Review

Current HER2 Testing Recommendations and Clinical Relevance as a Predictor of Response to Targeted Therapy Tarah J. Ballinger,1 Melinda E. Sanders,2 Vandana G. Abramson1,3 Abstract Clinical decision-making in the treatment of breast cancer depends on an accurate determination and understanding of human epidermal growth factor receptor 2 (HER2) status. The guidelines for HER2 testing were recently updated in late 2013, but limitations continue to exist in the interpretation and clinical application of results when the tumor specimens do not fall neatly into positive or negative categories with immunohistochemistry and fluorescence in situ hybridization testing. The issues, including discordance between pathologists or laboratories, polysomy, and genetic heterogeneity, present challenging situations that are difficult to translate into clinical significance. The present review discussed the changes in the updated American Society of Clinical Oncology/College of American Pathologists guidelines, the clinical relevance of complex issues in HER2 testing, and the implications of the results on the response to HER2-targeted therapies. Great advances have been made in the treatment of HER2-positive breast cancer; however, the challenge remains to determine the best testing analysis that will identify patients who will benefit the most from these therapies. Clinical Breast Cancer, Vol. -, No. -, --- ª 2014 Elsevier Inc. All rights reserved. Keywords: Breast cancer, Fluorescence in situ hybridization, Human epidermal growth factor receptor 2, Immunohistochemistry, Trastuzumab

Introduction Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor tyrosine kinase encoded by a region of chromosome 17 that results in the increased activity of the cellular processes responsible for tumor growth and progression when activated.1-3 HER2 is overexpressed and/or amplified in 15% to 20% of invasive breast cancer cases and is associated with an aggressive phenotype and a poor prognosis in patients who do not receive systemic HER2-directed therapy.4-8 Breast cancers can have
50 copies of the HER2 gene and a 40- to 100-fold increase in HER2 protein expression. HER2 gene amplification is the primary mechanism responsible for the overexpression of the HER2 protein in approximately 90% of HER2-positive (HER2þ) breast cancer cases.9,10 Trastuzumab is a humanized monoclonal antibody targeting HER2 that has been proved to significantly improve 1

Department of Medicine Department of Pathology, Microbiology, and Immunology 3 Breast Cancer Program, Vanderbilt-Ingram Cancer Center Vanderbilt University School of Medicine, Nashville, TN 2

Submitted: Aug 18, 2014; Revised: Nov 25, 2014; Accepted: Nov 25, 2014 Address for correspondence: Vandana G. Abramson, MD, Breast Cancer Program and Department of Hematology and Oncology, Vanderbilt-Ingram Cancer Center, 2220 Pierce Avenue, 777 PRB, Nashville, TN 37232 E-mail contact: [email protected]

1526-8209/$ - see frontmatter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clbc.2014.11.009

the disease-free and overall survival of women with tumors overexpressing HER2 in both early and metastatic breast cancer.11-14 Given the significance of HER2 status for prognosis and clinical decision-making, accurate and efficient modes of HER2 testing are necessary. Currently, the vast majority of HER2 testing is done using fluorescence in situ hybridization (FISH) to directly quantify the HER2 gene copy number or by first screening with immunohistochemistry (IHC), with confirmatory FISH for cases with equivocal results (grade 2þ expression). IHC evaluates the expression of HER2 at the cell surface using membranous staining, and FISH assesses the number of copies of the HER2 gene in the nucleus. Testing for HER2 amplification or overexpression is recommended for all primary and metastatic breast cancers to guide appropriate treatment decisions.15 Although the decision to use HER2-targeted therapies will be either “yes” or “no,” the results using current testing methods are not as black and white. The continuous spectrum of results creates a gray area that complicates the clinically imperative decision to use HER2-directed adjuvant and neoadjuvant therapy. The American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) released new guidelines in late 2013 to improve the accuracy and clinical utility of HER2 testing.15 The present review has detailed the new features of the updated testing guidelines and the continued shortcomings of IHC and FISH testing and examined the

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Current HER2 Testing Recommendations importance of accurate results as they pertain to predicting patients’ responses to HER2-targeted therapy.

Immunohistochemistry IHC is performed on formalin-fixed breast tumor tissue to detect the amount of HER2 protein at the cell membrane (Figure 1). The results are reported using a 4-tier scoring system: 0, the absence of membranous staining; 1þ, faint, partial membranous staining in > 10% of cancer cells, with rare or absent cells having circumferential staining; 2þ, weak, circumferential membranous staining in > 10% of cells; and 3þ, intense circumferential membranous staining in > 10% of cells. Absent or weak staining cells (0 or 1þ, respectively) equate to < 100,000 receptors and no detectable amplification of the HER2 gene. Strong, circumferential staining (3þ) correlates with > 2,300,000 receptors and a level of expression almost always associated with HER2 amplification.16 A positive result (3þ) has been newly defined by ASCO as > 10% of invasive tumor cells with complete and intense membrane staining. This reverts to the recommendation before 2007, after which the cutoff was increased to > 30% complete and intense staining (see the comparisons listed in Table 1).4 Equivocal results are scored as 2þ, with nonintense, but complete, membranous staining of > 10% of cells. The frequency of equivocal IHC results has been consistent across many studies, reported in about 15% to 20% of cases, regardless of whether the positive cutoff was 10% or 30%.17,18 It has been recommended that patients with such equivocal results undergo additional confirmatory testing using an alternate method, such as FISH. The false-negative rate of IHC has been

estimated at < 1% to 11% across various studies, and reflex FISH testing is not currently recommended for IHC-negative (IHC) tumors.19 However, concern exists surrounding the number of IHC tumors with positive HER2 amplification that would not undergo testing by FISH according to the guidelines. Therefore, some patients might not by considered for potentially lifesaving anti-HER2 therapy.20,21 The advantages of IHC testing include its wide availability, relatively low cost, simple use of routine microscopy, and an easier preservation of slides. However, difficulties with variable fixation and the preparation of tumor sections have been shown to create incorrect results, including false-positive results secondary to overstaining.22

Fluorescence In Situ Hybridization The widely accepted reference standard for determining HER2 positivity is FISH to detect HER2 gene amplification. Overexpression of the HER2 protein at the cell surface occurs in the absence of gene amplification in < 5% of cases.9,10,23 FISH can be performed using a dual- or single-probe technique on formalinfixed, paraffin-embedded tissue specimens (Figure 2). Dual-probe assays use fluorescent-labeled DNA probes to detect the relative copy number of HER2 gene signals per cell compared with the number of chromosome enumeration probe 17 (CEP17) gene signals. CEP17 is the centromere region of chromosome 17, the same chromosome on which HER2 is located and, therefore, serves as a control.24 Single-probe assays use only a HER2 gene probe, giving a direct copy number, rather than a ratio. Previously, the 2007 ASCO/CAP guidelines considered a specimen to be positive

Figure 1 Breast Cancer Tissue Staining by Immunohistochemistry (ICH). (A) IHC 0, Negative, Absence of Membrane Staining; (B) IHC 1D, Negative, Membranous Staining Is Weak and Incomplete; (C) IHC 2D, Equivocal, Complete Membranous Staining That Is Not Intense and Not Demonstrated in All Cells; and (D) IHC 3D, Positive, Intense, Circumferential, Membranous Staining

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Tarah J. Ballinger et al Table 1 Comparison of 2007 and 2013 ASCO/CAP Guidelines for HER2 Testing Results in Breast Cancer Variable HER2þ

HER2 equivocal

HER2

2007 Recommendations IHC 3þ (>30% of invasive tumor cells with intense membrane staining) or FISH positive HER2 gene copy >6 per nucleus by single probe or HER2/CEP17 ratio >2.2 IHC 2þ or FISH equivocal HER2 gene copy 4-6 per nucleus by single probe or HER2/CEP17 ratio of 1.8-2.2

IHC 0 (no staining) or IHC 1þ (any cells with weak and incomplete staining or 10% of invasive tumor cells with incomplete membrane staining or 6 will classify them as amplified regardless of the HER2/CEP17 ratio. Although not yet included in the ASCO/CAP guidelines, some investigators have recommended the use of additional probes, including SMS (17p11.2), RARA (17q21.2), TOP2 (17q21.3-22), and p53 (17p13.1), to better elucidate the presence and size of the HER2 amplicons, including the centromere.24,45,48

present diffusely, in clusters, mixed with nonamplified cells, or in a metastatic site (Figure 3).49 The prevalence of HER2 heterogeneity has varied from 5% to 40% in different reports.50-52 The round robin study by Perez et al21 of 3, large adjuvant trials (N9831, BCIRG-006, and BCIRG-005) offered heterogeneity as a plausible explanation for the discordant results between local and central HER2 testing. They noted that 22 tumors in the N9831 study were HER2þ locally but negative centrally; however, 5 of these were actually positive centrally on retrospective reassessment of a different tumor block.21 This suggests that even patients with nonequivocal tumors could benefit from having supplementary portions of the tumor tested, especially if HER2; however, whether this would be effective in practice remains to be studied. The guidelines issued in 2009 by CAP defined heterogeneity as a HER2/CEP17 ratio > 2.2, or copy number > 6, in 5% to 50% of tumor cells.50 The UK guidelines called for scanning the entire tumor section, counting the number of CEP17 and HER2 signals in 20 to 60 cells per field, and calculating the mean ratio, with HER2/CEP17 > 2.0 considered positive. The relevance of heterogeneity has continued to be debated and rests on whether it affects the final HER2 results, patient outcome, or response to HER2-targeted therapies. In a very important audit of cases from the adjuvant Tamoxifen versus Exemestane study, Bartlett et al51 found that 45% of tumors exhibited heterogeneity (33.5%) or amplification (11.7%) using the CAP definition, tripling the number of tumors considered amplified using the UK definition. Heterogeneity using the CAP definition of 5% to 50% had a negligible effect on the prognosis. Only patients with > 30% HER2-amplified cells showed a decrease in DFS compared with patients with no amplification. In the tumors with 30% to 50% amplified cells, DFS was decreased only in the presence of an identifiable amplified clone, but the difference was not statistically significant. Therefore, the investigators suggested that the proposed CAP definition using the percentage of amplified cells is not a valid definition of heterogeneity and usually represents difficulties in

Figure 3 Example of Fluorescence In Situ Hybridization Human Epidermal Growth Factor Receptor 2-Negative Breast Carcinoma Demonstrating Genetic Heterogeneity and Amplification in 33% of Cells

Genetic Heterogeneity Genetic heterogeneity is an additional phenomenon that can frequently result in discrepant or equivocal FISH and IHC results across different laboratories, pathologists, and tumor sections, affecting the accurate assessment of HER2 status and the determination of appropriate therapy. Heterogeneity refers to different amplification characteristics between tumor cell subpopulations or between a primary tumor and its metastases. Amplified cells can be

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pathologic interpretation.51 A large study by Chang et al53 also argued that this definition is not adequate, because the proportion of amplified clones varies in a linear and predictable pattern with the HER2/CEP17 ratio. Instead, Bartlett et al51 advocated the UK recommendations of scanning the entire slide and counting 20 to 60 cells in  3 areas. The tumor should only be regarded as heterogeneous if multiple areas of clearly amplified cells are present, with a mean ratio of > 2.0, and clear areas of nonamplified cells are also present.51 Seol et al52 examined a smaller sample size of 96 cases and found that genetic heterogeneity as defined by the CAP definition was associated with low-grade or equivocal HER2 amplification or expression and was a significant independent risk factor for decreased DFS. Currently, clinical studies evaluating the effect of intratumoral heterogeneity on the response to HER2-targeted therapies are lacking. A recent mouse model of heterogeneous tumors proposed by Song et al54 showed a negative correlation between the percentage of heterogeneity and primary tumor growth and overall survival in response to anti-HER2 therapy. Lee et al55 recently published a study of 112 patients with HER2þ metastatic breast cancer and showed that HER2 regional heterogeneity (differing amplification across areas of the tumor) correlated significantly with a poorer response to trastuzumab. In addition, all tumors with genetic or regional heterogeneity displayed low-level or equivocal HER2 amplification, which was associated with a significantly shorter interval to progression and overall survival.55 The results of their study suggest that information on heterogeneity and the percentage of cells showing a HER2/CEP17 ratio > 2.2 should be a part of the pathologic report and draw attention to the need for more studies on this topic. In addition to the unclear effect on DFS and the response to HER2-targeted therapy, studies have not confirmed that only HER2-amplified clones metastasize but rather that metastases have a heterogeneous population of both amplified and nonamplified cells similar to those of the primary tumor. Recently, Valent et al56 described an additional important phenomenon in which taxane therapy, commonly used for both HER2þ and HER2 cases, resulted in an increased HER2 copy number secondary to polyploidization. HER2 therapies have been approved for use in tumors that are positive secondary to gene amplification, not aberrant polyploidization; therefore, the investigators suggested performing additional testing of tumors found to have > 6 HER2 copies after taxane therapy.56 The 2013 ASCO/CAP guidelines for HER2 testing have not thoroughly addressed heterogeneity. However, they cited a review by Hanna et al39 for additional recommendations. They recommended a cutoff of > 10% of cells with HER2 amplification should be regarded as positive to better correlate with the FDA cutoff for IHC positivity of > 10%. In addition, they recommended that a single amplified clone should be considered positive and that scattered amplified cells should be characterized by a mean score. They also reiterated the similar recommendations from Bartlett et al51 to scan the entire slide for possible clusters of amplified cells, counting > 60 cells in multiple areas if evidence of scattered amplification is found.39 The cutoff of > 10% is in contrast to previous recommendations of > 50% and has not yet been confirmed by prognostic data. It is hoped that more information will be gleaned from the ongoing phase III trial

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NSABP B-47 of trastuzumab in cases of breast cancer with low HER2 expression.

Response to HER2-Targeted Therapy Most studies have shown HER2 overexpression as an independent predictor of worse prognosis in both lymph node-positive and lymph node-negative breast cancer.6,8 However, with the advent of targeted therapies, HER2 status is no longer recommended as a prognostic factor because of the great influence of neoadjuvant and adjuvant therapy on the prognosis. Studies have shown that some patients with HER2þ breast cancer who have been treated with trastuzumab had an improved prognosis compared with those with HER2 cancer.57 Trastuzumab was first approved by the FDA in 1998 for use in the metastatic setting and was established for adjuvant use with concurrent chemotherapy in a phase III trial by Slamon et al,11 published in 2001. The response rates were approximately 50% in HER2þ tumors, with greater response rates seen in those patients with tumors that were IHC 3þ than in those with tumors that were IHC 2þ. Trastuzumab later showed benefit, both alone and combined with chemotherapy, when used as adjuvant therapy in early-stage HER2þ breast cancer and, subsequently, when used as neoadjuvant therapy.58-62 The correct determination of whether to use trastuzumab is not only important according to the treatment benefits it provides, but also because of the toxicity and cost that can be avoided for the patients who would not benefit. Currently, trastuzumab has been recommended as adjuvant therapy for 1 year, the cost of which is $70,000 to $100,000 dollars per patient per year. In addition, albeit a small risk, cardiac failure (New York Heart Association class III or IV) or cardiac death has been reported in about 0.4% of patients receiving trastuzumab without an anthracycline.61 Given the continuous spectrum of HER2 expression and amplification, which clashes with the dichotomous determination of HER2 status as positive or negative, an interesting question becomes whether the quantitative level of expression or amplification influences the prognosis and response to HER2-directed therapy (the relevant studies are listed in Table 2). In the HERceptin Adjuvant trial, no difference in DFS or clinical response to trastuzumab was seen in HER2þ metastatic tumors when stratified by varying intensities of IHC staining.63 In addition, a retrospective study by Mass et al30 found the benefit from trastuzumab to be restricted only to FISHþ tumors and not to IHC 2þ or 3þ tumors that were FISH. In contrast to these findings, Toi et al64 found that greater protein expression levels found using IHC predicted better survival outcomes in response to trastuzumab in those with metastatic breast cancer, arguing for a quantitative, rather than qualitative, assessment by IHC. IHC results alone might not predict the response to trastuzumab; however, lower levels of protein expression in FISHþ tumors might predict a reduced response.65 Because trastuzumab is an antibody against the protein expressed by the HER2 gene, it would follow that patients with greater gene copy numbers found using FISH might derive more benefit from anti-HER2 therapy. A recently published study of trastuzumab in metastatic gastric cancer found that the level of HER2 gene copy number predicted sensitivity to therapy and overall survival.66 In metastatic breast tumors, Kim et al67 found a significant increase in

Tarah J. Ballinger et al Table 2 Summary of Referenced Publications Evaluating Effects of HER2 Expression or Amplification Levels on Response to HER2-Targeted Therapy Publication

Study Design

469 Patients, metastatic breast cancer and IHC 2þ or 3þ expression in >10% of cells, randomized to standard therapy with or without trastuzumab 1180 Patients in the HERA trial of adjuvant Zabaglo trastuzumab versus no trastuzumab in early et al,63 2013 invasive HER2þ breast cancer to evaluate DFS based on IHC staining Retrospective analysis, 765 patients from 3 Mass et al,30 2005 Genetech studies (H0658g, H0649g, H0650g) of metastatic breast cancer to determine relationship between FISH and clinical benefit from trastuzumab Slamon et al,61 2001

Toi et al,64 2010

75 Patients, HER2þ (by FISH or IHC) metastatic breast cancer treated with trastuzumab to assess relationship between quantitative HER2 protein expression (measured using HERmark assay) and outcomes

Lipton et al,65 102 Patients, metastatic, HER2þ (IHC 3þ or FISH2010 positive) breast cancer treated with trastuzumab, studied to correlate quantitative measurements of HER2 protein expression with clinical outcomes

Kim et al,67 2013

Desmedt et al,68 2009

Paik et al,69 2008

52 Patients, metastatic, HER2þ FISHþ breast cancer treated with first-line trastuzumab plus taxane, studied to determine clinical implications of HER2/CEP17 ratio 71 Patients, metastatic breast cancer treated with trastuzumab alone or combined with chemotherapy, studied to determine correlation between HER2 protein expression and clinical outcomes Retrospective tissue analysis of NSABP-B31 of adjuvant trastuzumab added to standard therapy for 1787 patients

Perez et al,44 2010

1888 Patients in N9831 trial of adjuvant trastuzumab for node-positive or high-risk breast cancer, studied to determine effect of HER2 expression or copy number on DFS

Denkert et al,36 2013

217 HER2þ tumors from GeparQuattro trial of neoadjuvant trastuzumab with chemotherapy were tested for correlation between HER2 mRNA levels and pCR

Pogue-Geile et al,13 2013

1579 Patients from NSABP-B31 trial of adjuvant trastuzumab added to standard chemotherapy for node-positive breast cancer, studied in discovery and confirmation groups to determine gene expression model to predict benefit from trastuzumab

Results

Conclusion

Significant increase in response rates, PFS, and OS in trastuzumab group (P < .001, P < .001, P ¼ .046)

Trastuzumab increases efficacy of standard chemotherapy in metastatic breast cancer expressing HER2; trastuzumab efficacy was greater for IHC 3þ versus 2þ Variability in HER2 staining intensity by IHC had no bearing on clinical management of adjuvant trastuzumab

IHC staining intensity correlated with HER2 copy number and HER2/CEP17 ratio (P < .001); no difference in DFS based on IHC staining intensity in either arm (OR, 1.017; 95% CI, 0.925-1.120) FISH-positive patients had significantly greater response rates to trastuzumab (H0648g, P < .0001; H0650g, FISH-positive, 34% vs. FISH-negative, 7%) and improved OS (H0648, FISH-positive, RR ¼ 0.71 vs. FISH-negative, RR ¼ 1.10; H0649g, FISH-positive, 14.2 mo vs. FISH-negative, 8.8 mo) Significant difference seen in OS based on HER2 protein expression levels (P ¼ .003), but not IHC subgroups (P ¼ .610); OS improved for greater HER2 expression in high-expressing group (HR ¼ 0.06; P ¼ .010; 95% CI, 0.01-0.51); in low HER2 expressing group, OS improved with lower HER2 protein values (HR ¼ 16.0; P ¼ .017, 95% CI, 1.64-155.9) HER2þ FISHþ patients with low HER2 protein expression had shorter TTP compared with HER2þ FISHþ with high HER2 expression (11.3 vs. 3.7 mo, HR ¼ 0.43; P ¼ .01); in FISHþ patients, HER2 protein expression was independent predictor of TTP (HR ¼ 0.29; P ¼ .0015) and OS (HR ¼ 0.19; P < .001) Patients with HER2/CEP17 ratio of 3.0 had longer PFS (P ¼ .002) than those with ratio of 2.2-2.9; this was not true for a small subset that had high HER2/CEP17 ratios but were only IHC 1þ Greater HER2 expression or HER2:HER2 dimer levels correlated with better OS (P ¼ .0058, P ¼ .016); patients with high levels seemed to benefit less from additional chemotherapy than patients with low levels (P ¼ .43, P ¼ .27) Patients HER2 on central review (n ¼ 174) still benefited from trastuzumab in DFS (RR ¼ 0.34; 95% CI, 0.14-0.8; P ¼ .014); this was confirmed by HER2 mRNA levels HER2þ patients by IHC or FISH, or both, benefited from trastuzumab (HR ¼ 0.46, 0.49, and 0.45, respectively; P ¼ .0001); polysomic HER2amplified tumors also benefited (HR ¼ 0.37; P ¼ .006); patients with HER2 tumors (n ¼ 103) benefited insignificantly from trastuzumab (HR ¼ 0.51; P ¼ .14); patients with HER2/CEP17 ratio of 2-15 had similar DFS Only centrally positive tumors (n ¼ 158) benefited from trastuzumab (P < .0005); mRNA expression level was associated with response in HER2þ/ESR1þ tumors (P ¼ .004) but not in HER2þ/ESR1 tumors Eight predictive genes associated with HER2 were used to discover a predictive model, confirmed by P < .001; the group with the least benefit from trastuzumab had intermediate ERBB2 expression and high ESR1 expression

Clinical benefit from trastuzumab restricted to FISH-positive tumors (HER2/CEP17 ratio 2.0) compared with FISH-negative tumors considered positive by IHC (2þ or 3þ) and, therefore, should be considered the preferred method Response to trastuzumab showed parabolic, not linear, relationship to HER2 protein expression; a quantitative measurement of HER2 expression might help better identify patients who will benefit most from trastuzumab therapy HER2þ patients with lower HER2 protein expression had reduced response to trastuzumab, similar to FISH patients

Level of HER2/CEP17 ratio might predict clinical outcomes after first-line trastuzumab plus taxane These data suggest that quantitative measurements of HER2 status might better predict clinical response

The benefit of adjuvant trastuzumab might not be restricted to patients with HER2 amplification These results confirmed that both IHC and FISH can appropriately select patients who will respond to trastuzumab; no linear dose effect was seen between HER2 amplification level and trastuzumab response

Response to trastuzumab according to mRNA level correlated only in ESR1þ tumors These data support that not all HER2þ patients will respond well to trastuzumab and confirm that HER2 patients could also benefit; gene expression was a predictive model for trastuzumab benefit

Abbreviations: CEP17 ¼ chromosome enumeration probe 17; CI ¼ confidence interval; DFS ¼ disease-free survival; ERBB2 ¼ HER2/neu, v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2; ESR1 ¼ estrogen receptor 1; FISH ¼ fluorescence in situ hybridization; HER2 ¼ human epidermal growth factor receptor 2; HERA ¼ HERceptin Adjuvant; HR ¼ hazard ratio; IHC ¼ immunohistochemistry; NSABP ¼ National Surgical Adjuvant Breast and Bowel Project; OR ¼ odds ratio; OS ¼ overall survival; pCR ¼ pathologic complete response; PFS ¼ progression-free survival; RR ¼ relative risk; TTP ¼ time to progression.

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progression-free survival in patients treated with trastuzumab plus taxane therapy if the HER2/CEP17 ratio was > 3.0 compared with patients with FISHþ results with a ratio < 3.0. Desmedt et al68 similarly found that increasing quantitative levels of HER2 expression correlated with increasing survival after trastuzumab treatment. They also found that those patients with greater levels of expression derived less benefit from the addition of concomitant chemotherapy.68 This suggests that the actual level of expression, rather than whether it is simply positive or negative, could help influence clinical decision-making—both whether to use trastuzumab and whether concurrent chemotherapy is necessary. However, a review of the NSABP-B31 trial (adjuvant doxorubicin and cyclophosphamide followed by 4 cycles of paclitaxel [ACT] vs. ACT plus trastuzumab) found no association between trastuzumab benefit and HER2 copy number. Even patients with HER2 results by both central review and mRNA levels derived a benefit in DFS, challenging the current guidelines.69 Similar results were found on review of the N9831 adjuvant trastuzumab trial.44 It is possible that some false-negative findings resulting from HER2 mutations or underappreciated heterogeneity could also explain these results. The NSABP-B31 and N9831 findings of a possible benefit for anti-HER2 therapy in the HER2 population might have been because even normal levels of HER2 can influence cancer growth in hormone receptor-positive tumors that do not necessarily overexpress HER2. A 2013 study by Ithimakin et al70 used breast cancer cell lines from primary or metastatic human tissue and mouse xenograft models to show that HER2 is overexpressed in bone metastases of estrogen receptorepositive (ERþ), HER2 luminal breast cancer, believed secondary to receptor activation of the Nf-kb-RANK ligand, rather than gene amplification. This HER2 overexpression was seen in stem cells, which represent only a small number of the total cancer cells and, therefore, would not be caught by our current methods of detection.70 Again, we anxiously await the results of the NSABP B-47 trial of trastuzumab in patients with low HER2 expression to add to this discussion. Although no clear, linear relationship has been defined between the level of the HER2 copy number using FISH and the response to chemotherapy or prognosis, some studies have suggested that a relationship exists between the level of HER2 mRNA expression and the response to chemotherapy. In the GeparQuattro trial of neoadjuvant trastuzumab, ERþ/HER2þ tumors showed a continuous and linear increase in those with a pathologic complete response with increasing levels of HER2 mRNA expression. This suggests that hormone receptor-positive tumors with greater levels of HER2 expression might be more dependent on the HER2 pathway than those with lower HER2 expression and, therefore, would respond more to HER2-targeted therapy. The same was not true for ER-negative (ER)/HER2þ tumors, a group in which the dichotomous positive or negative categories were adequate for the prediction of the response.36 Others have alluded to similar findings from the ongoing research of Paik et al36 and NSABP B-31, in which the degree of mRNA expression corresponded with the degree of trastuzumab benefit in terms of the 8-year DFS for only those tumors that were both ERþ and HER2þ.71 These findings might explain the conflicting conclusions of studies searching for correspondence between the HER2 copy number and therapy response that did not further characterize their findings by hormone receptor status.

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Additionally, it provides evidence that mRNA expression might be a more powerful predictor of the response than the gene copy number. From the provocative findings from the NSABP B-31 trial, PogueGeile et al13 were able to develop an RNA-based multiplex gene expression profile to accurately predict clinical benefit from trastuzumab. Patients with HER2 tumors belonged to the group with a moderate response, with a 40% reduction in the recurrence rate. In addition, the investigators identified 10% of patients with HER2þ tumors who would not benefit from the addition of trastuzumab to adjuvant chemotherapy or endocrine therapy, a subgroup with moderate expression of HER2 genes but high expression of estrogen receptor 1eassociated genes.13 This highlights the complex relationship of breast cancer pathways and the need for assays that account for this for the best clinical decisions.

Conclusion The prognosis of patients with HER2-overexpressing tumors has improved significantly since the development and approval of HER2-targeted therapies. However, we must now optimize their benefit and decrease both toxicities and cost. This is especially relevant because combinations of HER2-targeting agents are being used in an attempt to minimize the use of chemotherapy. Although it is clear that drugs such as trastuzumab are effective, the numerous nuances in the identification of patients who will benefit must be better understood. In clinical practice, significant confusion remains regarding the treatment of patients with equivocal results using all standard testing, especially in cases in which HER2 versus HER2þ findings could mean the use of endocrine therapy alone versus the combination of chemotherapy, trastuzumab, and endocrine therapy. The updated ASCO/CAP guidelines for HER2 testing in breast cancer have provided improved guidance on HER2 testing and updated recommendations on how to interpret the results. We next need to identify the test that can most clearly and consistently determine which patients will benefit from HER2-directed therapy. Second, clinical trials addressing the optimal cutoff for the use of trastuzumab in patients with genetic heterogeneity and other variations are needed. We have seen great advances in the treatment of HER2 overexpressing breast cancer in recent years. Our challenge now is to refine HER2 testing and the use of HER2-targeting therapies.

Disclosure The authors have stated that they have no conflicts of interest.

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33. 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; 94:852-4. 34. Roche PC, Suman VJ, Jenkins RB, et al. Concordance between local and central laboratory HER2 testing in the breast intergroup trial N9831. J Natl Cancer Inst 2002; 94:855-7. 35. McCullough AE, Dell’orto P, Reinholz MM, et al. Central pathology laboratory review of HER2 and ER in early breast cancer: an ALTTO trial [BIG 2-06/NCCTG N063D (Alliance)] ring study. Breast Cancer Res Treat 2014; 143: 485-92. 36. Denkert C, Huober J, Loibl S, et al. HER2 and ESR1 mRNA expression levels and response to neoadjuvant trastuzumab plus chemotherapy in patients with primary breast cancer. Breast Cancer Res 2013; 15:R11. 37. Starczynski J, Atkey N, Connelly Y, et al. HER2 gene amplification in breast cancer: a rogues’ gallery of challenging diagnostic cases: UKNEQAS interpretation guidelines and research recommendations. 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Current HER2 Testing Recommendations 61. Slamon D, Eiermann W, Robert N, et al. Adjuvant trastuzumab in HER2-positive breast cancer. N Engl J Med 2011; 365:1273-83. 62. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005; 353:1673-84. 63. Zabaglo L, Stoss O, Ruschoff J, et al. HER2 staining intensity in HER2-positive disease: relationship with FISH amplification and clinical outcome in the HERA trial of adjuvant trastuzumab. Ann Oncol 2013; 24:2761-6. 64. Toi M, Sperinde J, Huang W, et al. Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer. BMC Cancer 2010; 10:56. 65. Lipton A, Kostler WJ, Leitzel K, et al. Quantitative HER2 protein levels predict outcome in fluorescence in situ hybridization-positive patients with metastatic breast cancer treated with trastuzumab. Cancer 2010; 116:5168-78. 66. Gomez-Martin C, Plaza JC, Pazo-Cid R, et al. Level of HER2 gene amplification predicts response and overall survival in HER2-positive advanced gastric cancer treated with trastuzumab. J Clin Oncol 2013; 31:4445-52.

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67. Kim JW, Kim JH, Im SA, et al. HER2/CEP17 ratio and HER2 immunohistochemistry predict clinical outcome after first-line trastuzumab plus taxane chemotherapy in patients with HER2 fluorescence in situ hybridization-positive metastatic breast cancer. Cancer Chemother Pharmacol 2013; 72:109-15. 68. Desmedt C, Sperinde J, Piette F, et al. Quantitation of HER2 expression or HER2:HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully selected for trastuzumab treatment primarily by FISH. Diagn Mol Pathol 2009; 18:22-9. 69. Paik S, Kim C, Wolmark N. HER2 status and benefit from adjuvant trastuzumab in breast cancer. N Engl J Med 2008; 358:1409-11. 70. Ithimakin S, Day KC, Malik F, et al. HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: implications for efficacy of adjuvant trastuzumab. Cancer Res 2013; 73:1635-46. 71. Goldhirsch A, Wood WC, Coates AS, Gelber RD, Thurlimann B, Senn HJ. Strategies for subtypes—dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol 2011; 22:1736-47.