppl 2 S60–S64.
31. Rouzier R, Perou CM, Symmans WF, et al .Breast cancer molecular subtypes respond differently to preoperative chemotherapy.Clin Cancer Res. 2005; 11:5678–5685.
32. Simpson PT, Reis-Filho JS, Lambros MB, et al .Molecular profiling pleomorphic lobular carcinomas of the breast: evidence for a common molecular genetic pathway with classic lobular carcinomas.J Pathol. 2008; 215:231–244.
33. Oakley GJ III, Tubbs RR, Crowe J, et al .HER-2 amplification in tubular carcinoma of the breast.Am J Clin Pathol. 2006; 126:55–58.
34. Adair JD, Harvey KP, Mahmood A, et al .Recurrent pure mucinous carcinoma of the breast with mediastinal great vessel invasion: HER-2/neu confers aggressiveness.Am Surg. 2008; 74:113–116.
35. Jacquemier J, Padovani L, Rabayrol L, et al .Typical medullary breast carcinomas have a basal/myoepithelial phenotype.J Pathol. 2005; 207:260–268.
36. Kleer CG, van Golen KL, Braun T, et al .Persistent E-cadherin expression in inflammatory breast cancer.Mod Pathol. 2001; 14:458–464.
37. Wolber RA, Dupuis BA, Wick MR .Expression of c-erbB-2 oncoprotein in mammary and extramammary Paget’s disease.Am J Clin Pathol. 1991; 96:243–247.
38. Yonemori K, Hasegawa T, Shimizu C, et al .Correlation of p53 and MIB-1 expression with both the systemic recurrence and survival in cases of phyllodes tumors of the breast.Pathol Res Pract. 2006; 202:705–712.
39. Joshi MG, Lee AK, Loda M, et al .Male breast carcinoma: an evaluation of prognostic factors contributing to a poorer outcome.Cancer. 1996; 77:490–498.
40. Bloom KJ, Govil H, Gattuso P, et al .Status of HER-2 in male and female breast carcinoma.Am J Surg. 2001; 182:389–392.
41. Willmore C, Holden JA, Layfield LJ .Correlation of HER2 gene amplification with immunohistochemistry in breast cancer as determined by a novel monoplex polymerase chain reaction assay.Appl Immunohistochem Mol Morphol. 2005; 13:333–341.
42. Moreno A, Lloveras B, Figueras A, et al .Ductal carcinoma in situ of the breast: correlation between histologic classifications and biologic markers.Mod Pathol. 1997; 10:1088–1092.
43. Palacios J, Robles-Frias MJ, Castilla MA, et al .The molecular pathology of hereditary breast cancer.Pathobiology. 2008; 75:85–94.
44. Yarden Y .Biology of HER2 and its importance in breast cancer.Oncology. 2001; 61:suppl 2 1–13.
45. Vincent-Salomon A, Jouve M, Genin P, et al .HER2 status in patients with breast carcinoma is not modified selectively by preoperative chemotherapy and is stable during the metastatic process.Cancer. 2002; 94:2169–2173.
46. Tapia C, Savic S, Wagner U, et al .HER2 gene status in primary breast cancers and matched distant metastases.Breast Cancer Res. 2007; 9:R31–R38.
47. Lower EE, Glass E, Blau R, et al .HER-2/neu expression in primary and metastatic breast cancer.Breast Cancer Res Treat. 2009; 113:301–306.
48. Carlson RW, Moench SJ, Hammond ME, et al .HER2 testing in breast cancer: NCCN Task Force report and recommendations.J Natl Compr Canc Netw. 2006; 4:suppl 3 S1–22.
49. Cuadros M, Villegas R .Systematic review of HER2 breast cancer testing.Appl Immunohistochem Mol Morphol. 2009; 17:1–7.
50. Paik S, Bryant J, Tan-Chiu E, et al .Real-world performance of HER2 testing—National Surgical Adjuvant Breast and Bowel Project experience.J Natl Cancer Inst. 2002; 94:852–854.
51. Press MF, Hung G, Godolphin W, et al .Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression.Cancer Res. 1994; 54:2771–2777.
52. Hayes DF, Thor AD .c-erbB-2 in breast cancer: development of a clinically useful marker.Semin Oncol. 2002; 29:231–245.
53. Wolff AC, Hammond ME, Schwartz JN, et al .American Society of Clinical Oncology/College of American Pathologists Guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer.Arch Pathol Lab Med. 2007; 131:18–43.
54. Wolff AC, Hammond ME, Hicks DG, et al .Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update.J Clin Oncol. 2013; 31:3997–4013.
55. Hicks DG, Kulkarni S .HER2+ breast cancer: review of biologic relevance and optimal use of diagnostic tools.Am J Clin Pathol. 2008; 129:263–273.
56. Wang S, Saboorian MH, Frenkel EP, et al .Assessment of HER-2/neu status in breast cancer. Automated Cellular Imaging System (ACIS)-assisted quantitation of immunohistochemical assay achieves high accuracy in comparison with fluorescence in situ hybridization assay as the standard.Am J Clin Pathol. 2001; 116:495–503.
57. Dowsett M, Hanby AM, Laing R, et al .HER2 testing in the UK: consensus from a national consultation.J Clin Pathol. 2007; 60:685–689.
58. Hanna WM, Kwok K .Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm.Mod Pathol. 2006; 19:481–487.
59. Di Palma S, Collins N, Faulkes C, et al .Chromogenic in situ hybridisation (CISH) should be an accepted method in the routine diagnostic evaluation of HER2 status in breast cancer.J Clin Pathol. 2007; 60:1067–1068.
60. Zhao J, Wu R, Au A, et al .Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in archival breast carcinoma.Mod Pathol. 2002; 15:657–665.
61. Dietel M, Ellis IO, Hofler H, et al .Comparison of automated silver enhanced in situ hybridisation (SISH) and fluorescence ISH (FISH) for the validation of HER2 gene status in breast carcinoma according to the guidelines of the American Society of Clinical Oncology and the College of American Pathologists.Virchows Arch. 2007; 451:19–25.
62. Papouchado BG, Myles J, Lloyd RV, et al .Silver in situ hybridization (SISH) for determination of HER2 gene status in breast carcinoma: comparison with FISH and assessment of interobserver reproducibility.Am J Surg Pathol. 2010; 34:767–776.
63. Nitta H, Hauss-Wegrzyniak B, Lehrkamp M, et al .Development of automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence in situ hybridization (FISH).Diagn Pathol. 2008; 3:41–52.
64. Ross JS, Cronin M .Whole cancer genome sequencing by next-generation methods.Am J Clin Pathol. 2011; 136:527–539.
65. Lipson D, He J, Yelensky R, et al .Next-generation sequencing of FFPE breast cancers demonstrates high concordance with FISH in calling HER2 amplifications and commonly detects clinically relevant genomic alterations (poster abstract).Cancer Res. 2012; 72:suppl 3
Doi: 10.1158/0008-5472. SABCS12-PD02-07
66. Bose R, Kavuri SM, Searleman AC, et al .Activating HER2 mutations in HER2 gene amplification negative breast cancer.Cancer Discov. 2013; 3:224–237.
67. Bhargava R, Dabbs DJ .Oncotype DX test on unequivocally HER2-positive cases: potential for harm.J Clin Oncol. 2012; 30:570–571.
68. Hyun CL, Lee HE, Kim KS, et al .The effect of chromosome 17 polysomy on HER-2/neu status in breast cancer.J Clin Pathol. 2008; 61:317–321.
69. Krishnamurti U, Hammers JL, Atem FD, et al .Poor prognostic significance of unamplified chromosome 17 polysomy in invasive breast carcinoma.Mod Pathol. 2009; 22:1044–1048.
70. Torrisi R, Rotmensz N, Bagnardi V, et al .HER2 status in early breast cancer: relevance of cell staining patterns, gene amplification and polysomy 17.Eur J Cancer. 2007; 43:2339–2344.
71. Staaf J, Jonsson G, Ringner M, et al .High-resolution genomic and expression analyses of copy number alterations in HER2-amplified breast cancer.Breast Cancer Res. 2010; 12:R25–R42.
72. Yeh IT, Martin MA, Robetorye RS, et al .Clinical validation of an array CGH test for HER2 status in breast cancer reveals that polysomy 17 is a rare event.Mod Pathol. 2009; 22:1169–1175.
73. Marchio C, Lambros MB, Gugliotta P, et al .Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis.J Pathol. 2009; 219:16–24.
74. Moelans CB, de Weger RA, van Diest PJ .Absence of chromosome 17 polysomy in breast cancer: analysis by CEP17 chromogenic in situ hybridization and multiplex ligation-dependent probe amplification.Breast Cancer Res Treat. 2010; 120:1–7.
75. Downs-Kelly E, Yoder BJ, Stoler M, et al .The influence of polysomy 17 on HER2 gene and protein expression in adenocarcinoma of the breast: a fluorescent in situ hybridization, immunohistochemical, and isotopic mRNA in situ hybridization study.Am J Surg Pathol. 2005; 29:1221–1227.
76. Petroni S, Addati T, Mattioli E, et al .Centromere 17 copy number alteration: negative prognostic factor in invasive breast cancer? Arch Pathol Lab Med. 2012; 136:993–1000.
77. Watters AD, Going JJ, Cooke TG, et al .Chromosome 17 aneusomy is associated with poor prognostic factors in invasive breast carcinoma.Breast Cancer Res Treat. 2003; 77:109–114.
78. Krishnamurti U, Zarineh A, Atem FD, et al .Correlation of immunohistochemical expression of p53 with unamplified chromosome 17 polysomy in invasive breast carcinoma.Appl Immunohistochem Mol Morphol. 2011; 19:28–32.
79. Viale G .Be precise! The need to consider the mechanisms for CEP17 copy number changes in breast cancer.J Pathol. 2009; 219:1–2.
80. Hofmann M, Stoss O, Gaiser T, et al .Central HER2 IHC and FISH analysis in a trastuzumab (Herceptin) phase II monotherapy study: assessment of test sensitivity and impact of chromosome 17 polysomy.J Clin Pathol. 2008; 61:89–94.