Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

The HER-2 Receptor and Breast Cancer: Ten Years of Targeted Anti–HER-2 Therapy and Personalized Medicine

The HER-2 Receptor and Breast Cancer: Ten Years of Targeted Anti–HER-2 Therapy and Personalized... Downloaded from by DeepDyve user on 01 February 2022 The Oncologist CME Program is located online at To take the CME activity related to this article, you must be a registered user. The Oncologist Breast Cancer The HER-2 Receptor and Breast Cancer: Ten Years of Targeted Anti–HER-2 Therapy and Personalized Medicine a a b b JEFFREY S. ROSS, ELZBIETA A. SLODKOWSKA, W. FRASER SYMMANS, LAJOS PUSZTAI, b b PETER M. RAVDIN, GABRIEL N. HORTOBAGYI Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, New York, USA; Departments of Pathology, Breast Medical Oncology, and Biostatistics and Quantitative Sciences, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA Key Words. HER-2 � Trastuzumab � Lapatinib � IHC � FISH � CISH � Prognosis � Review Disclosures Jeffrey S. Ross: None; Elzbieta A. Slodkowska: None; W. Fraser Symmans: None; Lajos Pusztai: None; Peter M. Ravdin: Employment/leadership position: Adjuvant Inc.; Intellectual property rights: Adjuvant Inc.; Ownership interest: Adjuvant Inc.; Gabriel N. Hortobagyi: None. Section editor Kathleen I. Pritchard has disclosed no financial relationships relevant to the content of this article. The content of this article has been reviewed by independent peer reviewers to ensure that it is balanced, objective, and free from commercial bias. LEARNING OBJECTIVES 1. Contrast the current strengths and limitations of the three main slide-based techniques (IHC, FISH, and CISH) currently in clinical use for testing breast cancer tissues for HER-2 status. 2. Compare the efficacy of trastuzumab- and lapatinib-based regimens in the adjuvant and metastatic settings as reported in published clinical trials and regulatory approval databases. 3. Contrast the list of biomarkers that have been associated with clinical resistance to trastuzumab and lapatinib and describe their current level of validation. CME CME This article is available for continuing medical education credit at ABSTRACT The human epidermal growth factor receptor (HER-2) 39,730 patients, which produced an overall HER-2– oncogene encodes a transmembrane tyrosine kinase re- positive rate of 22.2% and a mean relative risk for over- ceptor that has evolved as a major classifier of invasive all survival (OS) of 2.74. The issue of HER-2 status in breast cancer and target of therapy for the disease. The primary versus metastatic breast cancer is considered validation of the general prognostic significance of along with a section on the features of metastatic HER- HER-2 gene amplification and protein overexpression 2–positive disease. The major marketed slide-based in the absence of anti–HER-2 targeted therapy is dis- HER-2 testing approaches, immunohistochemistry, flu- cussed in a study of 107 published studies involving orescence in situ hybridization, and chromogenic in situ Correspondence: Jeffrey S. Ross, M.D., Albany Medical College, Department of Pathology, Mail Code 81, 47 New Scotland Avenue, Albany, New York 12208, USA. Telephone: 518-262-5461; Fax: 518-262-8092; e-mail: Received October 21, 2008; accepted for publication February 15, 2009; first published in The Oncologist Express on April 3, 2009. ©AlphaMed Press 1083- 7159/2009/$30.00/0 doi: 10.1634/theoncologist.2008-0230 The Oncologist 2009;14:320 –368 Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 321 hybridization, are presented and contrasted in detail for the more recently approved small molecule HER- against the background of the published American So- 1/HER-2 kinase inhibitor lapatinib are also presented ciety of Clinical Oncology–College of American Pathol- along with a more limited review of markers of resis- ogists guidelines for HER-2 testing. Testing issues, such tance for this agent. Additional topics in this section as the impact of chromosome 17 polysomy and local include combinations of both anti–HER-2 targeted versus central HER-2 testing, are also discussed. therapies together as well as with novel agents includ- Emerging novel HER-2 testing techniques, including ing bevacizumab, everolimus, and tenespimycin. A mRNA-based testing by real-time polymerase chain series of novel HER-2–targeting agents is also pre- reaction and DNA microarray methods, HER-2 re- sented, including pertuzumab, ertumaxomab, HER-2 ceptor dimerization, phosphorylated HER-2 recep- vaccines, and recently discovered tyrosine kinase in- tors, and HER-2 status in circulating tumor cells, are hibitors. Biomarkers predictive of HER-2 targeted also considered. A series of biomarkers potentially as- therapy toxicity are included, and the review con- sociated with resistance to trastuzumab is discussed cludes with a consideration of HER-2 status in the with emphasis on the phosphatase and tensin homo- prediction of response to non–HER-2 targeted treat- logue deleted on chromosome ten/Akt and insulin-like ments including hormonal therapy, anthracyclines, growth factor receptor pathways. The efficacy results and taxanes. The Oncologist 2009;14:320 –368 SECTION ONE:BIOLOGY,PATHOLOGY,DIAGNOSIS, the family (EGFR, HER-2, HER-3, or HER-4). It may dimer- AND CLINICAL SIGNIFICANCE OF HER-2–POSITIVE ize with a like member of the family (homodimerization) or it BREAST CANCER may dimerize with a different member of the family (het- erodimerization). The specific tyrosine residues on the intra- Introduction and Background Biology cellular portion of the HER-2/neu receptor that are The human epidermal growth factor receptor 2 (HER-2, phosphorylated, and hence the signaling pathways that are ac- HER-2/neu,c-erbB-2) gene, first discovered in 1984 by tivated, depend on the ligand and dimerization partner. The Weinberg and associates [1], is localized to chromosome wide variety of ligands and intracellular crosstalk with other 17q and encodes a transmembrane tyrosine kinase receptor pathways allow for significant diversity in signaling. Although protein that is a member of the epidermal growth factor re- no known ligand for the HER-2 receptor has been identified, it ceptor (EGFR) or HER family (Fig. 1) [2]. This family of is the preferred dimerization partner of the other family mem- receptors is involved in cell– cell and cell–stroma commu- bers. HER-2 heterodimers are more stable [5, 6] and their sig- nication primarily through a process known as signal trans- naling is more potent [7] than receptor combinations without duction, in which external growth factors, or ligands, affect HER-2. the transcription of various genes, by phosphorylating or HER-2 gene amplification and/or protein overexpression dephosphorylating a series of transmembrane proteins and has been identified in 10%–34% of invasive breast cancers [1]. intracellular signaling intermediates, many of which pos- Unlike a variety of other epithelial malignancies, in breast can- sess enzymatic activity. Signal propagation occurs as the cer, HER-2 gene amplification is uniformly associated with enzymatic activity of one protein turns on the enzymatic HER-2 (p185neu) protein overexpression and the incidence of activity of the next protein in the pathway [3]. Major single copy overexpression is exceedingly rare [8]. HER-2 pathways involved in signal transduction, including the gene amplification in breast cancer has been associated with Ras/mitogen-activated protein kinase pathway, the phos- increased cell proliferation, cell motility, tumor invasiveness, phatidylinositol 3 kinase (PI3K)/Akt pathway, the Janus progressive regional and distant metastases, accelerated an- kinase/signal transducer and activator of transcription path- giogenesis, and reduced apoptosis [9]. When classified by rou- way, and the phospholipase C pathway, ultimately affect tine clinicopathologic parameters and compared with HER-2– cell proliferation, survival, motility, and adhesion. negative tumors, HER-2–positive breast cancer is more often Receptor activation requires three variables, a ligand, a re- of intermediate or high histologic grade, more often lacking ceptor, and a dimerization partner [4]. After a ligand binds to a estrogen receptors (ERs) and progesterone receptors (PgRs) receptor, that receptor must interact with another receptor of (ER and PgR negative), and featuring positive lymph node identical or related structure in a process known as dimeriza- metastases at presentation [1]. In the recent molecular classi- tion in order to trigger phosphorylation and activate signaling fication of breast cancer, positive HER-2 status does not con- cascades. Therefore, after ligand binding to an EGFR family stitute a unique molecular category and is identified in both the member, the receptor can dimerize with various members of “HER-2” and “luminal” tumor classes [10]. Downloaded from by DeepDyve user on 01 February 2022 322 HER-2 in Breast Cancer Figure 1. The human epidermal growth factor receptor (HER) gene family. This image depicts the complex crosstalk between members of the HER family of receptor tyrosine kinases and intracellular signaling. Activated HER receptors can function to both stimulate and inhibit downstream signaling of members of other biologic pathways. Note that HER-2 has no activating ligands and HER-3 lacks a tyrosine kinase domain. HER-2–mediated signaling is associated with cell proliferation, motility, resistance to apoptosis, invasiveness, and angiogenesis. The figure shows the complexity of signaling pathways initiated by, and influenced by, HER family protein receptors at the cell surface. Abbreviations: Amp, amphiregulin; -cel, -cellulin; EGF, epidermal growth factor; Epi, epinephrine; HB-GF, heparin- binding growth factor; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal–related kinase kinase; NRG, neuregulin; PI3K, phosphatidylinositol 3 kinase; SOS, son of sevenless; TGF, transforming growth factor ; VEGF, vascular endothelial growth factor. HER-2 Status and Prognosis in Breast Cancer used immunohistochemistry (IHC) on paraffin-embed- Both morphology-based and molecular-based tech- ded tissues as the HER-2/protein detection technique, niques have been used to measure HER-2/neu status in two (15%) used fluorescence in situ hybridization breast cancer clinical samples [11–117]. By a substantial (FISH), two (15%) used Southern analysis, and one (7%) majority, abnormalities in HER-2 expression at the gene, used a real-time polymerase chain reaction (RT-PCR) message, or protein level have been associated with ad- technique. Of the 15 studies that used the FISH tech- verse prognosis in both lymph node–negative and lymph nique, 13 (87%) showed univariate prognostic signifi- node–positive breast cancer. Of the 107 studies consid- cance of gene amplification, and 11 of these (85%) ering 39,730 patients listed in Table 1, 95 (88%) of the showed prognostic significance on multivariate analysis studies determined that either HER-2 gene amplification as well. The two studies that used chromogenic in situ or HER-2 (p185 neu) protein overexpression predicted hybridization (CISH) HER-2 gene amplification detec- breast cancer outcome on either univariate or multivari- tion techniques both found that HER-2 amplification was ate analysis. In 68 (73%) of the 93 studies that featured an independent predictor of outcome on multivariate multivariate analysis of outcome data, the adverse prog- analysis [100, 112]. However, interpretation of these nostic significance of HER-2 gene, message, or protein studies is complicated by the fact that most studies in- overexpression was independent of all other prognostic cluded patients who received variable types of systemic variables. In only 13 (12%) of the studies, no correlation adjuvant therapy; therefore, the pure prognostic value of between HER-2 status and clinical outcome was identi- HER-2 overexpression in the absence of any systemic fied. Of these 13 noncorrelating studies, eight (62%) adjuvant therapy is incompletely understood. Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 323 Table 1. HER-2 status and prognosis in breast cancer n of Method(s) of Univariate Multivariate Study Reference cases Specimen type analysis % positive significance significance RR Comment 1 Slamon et al. (1987) [11] 189 Frozen Southern blot 30 Yes Yes Not given HER-2 amplification predicted overall survival and time to relapse 2 Berger et al. (1988) [12] 51 Frozen/paraffin Southern blot/IHC 25 Yes Not done Not given HER-2 protein by IHC correlated with node status and tumor grade 3 van de Vijver et al. (1988) 189 Paraffin IHC 14 No No HR: RFS, 1.32; OS, Correlated with size, [13] 2.36 comedo-type carcinoma 4 Wright et al. (1989) [14] 185 Paraffin IHC 17 Yes Yes Overexpression correlated with high grade and negative ER, not size or node status 5 Heintz et al. (1990) [15] 50 Frozen Southern analysis 34 No No Not given Mitoses, ER/PR negative 6 Tsuda et al. (1990) [16] 176 Paraffin Southern analysis 18 Yes No Not given Impact on prognosis absorbed by grade 7 Borg et al. (1990) [17] 300 Fresh Western 17 Yes No Not given Gene amplification predicted relapse and death in node-negative patients on univariate analysis only 8 Paik et al. (1990) [18] 292 Paraffin IHC 21 Yes Yes Not given HER-2 protein overexpression second only to nodal status in predicting outcome 9 Battifora et al. (1991) [19] 245 Paraffin IHC 22 Yes Yes DFS, 2.7 Significant only for low-grade, low-stage cases 10 Kallioniemi et al. (1991) [20] 319 Paraffin IHC 23 Yes Yes 2.2 Independent predictor in node- negative and node- positive patients 11 Gullick et al. (1991) [21] 483 Paraffin IHC 21.3 Yes Yes Not given Overexpression correlated with grade, but not size or ER or nodal status 12 Clark and McGuire (1991) 362 Fresh Slot blot 33 No No Not given Amplification [22] marginally predictive in node- positive patients only 13 Lovekin et al. (1991) [23] 782 Paraffin IHC Stage 1–2 disease, Yes Yes Not given HER-2 15; stage 3–4 overexpression disease, 20 predicted outcome in early and advanced cases; grade more significant predictor 14 McCann et al. (1991) [24] 314 Paraffin IHC 17 Yes Yes Not given Predicted outcome in node-negative and node-positive patients 15 Dykins et al. (1991) [25] 178 Paraffin IHC 14 Yes Yes Not given Overexpression predicted shorter survival in node- negative but not node-positive patients 16 Rilke et al. (1991) [26] 1,210 Paraffin IHC 23 Yes No Not given Protein overexpression predicted outcome in node-positive patients only 17 Winstanley et al. (1991) [27] 465 Paraffin IHC 22 Yes Yes Not given HER-2 protein staining independently predicted survival 18 O’Reilly et al. (1991) [28] 172 Paraffin IHC 16 Yes Yes Not given HER-2 protein overexpression predicted outcome in node-positive but not node-negative disease (continued) Downloaded from by DeepDyve user on 01 February 2022 324 HER-2 in Breast Cancer Table 1. (Continued) n of Method(s) of Univariate Multivariate Study Reference cases Specimen type analysis % positive significance significance RR Comment 19 Paterson et al. (1991) [29] 115 Paraffin Slot blot 11.7 Yes Yes Not given Gene amplification predicted disease- free interval in node- negative patients 20 Toikkanen et al. (1992) [30] 209 Paraffin IHC 26 Yes Yes Not given Protein overexpression predicted shorter survival in node- positive patients 21 Molina et al. (1992) [31] 301 Paraffin IHC, Western blot Western blot, 16.6; Yes Yes Not given Both methods of IHC, 16.3 protein levels predicted worse overall survival 22 Noguchi et al. (1992) [32] 234 Paraffin IHC 30 Yes No Not given Only nodal status correlated independently 23 Allred et al. (1992) [33] 613 Paraffin IHC 14.3 Yes Not done Not given Significant for node- negative, low-risk only 24 Babiak et al. (1992) [34] 78 Paraffin Slot blot 10 Yes Not done Not given Only when combined with aneuploid status 25 Tiwari et al. (1992) [35] 61 Frozen Southern 28 Yes Not done Not given HER-2 amplification associated with nodal metastasis 26 Gusterson et al. (1992) [36] 1,506 Paraffin IHC Node-positive, 16; Yes Yes Not given Predicted outcome in node-negative, 19 node-positive patients only 27 Bianchi et al. (1993) [37] 230 Paraffin IHC 20.9 Yes No Not given Only diffuse intense staining correlated 28 Press et al. (1993) [38] 210 Paraffin IHC Polyclonal R60–50, Yes Yes Not given Predicts relapse in monoclonal 3B5–25 node-negative patients 29 Seshadri et al. (1993) [39] 1,056 Fresh Slot blot 21 Yes Yes DFS, 1.62; OS, 1.39 Independent for both node-negative and node-positive patients 30 Descotes et al. (1993) [40] 199 Frozen Slot blot, Southern 16.6 Yes Yes Not given Independent blot predictor in ER- positive, node- negative patients 31 Giai et al. (1994) [41] 159 Paraffin Western blot 41 Yes Yes Not given Independent predictor in node- negative patients 32 Muss et al. (1994) [42] 442 Paraffin IHC 29 Yes Not done DFS, 1.56; OS, 1.61 Predicts response to chemotherapy 33 Têtu and Brisson (1994) [43] 888 Paraffin IHC 16.1 Yes Yes HR: DMFS, 2.05; Survival in node- OS, 1.8 positive patients, only membranous pattern correlates 34 Hartmann et al. (1994) [44] 340 Paraffin IHC 20 Yes No Not given Predicts lymph node status 35 Jaquemier et al. (1994) [45] 81 Paraffin IHC 23 No No Not given No correlation with therapy response 36 Marks et al. (1994) [46] 230 Paraffin IHC 17 Yes Yes OS, 3.4 p53 and node status also independent 37 Rosen et al. (1995) [47] 440 Paraffin IHC 44 No No Not given Medullary carcinoma is negative 38 Quénel et al. (1995) [48] 942 Paraffin IHC 24 Yes Yes Node-negative only: HER-2 predicted RFS, 1.8, MFS, 1.7 RFS and MFS 39 Sundblad et al. (1996) [49] 271 Paraffin IHC 22.5 Yes Yes Not given Node positive, HER- 2/neu negative, CEA positive, and bcl-2 positive had the best prognosis 40 O’Malley et al. (1996) [50] 107 Paraffin IHC Not given Yes Yes DFS, 2.78; OS, 3.38 Both p53 and HER- 2/neu staining independently predicted outcome 41 Hieken et al. (1996) [51] 100 Paraffin, cytosol IHC, ELISA 74 No No Not given HER-2 protein by ELISA, not IHC, predictive (continued) Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 325 Table 1. (Continued) n of Method(s) of Univariate Multivariate Study Reference cases Specimen type analysis % positive significance significance RR Comment 42 Xing et al. (1996) [52] 37 Paraffin FISH 68 Yes Not done Not given HER-2 amplification more predictive than nodal status in young patients 43 Dittadi et al. (1997) [53] 115 Cytosol ELISA 2,150 U/mg, 25.7; Yes Yes 2.74 HER-2 protein levels 3,000 u/mg, 13.3 predicted DFS 44 Fernández-Acenero et al. 112 Paraffin IHC 30.3 Yes No Not given Only TNM stage (1997) [54] independent predictor 45 Eissa et al. (1997) [55] 100 Cytosol ELISA 28 Yes Yes 6.3 HER-2 strongest predictor of recurrence in node- negative patients 46 Charpin et al. (1997) [56] 148 Frozen IHC 45 Yes Yes Not given OS and DFS independent of nodal status 47 Press et al. (1997) [57] 324 Paraffin FISH 35 Yes Yes Early recurrence, 3.1; HER-2/neu gene recurrence, 2.9; amplification disease-related death, predicts recurrence 5.5 and death 48 Ross et al. (1998) [58] 224 Paraffin FISH Not given Yes Yes Not given 3-tiered amplification system 49 Depowski et al. (1999) [59] 123 Paraffin FISH 47.2 Yes Yes Not given HER-2 amplification predicted death independent of nodal status 50 Querzoli et al. (1998) [60] 164 Paraffin IHC 29.3 Yes Yes Not given HER-2 overexpression independently predicted recurrence and survival 51 Andrulis et al. (1998) [61] 580 Fresh Southern 20 Yes Yes RR for recurrence, HER-2 amplification 2.36 independently predicted recurrence in node-negative patients; impact greater in adjuvant- treated cases 52 Sjögren et al. (1998) [62] 315 Paraffin IHC 19 Yes Yes Relative hazard, 2.7 CB-11 antibody IHC predicted OS independent of age, node status, size, grade, hormone receptor status, S phase, p53 status, and adjuvant therapy 53 Harbeck et al. (1999) [63] 112 Paraffin FISH, IHC FISH, 31; IHC, 41 Yes Yes DFS, 2.7; OS, 4.8 HER-2 Amplification by FISH and plasminogen protease (uPA, PAI- 1) levels were both independent predictors in node- negative patients 54 Scorilas et al. (1999) [64] 136 Fresh Southern 29.7 Yes No OS: node positive, Cathepsin D was an 2.54; node negative, independent 6.93 predictor, but HER-2 was not 55 Rudolph et al. (1999) [65] 356 Paraffin IHC 37.1 Yes No Not given HER-2 overexpression not an independent predictor of prognosis 56 Reed et al. (2000) [66] 385 Paraffin IHC 10 No No Not given HER-2–positive rate was only 10% in noncorrelating IHC study 57 Pauletti et al. (2000) [67] 189 Paraffin FISH, IHC FISH, 19; IHC, Yes Yes DFS, 1.3; OS, 1.45 Amplification 17.2 detected by FISH was consistently predictive of outcome, but IHC testing was not (continued) Downloaded from by DeepDyve user on 01 February 2022 326 HER-2 in Breast Cancer Table 1. (Continued) n of Method(s) of Univariate Multivariate Study Reference cases Specimen type analysis % positive significance significance RR Comment 58 Kakar et al. (2000) [68] 117 Paraffin FISH, IHC FISH, 19; IHC, 28 Yes Not done Not given IHC 3 positive and FISH-positive cases correlated with outcome; IHC 2 positive cases did not 59 Agrup et al. (2000) [69] 110 Paraffin IHC 27 Yes Yes Not given HER-2 overexpression predicted survival in node-positive disease in young women independent of adjuvant therapy 60 Umekita et al. (2000) [70] 159 Paraffin IHC 26 Yes No Not given Coexpression of TGF- and EGFR had worst prognosis 61 Pawlowski et al. (2000) [71] 365 Fresh RT-PCR 25.1 No No DFS, 1.61; OS, 1.56 HER-2 mRNA levels correlated with grade and ER/PgR status but not survival; increased HER-4 levels were a favorable prognostic finding 62 Volpi et al. (2000) [72] 286 Paraffin IHC 42.8 Yes No Not given HER-2 status predictive of short- term prognosis only and not an independent predictor 63 Carr et al. (2000) [73] 190 Paraffin FISH 43 Yes Yes Not given HER-2 amplification predicted recurrence independent of ER and lymph node status 64 Ferrero-Pou ¨ s et al. (2000) 488 Fresh ELISA 13.3 Yes Yes OS, 1.87 (1.82 in HER-2 [74] node negative); DFS, overexpression also 1.65 (1.79 in node predicted reduced positive) response to tamoxifen 65 Platt-Higgins et al. (2000) 349 Paraffin IHC 22 Yes No 1.69 S1004A protein [75] marker was an independent predictor; HER-2 expression was significant on univariate analysis only 66 Eppenberger-Castori et al. 1,123 Fresh ELISA 12 Yes Yes Not given HER-2 levels (2001) [76] predicted survival independent of hormonal or cytotoxic therapy 67 Jukkola et al. (2001) [77] 650 Fresh Southern 19 Yes Yes Not given HER-2–positive patients also had poorer responses to both hormonal and cytotoxic therapies 68 Gaci et al. (2001) [78] 100 Fresh ELISA 37 Yes Yes Not given HER-2 and cathepsin D expression were both independent predictors of OS 69 Rudolph et al. (2001) [79] 261 Paraffin IHC 35.2 Yes Yes DFS, 3.01; OS, 4.27 Cell cycling ratio was most predictive factor in multivariate analysis 70 Beenken et al. (2001) [80] 90 Paraffin IHC 31.1 Yes Yes Not given HER-2 and p53 coexpression had the worst prognosis 71 Pinto et al. (2001) [81] 306 Paraffin IHC 14.6 Yes Yes DFS, 2.16; OS, 2.45 HER-2–positive ER- positive patients had lower response rate to tamoxifen (continued) Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 327 Table 1. (Continued) n of Method(s) of Univariate Multivariate Study Reference cases Specimen type analysis % positive significance significance RR Comment 72 Riou et al. (2001) [82] 172 Fresh Southern 18 Yes Yes 2.2 HER-2 amplification independently predicted survival; no correlation with survival for IHC- positive cases with no gene amplification 73 Horita et al. (2001) [83] 76 Paraffin IHC 22.4 Yes Yes Not given HER-2 expression correlated with PCNA expression 74 Suo et al. (2002) [84] 100 Paraffin IHC 27 Yes Yes 4.71 Coexpression of HER-2 with EGFR (HER-1) had worse DFS 75 Ristimäki et al. (2002) [85] 1,576 Paraffin FISH 50 Yes Yes 1.46 HER-2 amplification was an independent predictor of survival and correlated with COX-2 expression 76 Rosenthal et al. (2002) [86] 177 Paraffin FISH ILC, 13; IDC, 48 Yes No Not given ILC had lower HER- 2 amplification rate (10%) than IDC and reached near significance as an independent predictor of OS 77 Tsutsui et al. (2002) [87] 698 Paraffin IHC 17.2 Yes Yes Node negative: DFS, Combined HER-2– 3.31; OS, 2.53; node positive and ER- positive: DFS, 2.23; negative patients had OS, 2.34 the worst outcome 78 Spizzo et al. (2002) [88] 205 Paraffin IHC 19.5 Yes Yes Not given Both HER-2 and EpCAM expression were independent predictors of OS 79 Kato et al. (2002) [89] 408 Paraffin IHC 30.5 Yes No RFS, 1.4; OS, 1.8 HER-2 status was an independent predictor in some, but not all, patient cohorts 80 el-Amady et al. (2002) [90] 94 Fresh ELISA 51 Yes Yes Not given HER-2 protein level by ELISA most predictive factor for short-term prognosis 81 Taucher et al. (2003) [91] 923 Paraffin IHC, FISH 17.3 Yes Yes Not given HER-2 overexpression associated with negative receptor status, grade 3 lesions, and young age; lack of correlation between node-positive disease and HER-2 82 Tsutsui et al. (2003) [92] 242 Paraffin IHC 13.6 Yes Yes 2.93 Breast cancer patients coexpressing both HER-2 and p53 have worse outcome 83 Joensuu et al. (2003) [93] 852 Paraffin IHC, CISH 12 Yes Yes 2.56 HER-2 status superior to ER as a prognostic factor in early breast cancer 84 Lal et al. (2005) [94] 3,655 Paraffin IHC, FISH IHC, 26.89; FISH, Yes Yes Not given Substantial portion 14.69; overall, of HER-2–positive 15.65 tumors express ER and PgR 85 Huang et al. (2005) [95] 1,362 Paraffin IHC 10.9 Yes Yes Not given In ER-positive tumors, expression of PgR affects likelihood of HER-2 overexpression; PgR-negative tumors more likely to be of higher grade (continued) Downloaded from by DeepDyve user on 01 February 2022 328 HER-2 in Breast Cancer Table 1. (Continued) n of Method(s) of Univariate Multivariate Study Reference cases Specimen type analysis % positive significance significance RR Comment 86 Knoop et al. (2005) [96] 805 Paraffin IHC, FISH IHC, 30.6; FISH, 21 Yes Yes Not given HER-2 positivity and TOP2A amplification or deletion associated with shorter survival; HER-2 status insufficient as a screening tool for TOP2A changes 87 Ariga et al. (2005) [97] 518 Paraffin FISH 15 Yes Yes Not given Correlation between HER-2 positivity and high grade, ER positivity, PR positivity, high MIB1, and IDC 88 Fritz et al. (2005) [98] 225 Paraffin IHC 19.1 Yes Yes Not given c-erbB-2 and TOP2A expression are independent prognostic factors 89 Prati et al. (2005) [99] 199 Paraffin IHC, FISH FISH, 19.1 Yes No Not given HER-2 associated with p53 90 Tanner et al. (2006) [100] 391 Paraffin CISH 32.7 Yes Yes Not given High sensitivity of HER-2/neu and TOP2A-coamplified tumors to anthracyclines 91 Diallo et al. (2006) [101] 106 Paraffin IHC, FISH 29.2 Yes Yes Not given Coexpression of HER-2 and c-Kit in high-grade DCIS 92 Lee et al. (2007) [102] 333 Paraffin FISH, IHC FISH, 24.6; IHC, Yes Yes OS HR, 1.61 Higher expression of 23.4 cyclin D1 associated with better prognosis in patients with c- erbB-2 overexpression 93 Lee et al. (2007) [103] 151 Paraffin IHC 19.9 Yes Yes Not given c-erbB-2 overexpression an independent indicator of poorer DFS 94 Ko et al. (2007) [104] 326 Paraffin IHC 40.5 No No Not given c-erbB-2 and p53 have no prognostic value in early node-negative breast cancer 95 Logullo et al. (2007) [105] 92 Paraffin IHC 18.5 Yes Not done Not given c-erbB-2 a marker for aggressive clinical behavior in early stages 96 Altundag et al. (2007) [106] 248 Paraffin IHC, FISH 23.1 Yes Not done Not given Statistically significant difference in OS in patients with CNS metastases and HER- 2–positive versus HER-2–negative tumors 97 Cao et al. (2007) [107] 263 Paraffin IHC 19.8 Yes Yes Not given High-level SLP2 associated with HER-2 positivity and shorter OS 98 Badve et al. (2007) [108] 438 Paraffin IHC Not given No Not done Not given HER-2 did not affect cancer-free survival; FOXA1 did not correlate with HER- 2 expression 99 Sandri et al. (2007) [109] 135 Fresh IA 21.48 Yes Yes Not given HER-2 predicts response to treatment and long-term clinical outcome 100 von Minckwitz et al. (2008) 250 Paraffin IHC 23.7 No No Not given bcl-2 predictor of [110] pathological complete response after preoperative chemotherapy (continued) Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 329 Table 1. (Continued) n of Method(s) of Univariate Multivariate Study Reference cases Specimen type analysis % positive significance significance RR Comment 101 Sunami et al. (2008) [111] 130 Paraffin IHC 27 Yes Yes Not given Epigenetic differences between HER-2–positive and HER-2–negative tumors; HER-2 independent of ER status 102 Beeghly-Fadiel et al. (2008) 313 Paraffin FISH, CISH 34.25 Yes Yes OS HR, 1.61 HER-2 significantly [112] associated with a greater hazard for death 103 Ihemelandu et al. (2008) 372 Paraffin IHC 11.6 Yes Not done Not given Correlation between [113] HER-2 positivity and DMFS; basal cell- like subtype associated with worse prognosis 104 Zhang et al. (2008) [114] 263 Paraffin IHC 19.77 Yes Yes Not given MMP13 expression correlates with HER- 2 and shorter OS 105 Bektas et al. (2008) [115] 250 Paraffin IHC 10.5 No Not done Not given Positive correlation between FOXM1 expression and HER- 2 status 106 Ludovini et al. (2008) [116] 256 Fresh, paraffin ELISA, FISH, ELISA, 9; IHC/ Yes Yes DFS HR, 3.25 High HER-2 IHC FISH, 16.4 associated with worse prognosis in early breast cancer 107 Kim et al. (2008) [117] 338 Paraffin FISH 26.3 Yes Yes DFS, 1.67; OS, 2.39 HER-2 amplification was an independent predictor of OS only in stage III patients Abbreviations: CEA, carcinoembryonic antigen; CISH, chromogenic in situ hybridization; CNS, central nervous system; COX-2, cyclo-oxygenase 2; DCIS, ductal carcinoma in situ; DFS, disease-free survival; DMFS, distant metastasis-free survival; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; EpCAM, epithelial cell adhesion molecule; ER, estrogen receptor; FISH, fluorescence in situ hybridization; FOX, forkhead box; HER-2, human epidermal growth factor receptor 2; HR, hazard ratio; IA, image analysis; IDC, invasive ductal carcinoma; IHC, immunohistochemistry; ILC, invasive lobular carcinoma; MFS, metastasis-free survival; MIB1, mindbomb homolog 1; MMP13, matrix metalloproteinase 13; OS, overall survival; PCNA, proliferating cell nuclear antigen; PgR, progesterone receptor; RFS, relapse-free survival; RR, relative risk; RT-PCR, real-time polymerase chain reaction; SLP2, stomatin-like protein 2; TGF-, transforming growth factor ; TNM, tumor–node–metastasis; TOP2A, topoisomerase II. HER-2 Positivity Rates was 2.33; for disease-free survival (DFS), the mean RR was The frequency of HER-2 positivity in all of the studies pre- 2.04 (range, 1.30 –3.01) and the median was 1.8. In several sented in Table 1 was 22.2%, with a range of 9%–74%. The studies, the RR was estimated with a hazard ratio (HR) HER-2–positive rate was similar for IHC, at 22% (range, model. The mean HR was 2.12 (range, 1.6 –2.7) and the me- 10%–74%), and FISH, at 23.9% (range, 14.7%– 68%). In dian was 2.08. current practice, HER-2–positive rates have trended below 20%, with most investigators currently reporting that the HER-2 Expression and Breast Pathology true positive rate is in the range of 15%–20%. The HER-2– The association of HER-2–positive status with specific positive rate may be higher when metastatic lesions are pathologic conditions of the breast is summarized in Table tested, and tertiary hospitals and cancer centers report 2. HER-2 overexpression has been consistently associated slightly higher rates than community hospitals and national with higher grades and extensive forms of ductal carcinoma reference laboratories. in situ (DCIS) and DCIS featuring comedo-type necrosis [118 –121]. The incidence of HER-2 positivity in DCIS has Relative Risk and Hazard Ratio varied in the range of 24%–38% in the published literature, In Table 1, a number of studies provided data as to the rel- which appears to be slightly higher than that for invasive ative risk (RR) of untreated HER-2–positive breast cancer breast cancer [118 –121]. Routine testing for HER-2 status being associated with an adverse clinical outcome. For OS, in DCIS is not widely performed. However, should anti– the mean RR was 2.74 (range, 1.39 – 6.93) and the median HER-2 targeted therapies directed at HER-2–positive DCIS Downloaded from by DeepDyve user on 01 February 2022 330 HER-2 in Breast Cancer Table 2. HER-2 status and breast pathology Pathologic condition HER-2 status DCIS High–grade subtype, multifocal disease, and cases with comedonecrosis are positive ILC versus IDC ILC lower HER-2–positive rate (10%) restricted to the pleomorphic ILC subtype Tumor grade in IDC and ILC HER-2–positive status in low-grade tumors extremely rare Paget’s disease Virtually all Paget’s disease cases are HER-2 positive Inflammatory carcinoma No association between HER-2 status and inflammatory carcinoma confirmed to date Mucinous (colloid) carcinoma Rare HER-2–positive mucinous carcinomas pursue an aggressive clinical course Medullary carcinoma Typical medullary carcinomas are HER-2 negative Primary versus metastatic carcinoma A near uniform consensus of multiple published studies states that the HER-2 status of matched primary and metastatic breast cancer samples maintain the same HER-2 status throughout the course of the disease in (at least) 70%–80% of cases BRCA1/BRCA2 mutation–associated carcinomas Hereditary breast cancer consistently features a lower incidence of HER-2 positivity than sporadic disease Breast sarcomas These tumors are HER-2 negative Male breast carcinoma No consistent association of HER-2 status or response to anti–HER-2 targeted therapy for male breast cancer; the low number of cases limits confidence in these observations Benign breast conditions A near significant association with low-level HER-2 expression in benign breast biopsies with subsequent development of invasive breast cancer has been reported Abbreviations: DCIS, ductal carcinoma in situ; HER-2, human epidermal growth factor receptor 2; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma. result in a reduction in the development of invasive disease, and HER-2 overexpression have been associated with ad- the widespread use of HER-2 testing in DCIS would be verse outcome in some studies of male breast carcinoma adopted. Finally, the invasive carcinoma that develops in [126 –129], but not in others [130 –132]. The incidence of association with HER-2–positive DCIS may, on occasion, HER-2 positivity appears to be lower in male breast cancer not feature a HER-2–positive status, a finding that has led than in female breast cancer [126 –132]. Documented re- investigators to believe that HER-2 gene amplification may sponses in male breast cancer to HER-2–targeting agents not be required for the local progression of breast cancer have been described, and therefore treatment with trastu- [122]. Compared with invasive ductal carcinoma (IDC), zumab is an acceptable option for these patients, but the true HER-2 gene amplification occurs at a significantly lower activity rate remains uncertain [133]. The rate of HER-2 rate in invasive lobular carcinoma (ILC) (10%), but has overexpression in mucinous (colloid) breast cancers is ex- also been linked to an adverse outcome [85]. HER-2 posi- tremely low, although, on occasion, it has been associated tivity is linked exclusively to the pleomorphic variant of with aggressive disease [134 –136]. In medullary breast ILC and is not encountered in classic ILC [123]. HER-2 am- carcinoma, HER-2 testing has consistently found negative plification is strongly correlated with tumor grade in both results [137]. Similarly, HER-2 positivity is extremely rare IDC and ILC. For example, in one study, only one of 73 in cases of tubular carcinoma [138]. HER-2 status has not grade I IDC cases and one of 67 low-grade classic ILC cases been consistently linked to the presence of inflammatory showed HER-2 amplification detected by FISH [86]. breast cancer [139, 140]. Molecular studies of hereditary HER-2 overexpression and HER-2 amplification have been breast cancer including cases with either BRCA1 or BRCA2 a consistent feature of both mammary and extramammary germline mutations have found a consistently lower inci- Paget’s disease [124, 125] (Fig. 2). HER-2 amplification dence of HER-2–positive status for these tumors [141]. Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 331 Figure 2. Human epidermal growth factor receptor (HER)-2–positive Paget’s disease of the nipple. In this patient, who presented with HER-2–positive invasive duct carcinoma, classic clinical features of Paget’s disease of the nipple were present. A section of the nipple from the mastectomy specimen shows 3 continuous cell membrane immunoreactivity for HER-2 protein using the Ventana Pathway immunohistochemistry assay (Ventana Medical Systems, Inc., Tucson, AZ). Nearly 100% of Paget’s disease of the breast cases are HER-2 positive (see text). Breast sarcomas and phyllodes tumors have consistently directed therapy, but also happens in the absence of such been HER-2 negative [142]. Finally, low-level HER-2/neu treatment. In cases where the original primary HER-2 test overexpression has been identified in benign breast disease result is questioned because of technical or interpretive is- biopsies and is associated with a greater risk for subsequent sues and in patients where there has been an unusually long invasive breast cancer [143]. (i.e., 5-year) interval between the primary occurrence and the detection of metastatic disease, retesting of a metastatic HER-2 Status in Primary Versus Metastatic lesion may be warranted. Thus, although routine HER-2 Breast Cancer testing of metastatic disease is advocated by some investi- The majority of studies that have compared the HER-2 sta- gators, the preponderance of data indicates that the HER-2 tus in paired primary and metastatic tumor tissues have status remains stable and that routine retesting of HER-2 found an overwhelming consistency in the patient’s status may not be needed for most patients with metastatic dis- regardless of the method of testing (IHC versus FISH) ease. [144 –151]. However, several recent studies indicated 20%–30% discordance rates between the HER-2 status of Features of Metastatic HER-2–Positive primary and metastatic lesions. Some of these studies have Breast Cancer featured relatively high HER-2–positive rates on both Metastatic HER-2–positive breast cancer retains the pheno- paired specimens (35% positive), which has created con- type of the primary tumor not only in HER-2 status, but also cern about the conclusions of these reports [152]. Also, con- is typically ER/PgR negative, moderate to high tumor sidering that 10%–30% discordance rates have been grade, DNA aneuploid with high S phase fraction, and fea- reported even when the same tumor is tested repeatedly, it turing ductal rather than lobular histology. In the era prior to remains uncertain if the discordance rates seen between pri- the initiation of HER-2–targeted therapy, HER-2–positive mary and metastatic sites is higher than expected by the less breast cancer was more likely to spread early to major vis- than perfect reproducibility of the various HER-2 assays. ceral sites including the axillary lymph nodes, bone mar- Increasingly, emerging data suggest that there are changes row, lungs, liver, adrenal glands, and ovaries [153]. In the in HER-2 expression between primary and metastatic dis- post–HER-2 targeted therapy era, the incidence of progres- ease. This is particularly true after intervening HER-2– sive visceral metastatic disease in HER-2–positive tumors Downloaded from by DeepDyve user on 01 February 2022 332 HER-2 in Breast Cancer has diminished and has frequently been superseded by the ing (1), cells containing approximately 500,000 receptors development of clinically significant central nervous sys- would show light to moderate complete membrane staining tem (CNS) metastatic disease [154 –157]. It is widely held in 10% of the cells (2), and cells containing approxi- that the success in the control of visceral disease with tras- mately 2,300,000 receptors would show strong, complete tuzumab has unmasked previously occult CNS disease and, membrane staining in 10% of the cells (3). Studies have because of the inability of the therapeutic antibody to cross shown that when a standardized IHC assay is performed on the blood– brain barrier, allowed brain metastases to specimens that are carefully fixed, processed, and embed- progress during the extended OS duration of treated pa- ded, there is good to excellent correlation between gene tients [154, 155]. The small-molecule drug lapatinib has copy status and protein expression levels [162–165]. How- shown some promise for targeting HER-2–positive CNS ever, the ability to accurately determine HER-2 protein ex- metastases that are resistant to trastuzumab-based therapies pression status by IHC can be significantly impacted by in initial studies [158]. technical issues accentuated by the tissue fixation in form- aldehyde, tissue processing, and embedding procedure in Interaction of HER-2 Expression with Other heated paraffin wax. Advantages of IHC testing include its wide availability, relatively low cost, easy preservation of Prognosis Variables HER-2 gene amplification and protein overexpression have stained slides, and use of a familiar routine microscope. been associated consistently with high tumor grade, DNA Disadvantages of IHC include the impact of preanalytic is- aneuploidy, high cell proliferation rate, negative assays for sues including storage, duration and type of fixation, inten- nuclear protein receptors for estrogen and progesterone, sity of antigen retrieval, type of antibody (polyclonal versus p53 mutation, topoisomerase IIa amplification, and alter- monoclonal), lack of a positive internal control signal, vari- ations in a variety of other molecular biomarkers of breast ability in system control samples, and, most importantly, cancer invasiveness and metastasis [159 –161]. the difficulties in applying a semiquantitative subjective slide-scoring system. The IHC detection rates for HER-2 HER-2 Testing Techniques protein can vary considerably based on the antibody cho- A series of morphology-driven, slide-based assays de- sen, as shown in a study using a large tissue block contain- signed to detect HER-2 amplification and HER-2 overex- ing multiple breast tumors [166]. Problems with IHC pression and a group of in vitro laboratory HER-2 standardization in slide scoring have been emphasized in diagnostics performed on breast tumors and peripheral studies of patient response to trastuzumab [167]. Slide scor- blood are summarized in Table 3. ing can be improved by avoiding overinterpretation of spec- imen edges, retraction artifacts, under- or overfixation Slide-Based Assays artifacts, cases with significant staining of benign ductal and lobular cells, cytoplasmic tumor cell staining, and IHC. IHC was used as the clinical trial assay (CTA) in the membranous tumor cell staining that lacks a complete cir- phase III trial that led to the U.S. Food and Drug Adminis- cumferential staining pattern (the so-called “chicken wire” tration (FDA) approval of trastuzumab for the treatment of appearance). Data presented by the National Surgical Ad- HER-2– overexpressing metastatic breast cancer (MBC). juvant Breast and Bowel Project (NSABP) initially favored IHC staining remains the most frequent initial test for the idea that laboratories performing high-volume HER-2 HER-2 status and is performed on approximately 80% of testing produced a higher concordance between IHC and newly diagnosed breast cancers in the U.S. Unlike most FISH results (approaching 98% interlaboratory concor- IHC assays, the assessment of HER-2 status is quantitative dance when tumors assessed as 3 were reanalyzed by both rather than qualitative, because HER-2 is expressed in all IHC and FISH) when compared with central laboratory breast epithelial cells. In order to provide a meaningful in- testing at the NSABP laboratory [168]. Because most of the terpretation of a HER-2 immunostain, it was necessary to submitting laboratories were reference laboratories that establish a relationship between the number of HER-2 re- cannot control tissue fixation or storage, it has been sug- ceptors on a cell’s surface and the distribution and intensity gested that preanalytical issues may not be the major cause of the immunostain (Fig. 3A). Using cell lines, it was pos- of interlaboratory variability. In the United Kingdom, it has sible to establish a standardized IHC procedure and scoring been recommended that HER-2 testing be restricted to lab- system in which cells containing 20,000 receptors would oratories undertaking an annual minimum of 250 IHC tests show no staining (0), cells containing approximately (and/or 100 FISH tests) [169]. Results from the United 100,000 receptors would show partial membrane staining Kingdom National External Quality Assessment Scheme with 10% of the cells showing complete membrane stain- for Immunohistochemistry also suggested that the lack of Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 333 Table 3. Summary of HER-2 tests for breast cancer Included ASCO–CAP Year Current in drug Test FDA test result recommended test Test Manufacturer introduced FDA status commercial status label Method target Test format report result report IHC CTA Genentech, South San Francisco, 1995 Not submitted NA No IHC (CB-11  4D5 Protein Clinical trial only NA NA CA, antibodies) IHC HercepTest™ Ventana Medical Systems, 1998 Approved On the market Yes IHC (polyclonal goat Protein Decentralized in clinical 2 and 3 staining, 0, 1 staining, negative; Tucson, AZ, http:// (PMA) antibody) and commercial labs positive 2 staining, equivocal; 3 staining, positive IHC Pathway™ Ventana Medical Systems, 2002 Approved On the market No IHC (CB-11 replaced Protein Decentralized in clinical 2 and 3 staining, 0, 1 staining, negative; Tucson, AZ, http:// (PMA) with 4B5 in 2008) and commercial labs positive 2 staining, equivocal; 3 staining, positive FISH Inform™ Ventana Medical Systems, 1997 Approved On the market No FISH (HER-2 probe) Gene Decentralized in clinical 4.0 HER-2 gene 4.0 HER-2 gene Tucson, AZ, http:// (PMA) and commercial labs signals/nucleus, signals, negative; 4.0– positive 6.0 HER-2 gene signals, equivocal; 6.0 HER-2 gene signals, positive FISH Abbott Molecular, Abbott Park, 2002 Approved On the market Yes FISH (HER-2  Gene Decentralized in clinical 2.0 ratio of HER-2 2.0 HER-2/CEP17 PathVysion™ IL, http:// (PMA) CEP17 probes) and commercial labs gene signals/CEP17, ratio, negative; 2.0–2.2 positive HER-2/CEP17 ratio, equivocal; 2.2 HER-2/ CEP17 ratio, positive FISH Her2 Dako, Golstrup, Denmark, http:// 2005 Approved On the market No FISH (HER-2  Gene Decentralized in clinical 2.0 ratio of HER-2 Not included PharmDx™ (PMA) CEP17 probes) and commercial labs gene signals/CEP17, positive CISH SpotLight Invitrogen, Carlsbad, CA, http:// 2008 Approved On the market No FISH (HER-2 probe) Gene Decentralized in clinical 5.0 HER-2 gene 5.0 gene signals, (PMA) and commercial labs signals/nucleus, negative; 5.0–10.0 gene positive signals, positive (low amplification); 10.0 gene signals, positive (high amplification) SISH EnzMet™ Ventana Medical Systems, 2008 Pending In development No FISH (HER-2  Gene Pending NA Two-probe system Tucson, AZ, http:// CEP17 probes) pending; 6.0 gene signals, negative; 6.0 gene signals, positive mRNA Genomic Health, Redwood City, 2005 Not approved On the market No RT-PCR (paraffin) mRNA Centralized at company NA NA OncotypeDX™ CA, http:// headquarters mRNA Various 2005 Not approved Homebrews No RT-PCR (fresh/frozen); mRNA Decentralized in clinical NA NA microarray and commercial labs (fresh/frozen) Dimerization Monogram Biosciences, South 2008 Not approved On the market No VeraTag™; capillary Protein Decentralized in clinical NA NA HERmark™ San Francisco, CA, electrophoresis dimers and commercial labs ELISA serum Siemens Healthcare Diagnostics, 2002 Approved On the market No Sandwich Protein Decentralized in clinical 15 ng/ml NA HER-2 Advia Deerfield, IL, http:// (PMA) immunoassay (serum) and commercial labs Centaur™ Multiple HER-2 tests are included in the trastuzumab label. The lapatinib label does not include specific recommendations as to which HER-2 test must be used. Abbreviations: ASCO–CAP, American Society of Clinical Oncology–College of American Pathologists; CEP17, chromosome 17 centromere probe; CISH, chromogenic in situ hybridization; CTA, clinical trial assay; ELISA, enzyme-linked immunosorbent assay; FISH, fluorescence in situ hybridization; HER-2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; NA, not applicable; PMA, premarket approval; RT-PCR, real-time polymerase chain reaction; SISH, silver in situ hybridization. Downloaded from by DeepDyve user on 01 February 2022 334 HER-2 in Breast Cancer Figure 3. Human epidermal growth factor receptor (HER)-2 testing. (A): Immunohistochemistry (IHC). This panel depicts the four categories of HER-2 IHC staining including 0 and 1 (negative), 2 (equivocal), and 3 (positive) using the American Society of Clinical Oncology–College of American Pathologists guidelines for HER-2 IHC scoring. (B): Fluorescence in situ hybridization (FISH). This panel demonstrates a case of invasive duct carcinoma, on the left, negative for HER-2 gene amplifi- cation (gene copy number 4) and a case of HER-2 gene–amplified breast cancer (gene copy number 6), on the right, using the Ventana Inform single probe system (Ventana Medical Systems, Inc., Tucson, AZ). (C): True negative HER-2 IHC. In this image, the patient’s tumor is negative (0) for HER-2 by IHC (Ventana Pathway™, Ventana Medical Systems, Inc., Tucson, AZ). Note the 3 positive control section from another patient with known HER-2 3 positive disease in the red control box to the left confirming that the staining procedure for the current patient was performed properly. (D): Pitfall in HER-2 gene amplification testing by FISH. In this panel, the central portion of the mixed ductal carcinoma in situ (DCIS) and invasive breast cancer is HER-2 gene amplified (inset to the left). However, this area is the in situ carcinoma component and should not be scored or reported for HER-2 gene copy number. The invasive portion of the tumor seen to the right, where the HER-2 gene copy number should be calculated, is HER-2 unamplified (inset to the right). Cases such as this one can, on occasion, lead to a false conclusion that HER- 2–amplified breast cancer may frequently metastasize as HER-2– unamplified disease when, in fact, the invasive carcinoma was not HER-2 amplified to begin with. (E): Chromogenic in situ hybridization (CISH). This image depicts an invasive duct carcinoma with significant HER-2 gene amplification determined by the Invitrogen SpotLight CISH assay (Invitrogen, Inc., Carlsbad, CA). Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 335 reproducibility of HER-2 scoring between laboratories was IHC Versus FISH. Although the FISH method is more ex- pensive and time-consuming than IHC, numerous studies not the result of tumor heterogeneity or differences in fixa- have concluded that this cost is well borne by the greater tion or processing but rather the result of how the scoring accuracy and more precise use of anti–HER-2 targeted ther- system was applied [170]. The use of a quantitative image apies [179 –180, 182–183]. FISH is considered to be more analysis system can reduce slide-scoring variability among objective and reproducible in a number of systematic re- pathologists, especially in 2 cases [171]. When 130 HER- views [165, 180, 183–186]. In one study, the concordance 2–immunostained slides were reviewed by 10 pathologists rates between IHC and FISH were highest in tumors scored and then were later reviewed with the aid of image analysis, by IHC as 0 and 1 and lowest for 2 and 3 cases [183]. the use of image analysis eliminated most of the interob- Currently, the majority (approximately 80%) of HER-2 server variability that was significant by routine micros- testing in the U.S. commences with a screen by IHC, with copy [172]. Thus, in routine clinical practice, errors in results of 0 and 1 considered negative, 2 considered HER-2 testing by the IHC method are caused by both vari- equivocal and referred for FISH testing, and 3 considered ables associated with antigen retrieval and the reagents and positive. In a pharmacoeconomic study of patients being staining protocol and variation in the actual slide scoring considered for trastuzumab-based treatment for HER-2– [173, 174]. Two commercially available HER-2 IHC kits, positive tumors, FISH was found to be a cost-effective di- the Dako HercepTest™ (Dako Corporation, Glostrup, Den- agnostic approach “from a societal perspective” [187]. mark) and the Ventana Pathway™ (Ventana Medical Sys- tems, Tucson, AZ), are approved by the FDA for Pitfalls in IHC and FISH Test Interpretation. In addi- determining the eligibility of patients to receive trastu- tion to the preanalytic variables and issues with the subjec- zumab therapy (Table 3). tive scoring system for IHC, a number of additional pitfalls in IHC test interpretation must be considered. In order to FISH. The FISH technique (Fig. 3B), like IHC, is a mor- avoid false-positive IHC results, pathologists must learn to phology-driven slide-based DNA hybridization assay using avoid scoring specimen edges, areas of tissue thermal in- jury from cautery, cases with cytoplasmic staining, fibro- fluorescent-labeled probes. Both the hybridization steps cystic disease with apocrine metaplasia, and intraductal and the slide scoring can be automated. FISH has the ad- (DCIS) foci. Although some investigators have favored a vantages of a more objective scoring system and the pres- normalization approach, most experts hold that, when be- ence of a built-in internal control consisting of the two nign elements stain for HER in IHC procedures, either the HER-2 gene signals present both in benign cells and in ma- antigen retrieval process was overly intense or the anti– lignant cells that do not feature HER-2 gene amplification. HER-2 diagnostic antibody concentration was excessive The disadvantages of FISH testing include the higher cost [188]. A major cause of false-negative IHC staining is ei- of each test, longer time required for slide scoring, require- ther reagent failure or failure of the antibody to be applied to ment of a fluorescent microscope, inability to preserve the the tissue. The most successful approach to avoiding this slides for storage and review, and greater difficulty in as- problem is to stain the newly diagnosed breast cancer on a sessing background morphology such as in distinguishing slide that has had a 3 HER-2–positive breast cancer from in situ from invasive tumor. Three versions of the FISH as- another patient (positive control) previously placed on the say are FDA approved. The single-probe Ventana Inform™ same slide (Fig. 3C). test (Ventana Medical Systems) that measures only HER-2 Given the inability to recognize detailed background gene copies is approved as a prognostic test. The two dual- morphology during signal counting, a potential cause of probe (HER-2 probe plus chromosome 17 centromere false-positive FISH testing is the scoring of HER-2–ampli- probe) kits, the Abbott-Vysis PathVysion™ test (Abbott fied areas of DCIS in a tumor whose invasive carcinoma ar- Laboratories. Abbott Park, IL) and the Dako Cytomation eas lack HER-2 amplification (Fig. 3D). In that the Her2 PharmDx™ test (Dako Corporation), are approved technique features a built-in internal control system, false- both as prognostic tests and for the selection of patients for negative FISH results are rare but may occur when the slide trastuzumab-based therapies. Published studies indicate scorer fails to identify the amplified regions in a tumor with that the single-probe and dual-probe assays are highly cor- heterogeneity in HER-2 gene amplification. HER-2 gene related [175]. Although controversial, a group of investiga- amplification can be heterogeneous in a significant subset tors strongly favors FISH as being more accurate and of HER-2–positive invasive breast cancers, requiring dili- reliable than IHC in the classification of HER-2 status for gence and care on the part of the slide scorer when scanning breast cancer [176 –181]. the case at low magnification [189]. Downloaded from by DeepDyve user on 01 February 2022 336 HER-2 in Breast Cancer CISH and Silver In Situ Hybridization. The CISH trials [168 –170, 184, 211]. In general, these studies have method (Fig. 3E) and silver in situ hybridization (SISH) indicated that laboratories performing high-volume testing, method feature the advantages of both IHC (routine micro- that is, 10 tests per week, provide more accurate HER-2 scope, lower cost, familiarity) and FISH (built-in internal results based on concordance with central laboratory testing control, subjective scoring, the more robust DNA target) results. Although this issue has likely been significantly [190, 191]. The CISH technique uses a single HER-2 probe, mitigated by the incorporation of the American Society of detects HER-2 gene copy number only, and was recently Clinical Oncology–College of American Pathologists approved by the FDA to define patient eligibility for tras- (ASCO-CAP) HER-2 testing guidelines program (see be- tuzumab treatment. The SISH method employs both HER-2 low), it should be noted that the central laboratory HER-2 and chromosome 17 centromere probes hybridized on sep- test result may not always be the correct one. For example, arate slides and is currently under review by the FDA. Nu- when the central laboratory exclusively performs ratio- merous studies have confirmed a very high concordance based FISH testing, discrepancies may be caused by HER-2 between CISH and FISH, typically in the 97%–99% range overexpression associated with chromosome 17 polysomy. [191–203]. Similar to FISH, CISH has its highest correla- Also, the central laboratory may not always receive an ap- tion with IHC 0, 1, and 3 results and lowest correlation propriate sample to retest and the preparation and shipment with IHC 2 staining. of the patient’s tissue, paraffin block, or unstained slides may inadvertently expose the tissue to factors that can de- Chromosome 17 Polysomy. The incidence of chromo- grade the HER-2 signal. some 17 polysomy has varied from as low as 4% to as high as 30% in studies of invasive breast cancer [204 –208]. The 2007 ASCO-CAP Guidelines. In early 2007, a com- This may reflect differences in the definition of polysomy bined task force from ASCO and the CAP issued a series of ranging from a low-level definition of more than two copies recommendations designed to improve the accuracy of tis- per cell to a high of more than four copies per cell of the sue-based HER-2 testing in breast cancer [212]. A sum- chromosome. Most studies have linked chromosome 17 mary of the ASCO-CAP guidelines is provided in Table 4. polysomy with greater HER-2 protein overexpression Highlights of these recommendations include (a) standard- [204 –207], but some have found that protein overexpres- izing fixation in neutral-buffered formalin for no less than 6 sion only occurs in the presence of selective HER-2 gene hours and no more than 48 hours, (b) unlike their respective amplification [204]. In one study, 27% of cases featured FDA-approval specifications, defining equivocal zones for chromosome 17 polysomy, and 35% of these patients re- the IHC, FISH, and CISH tests, (c) establishing a standard- sponded to trastuzumab-based treatment [208]. The re- ized quality assurance program for testing laboratories, and sponding patients were restricted to cases that also had 3 (d) requiring the participation of these laboratories in a pro- IHC staining. Another clinical outcome study, in patients ficiency testing program [212]. The published guidelines with chromosome 17 copy numbers 2.2 detected by FISH were designed to improve the overall precision and reliabil- and HER-2/chromosome 17 ratios 2.0 (HER-2 unampli- ity of all types of slide-based HER-2 tests and remained fied), nonetheless featured a significant response rate to a neutral as to the relative superiority of one test over the trastuzumab-based regimen [209]. However, this associa- others. tion could not be confirmed for a lapatinib-based clinical trial [210]. Thus, at least for trastuzumab-based treatment Non–Slide-Based Assays of MBC, chromosome 17 polysomy may be a significant cause of the observation that some patients may test nega- Southern and Slot Blotting. These techniques, which tively for HER-2 gene amplification by ratio-based FISH measure the relative HER-2 DNA extracted from fresh tu- analysis and still respond to the drug. Finally, although not mor samples, were the original methods used to confirm as yet validated in large prospective trials, it should be that HER-2 amplification was an adverse prognostic factor noted that positive responses to HER-2–targeted therapy in in breast cancer [11]. patients with tumors that are polysomic for chromosome 17 appear to be restricted to tumors that are 3 by IHC testing. RT-PCR. Relative HER-2 mRNA levels measured by the RT-PCR technique (Fig. 4) have shown better correlation Central Versus Local Laboratory Testing. As mentioned with HER-2 gene amplification results detected by FISH above, several studies have considered the impact of local than with HER-2 protein levels determined by IHC [213]. laboratory HER-2 testing in comparison with results ob- To date, large clinical outcome studies have not been per- tained at central laboratories associated with major clinical formed to confirm that the RT-PCR method can reliably Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 337 mRNA by Microarray. In the original classification of Table 4. ASCO–CAP guidelines for HER-2 testing: breast cancer by molecular portraits using dense DNA mi- sources of HER-2 testing variation croarray– based relative mRNA measurements, HER-2– Preanalytic variables positive tumors fell into multiple classes, including the Type and time of fixation HER-2 and luminal groups, but not the normal or basal (tri- Method of tissue processing ple-negative) categories [10]. The HER-2 gene is typically Analytic variables amplified as part of an amplicon that includes multiple ad- Assay validation jacent genes (Fig. 5). In the various multigene predictor as- Equipment calibration says that have been commercialized for use in breast cancer Training and competency of staff management, the Oncotype DX™ test uses a direct mea- Type of antigen retrieval surement of the HER-2 mRNA level using RT-PCR [218, Test reagents 219]. The recently developed TargetPrint™ assay (Agendia Use of standardized control materials BV, Amsterdam, The Netherlands) measures ER, PgR, and Use of automated methods HER-2 mRNA levels on a custom microarray. Other mul- tigene predictors may use the expression of other genes di- Postanalytic variables rectly related to HER-2 expression (HER-2 pathway genes) Interpretation criteria to determine breast cancer risk [219]. In a recent microar- Use of image analysis ray-based study using fresh tissues and the U-133 genechip Reporting elements (Affymetrix, Santa Clara, CA), it was concluded that both Quality assurance procedures ER and HER-2 mRNA could be easily and reliably deter- Laboratory accreditation mined by this method [220]. Thus, like the multiplex RT- Proficiency testing PCR technique, genomic microarrays hold promise as Pathologist competency assessment potential multigene assays that can deliver routine prognos- Abbreviations: ASCO–CAP, American Society of tic and complex pharmacogenomic information for individ- Clinical Oncology–College of American Pathologists; HER-2, human epidermal growth factor receptor 2. ualized patient management. Dimerization Assays. In several recent studies, the use of a predict response to HER-2–targeting agents. Nonetheless, method for determining the HER-2 receptor dimerization there is growing interest in using mRNA levels to measure status by quantifying the number of HER-2 homodimers HER-2 status in breast cancer patients. RT-PCR is a rela- has predicted potential resistance to trastuzumab [221, tively low-cost technique that could be used as a rapid 222]. This approach in combination with a direct measure- screening method for establishing HER-2 mRNA status in ment of HER-2 receptor number has recently been commer- concert with measurements of ER, PgR, and cell prolifera- cialized (HERmark™; Monogram Biosciences, South San tion (Ki-67). However, being a non–morphology-driven Francisco, CA). non–slide-based approach, RT-PCR must be performed carefully on suitable areas of intact invasive cancer guided Phosphorylated HER-2 Receptors. Activation of the by examination of slides to confirm sample suitability HER-2 receptor by autophosphorylation has not been [214 –217]. HER-2 mRNA levels can be readily assessed widely studied in clinical breast cancer samples. Monoclo- on formalin-fixed, paraffin-embedded breast cancer sam- nal antibodies have been developed to detect autophosphor- ples as evidenced by the Oncotype DX™ (Genomic Health, ylated HER-2 by IHC [223]. In invasive breast cancer with Redwood City, CA) multigene predictor RT-PCR assay HER-2 overexpression, the receptor appears to be activated [218, 219]. HER-2 is one of the 21 gene expression mea- only in a small subset (12%) of patients [223, 224]. Inter- surements in the Oncotype DX™ test, and HER-2 mRNA estingly, the proportion of cases with phosphorylated individual determination has, on occasion, been used sepa- (phospho)–HER-2 appears to be greater (58%) in DCIS rately to assist in the resolution of cases in which initial [225]. In one large study of 800 cases of invasive breast HER-2 testing by IHC, FISH, and CISH has not been con- cancer with HER-2 overexpression, only cases with phos- clusive as to the true HER-2 status of the tumor. However, pho–HER-2 displayed an adverse prognosis [224]. Cases this approach has not been validated in a prospective trial with overexpressed but unphosphorylated receptor had a and the response rate to HER-2–targeted therapies in pa- prognosis as favorable as non–HER-2 overexpressing tients whose HER-2 status is determined in this manner is cases, which supports the concept that phospho–HER-2 not currently known. may be a more powerful prognostic marker than overall Downloaded from by DeepDyve user on 01 February 2022 338 HER-2 in Breast Cancer Figure 4. Real-time polymerase chain reaction (RT-PCR). In this RT-PCR assay using the Taqman RT-PCR System (Applied Biosystems Inc., Foster City, CA), note the detection of increased human epidermal growth factor receptor(HER)-2 mRNA ex- pression in green detected at lower numbers of amplification cycles compared with the two housekeeping genes shown in red and blue. Figure 5. DNA microarray. In this image, increased expression of human epidermal growth factor receptor (HER)-2 mRNA has been detected using a proprietary DNA microarray system (Millennium Pharmaceuticals, Inc., Cambridge, MA). The microarray demonstrates the coexpression of seven genes (HER-2 is second from the bottom) related to the amplification of HER-2 DNA in this case of HER-2–positive breast cancer. HER-2 protein overexpression. Activated HER-2 status has damage associated with fixation, embedding, and uncon- been associated with resistance to taxane-based therapies trolled storage. In the six published studies listed in Table 1, [226]. When IHC is used as the method of detection of ELISA-based measurements of HER-2 protein in tumor cy- HER-2 receptor phosphorylation, excess antibody concen- tosols, mostly performed in Europe, are uniformly corre- tration or overintense antigen retrieval exposure can cause lated with disease outcome. However, the small size of the antiphospho–HER-2 antibody to lose specificity and be- breast cancers associated with expanded screening pro- gin to detect wild-type HER-2 receptor. The role of phos- grams in the U.S. generally precludes tumor tissue ELISA pho–HER-2 as a predictor of trastuzumab therapy response methods because insufficient tumor tissue is available to is currently unknown. produce a cytosol. The serum ELISA test measures the concentration of the Tissue and Serum Enzyme-Linked Immunosorbent extracellular domain (ECD) of the HER-2 protein Assay. The tissue enzyme-linked immunosorbent assay (p185neu) in circulation. This assay has been approved by (ELISA) technique when performed on tumor cytosols the FDA for the monitoring of HER-2–positive breast can- made from fresh tissue samples avoids the potential antigen cers, including the identification of disease relapse and on- Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 339 going response to HER-2–targeted therapies [227]. the primary tumor was HER-2 negative [257, 258]. The Attempts to use this serum-based test as the sole classifier methodological differences in assessing HER-2 status in of HER-2 status for newly diagnosed cases have not been the primary tumor versus in CTCs may at least partially ac- widely accepted, although fairly good correlation exists be- count for these discrepant results. The different CTC tech- tween serum HER-2 ECD levels and the results of IHC and niques have influenced the capability of performing HER-2 FISH assessments on primary tumor tissues [228]. It has testing with the immunomagnetic bead cell capture tech- been recommended that a 37g/l serum HER-2 ECD cutoff nique, requiring slide-based assays such as the FISH and be used, which can achieve 95% specificity but low sensi- RT-PCR techniques, with or without immunomagnetic- tivity for HER-2–positive status determined on primary tu- based cellular enrichment, claiming an enhanced sensitivity mors [229]. Studies of breast cancer prognosis based on based on relative HER-2 mRNA measurements. Novel the serum ECD test have been conflicting, with some techniques are being developed to increase the yield of finding significant correlation [116] and others finding CTCs in a typical blood sample in order to facilitate more weak or no correlation [230]. In 22 published studies on accurate biomarker testing and the use of additional assess- 4,088 patients, 16 (73%) studies involving 3,458 (85%) ment techniques including transcriptional profiling [256, of the patients reported a significant correlation of serum 259 –261]. HER-2 protein levels with disease recurrence, metasta- sis, or shorter survival [231–247]. Two studies involving SECTION TWO: HER-2–TARGETED THERAPY AND 379 patients reported no significant association of serum THE TREATMENT OF HER-2–POSITIVE levels with prognosis [248, 249]. Of the 11 studies in BREAST CANCER which serum HER-2 protein levels were tested for their ability to predict response to therapy, eight (73%) of the Trastuzumab: HER-2 Testing and the Prediction studies found that elevated serum HER-2 protein levels of Response to Trastuzumab Therapy predicted therapy resistance [234, 242–244, 247–249], Using recombinant technologies, trastuzumab (Hercep- whereas three additional studies did not demonstrate this tin; Genentech, South San Francisco, CA), a monoclo- association [234, 249, 250]. Serum HER-2 levels have nal IgG class humanized murine antibody, was been correlated with shorter survival and the absence of developed by the Genentech Corporation to specifically clinical response to hormonal therapy in ER-positive tu- bind the extracellular portion of the HER-2 transmem- mors in some studies [239, 247], but not in others [249]. brane receptor. This antibody therapy was initially tar- Serum HER-2 protein measurements have successfully geted specifically for patients with advanced relapsed predicted resistance to high-dose chemotherapy [242– breast cancer that overexpresses HER-2 protein [262]. 244], bone marrow transplantation [243], and response Since its launch in 1998, trastuzumab has become an im- to trastuzumab single-agent and combination treatment portant therapeutic option for patients with HER-2–pos- for metastatic HER-2–positive disease [251, 252]. In itive breast cancer and is widely used for its approved general, the test is advocated by some oncologists for the indications in both the adjuvant and metastatic settings continuous monitoring of patients with HER-2–positive (Fig. 6) [185, 263–265]. Although trastuzumab is ap- disease undergoing anti–HER-2 targeted therapy [231]. proved as a single-agent regimen, most patients are Nonetheless, the HER-2 serum ELISA test continues to treated with trastuzumab plus cytotoxic agents. Table 5 be regarded as “under investigation” and has not, to date, summarizes the significant clinical trials that contributed been validated as a biomarker in large prospective clini- to the regulatory approvals of trastuzumab. cal trials. Metastatic Disease Setting Circulating Tumor Cells. The counting of circulating tu- Using a clinical trial IHC assay to select patients for the mor cells (CTCs) as a predictor of response to breast cancer phase III pivotal trial, the addition of trastuzumab to che- chemotherapy in the metastatic disease setting has been motherapy (either an anthracycline plus cyclophosphamide consistently validated in prospective studies [253–256]. or a taxane) was associated with a longer time to disease The use of captured CTCs for the purpose of determining progression (median, 7.4 versus 4.6 months; p  .001), a HER-2 status, however, has been controversial [257, 258]. higher rate of objective response (50% versus 32%; p Some studies have found that CTCs maintain the same .001), a longer duration of response (median, 9.1 versus 6.1 HER-2 status, typically assessed by the FISH technique, as months; p  .001), a lower rate of death at 1 year (22% ver- the primary tumor assay, whereas other reports have sus 33%; p  .008), a longer survival duration (median sur- claimed that CTCs may be HER-2 positive in cases where vival time, 25.1 versus 20.3 months; p  .01), and a 20% Downloaded from by DeepDyve user on 01 February 2022 340 HER-2 in Breast Cancer 1983 The "neu" oncogene is discovered by Weinberg and associates and associated with the erb family of tyrosine kinase receptors 1987 Slamon links amplification of the "HER-2/neu" gene to adverse prognosis in node negative and node positive breast cancer 1995 Trastuzumab Phase III Pivot Clinical Trial for patients with Metastatic Breast cancer initiated TM 1997 FDA approved first HER2 diagnostic test: The Oncor/Ventana Inform FISH test for HER2 gene amplification 1998 FDA approves Trastuzumab for the treatment of metastatic breast cancer TM 1998 FDA approves the Dako Herceptest IHC assay to identify patients with breast cancers that over-express HER2 protein for potential trastuzumab therapy 2000 ASCO guidelines recommend that HER2 testing be performed on all newly diagnosed primary invasive breast cancers TM 2002 FDA approves Ventana Pathway IHC assay TM 2002 FDA approves the Siemens' Advia Centaur serum HER-2 assay TM 2002 FDA approves the Abbott/Vysis Pathvysion FISH assay to identify patients with breast cancers that feature HER2 gene amplification for potential trastuzumab therapy 2006 FDA approves Trastuzumab for treatment of lymph node positive breast cancer in the adjuvant setting 2007 FDA approves Lapatinib for patients with metastatic breast cancer that over-express the HER2 protein 2008 FDA approves Trastuzumab for treatment of lymph node negative breast cancer in the adjuvant setting 2008 FDA approves the Invitrogen Spot-LightTM CISH assay to identify patients with breast cancers that feature HER2 gene amplification for potential trastuzumab therapy Figure 6. Highlights in the development of human epidermal growth factor receptor (HER)-2 targeted therapy for breast cancer. This figure highlights the discovery and development of HER-2 targeted therapy. Abbreviations: ASCO, American Society of Clinical Oncology; CISH, chromogenic in situ hybridization; FDA, U.S. Food and Drug Administration; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry. lower risk for death [266]. Cardiac dysfunction occurred in binding murine component of Herceptin and is not com- 27% of the anthracycline and cyclophosphamide plus tras- mercially available, and (b) CB-11, a monoclonal antibody tuzumab treated group, compared with 8% of the group directed toward the internal domain of the p185neu recep- given an anthracycline and cyclophosphamide alone [266]. tor, which is commercially available both as a research re- Class III or IV cardiac dysfunction occurred in 16% of pa- agent and as an FDA-approved diagnostic (Ventana tients who received trastuzumab plus an anthracycline, ver- Pathway™). The original CTA was succeeded by the FDA- sus 2% of patients treated with trastuzumab plus paclitaxel approved polyclonal HercepTest™. There was moderate [267]. In a subsequent, randomized, multicenter trial, the concordance between the CTA and HercepTest™, although combination of trastuzumab and docetaxel produced addi- 58 of the 274 tumors that were scored as positive with the tional strong positive results in terms of OS, response rate, CTA were scored as negative with the HercepTest™ and 59 duration of response, and time to treatment failure com- of the 274 tumors that were scored as negative with the pared with docetaxel treatment alone [268]. CTA were scored as positive with the HercepTest™ [269]. The original IHC technique used in the trastuzumab piv- After its FDA approval and launch, the HercepTest™ assay otal trial was the CTA, which consisted of two antibodies: was initially criticized for yielding false-positive results (a) 4D5, the monoclonal antibody that is the actual antigen- [270], although better performance was ultimately Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 341 Table 5. Major clinical trials in HER-2–targeted therapy for breast cancer Median Trial status and Enrollment n of duration of FDA HER-2 testing completion patients Trial design and Primary Secondary ORR (HR; response, Median OS, Median DFS, approval Trial name entry criteria date enrolled agents used endpoint endpoint p-value) mos mos TTP, mos TTF, mos mos pCR, % impact Reference Metastatic breast cancer Trastuzumab Randomized, phase 1998 469 First-line, 2-arm, OS RR, TTP, 50% (p  .001) 9.1 (p .001) 25.1 (HR, 7.4 (HR, 0.51 6.9 (HR, 0.58 Trastuzumab [266] trials H0468g III; HER-2 positive randomized, phase III trial duration of 0.80 0.64– 0.41–0.63 ; 0.47–0.70 ; approved in pivot trial defined as either comparing CT alone response, 1.00 ; p  p  .001) p  .001) 1998 for 2 or 3 staining (either doxorubicin or TTF .046) MBC by IHC using CTA epirubicin (4D5 and CB-11 cyclophosphamide or IHC antibodies) single-agent paclitaxel) with CT  trastuzumab M77001 Randomized, phase 2002 186 Randomized, multicenter, OS RR, TTP, 61% (p  .002) 11.7 (p  31.2 (p  11.7 (p  9.8 (p  [268] II; HER-2 positive multinational trial duration of .009%) .0325) .0001) .0001) defined as either comparing efficacy and response, FISH-positive for safety of first-line TTF amplification or trastuzumab plus IHC 3 for docetaxel with docetaxel overexpression alone in patients with HER-2–positive MBC Lapatinib Randomized, phase 2006 399 Capecitabine with or OS RR, TTP, 24% (HR, 1.9 7.4 (p  NS) 15.6 (HR, 6.2 (HR, 0.57 Lapatinib [332, 333] trials GSK- III; HER-2 positive without lapatinib in duration of 1.1–3.4 ; p  0.78 0.55– 0.43–0.77 ; approved in EGF100151, defined as either treating women with response .017) 1.12 ; p  p  .00013) 1997 for UCLA- FISH positive for locally advanced breast .177) MBC 0403074–01, amplification or cancer or MBC that has NCT00078572 IHC 3 for not responded to overexpression previous therapy Burstein et al. Phase II; HER-2 2006 140 Lapatinib monotherapy RR TTP, PFS 4.30% [334] positive defined by for patients who had 3 IHC or FISH progressed on positive trastuzumab Adjuvant treatment of breast cancer Trastuzumab Trial completed; joint 2004 3,351 NSABP B-31 compared DFS OS, NA NA 91% alive at 4 NA NA 85% disease-free Trastuzumab [280] trials NSABP analysis of 2 doxorubicin and toxicity yrs (HR, 0.67 at 4 yrs (HR, approved in B-31, NCCTG randomized adjuvant cyclophosphamide 0.48–0.93 ; 0.48 0.38–0.59 ; 1995 for N9831 trials; basis for FDA followed by paclitaxel p  .015) p  .0001) adjuvant approval of every 3 weeks (group 1) treatment of trastuzumab in the with the same regimen lymph node– adjuvant setting for plus 52 weeks of positive node-positive trastuzumab beginning breast cancer patients; HER-2 with the first dose of positive defined as paclitaxel (group 2); either FISH positive NCCTG N9831 compared for amplification or 3 regimens: doxorubicin IHC 3 for and cyclophosphamide overexpression in followed by weekly local or central lab paclitaxel (group A), the for NCCTG 9831 same regimen followed by and by approved 52 wks of trastuzumab reference lab only for after paclitaxel (group B), NSABP B-31 and the same regimen plus 52 wks of trastuzumab initiated concomitantly with paclitaxel (group C) (continued) Downloaded from by DeepDyve user on 01 February 2022 342 HER-2 in Breast Cancer Table 5. (Continued) Median Trial status and Enrollment n of duration of FDA HER-2 testing completion patients Trial design and Primary Secondary ORR (HR; response, Median OS, Median DFS, approval Trial name entry criteria date enrolled agents used endpoint endpoint p-value) mos mos TTP, mos TTF, mos mos pCR, % impact Reference HERA Trial completed; 2004 5,090 Patients received CT DFS Time to NA NA 96% alive at 2 NA NA 86% disease-free Trastuzumab [281] based in Europe; and radiation therapy distant yrs (HR, 0.76 at 2 yrs (HR, approved in HER-2 positive prior to enrollment; recurrence, 0.47–1.23 ; 0.54 0.43–0.67 ; 1996 for defined as either subsequently, patients OS, p  .26) p  .0001) adjuvant FISH positive for were randomized to toxicity treatment of amplification or IHC single-agent lymph node– 3 for trastuzumab or negative overexpression; local observation breast cancer lab with central lab verification; basis of FDA approval for single-agent trastuzumab (after CT and radiation) use in the adjuvant setting for lymph node–negative patients BCIRG 006 Trial ongoing; first 2009 3,222 Adjuvant trial comparing DFS OS, ACTH: 39% ACTH risk for [282] trial evaluating a (projected) doxorubicin plus toxicity, fewer deaths relapse at 23 nonanthracycline cyclophosphamide topoisomerase at second mos, 51% (35%– regimen in followed by docetaxel II status interim 63%) (HR, 0.61 combination with (ACT) to 2 analysis (HR, 0.48–0.76 ; p trastuzumab; HER- trastuzumab-based 0.59 0.42– .001); TCH risk 2 positive defined regimens: doxorubicin 0.85 ; p  for relapse at 23 by IHC 3 or plus cyclophosphamide .004); TCH: mos, 39% (21%– FISH positive by followed by both 26% fewer 53%) (HR, 0.67 central lab only; docetaxel and trastuzumab deaths at 0.54–0.83 ; p also testing impact (ACTH) and docetaxel second interim .003) of topoisomerase plus platinum salts plus analysis (HR, II amplification trastuzumab (TCH); 0.67 0.54– on response to ACTH approximately 0.83 ; p anthracycline doubled the cardiac .003) regimen; data not toxicity incidence of the mature ACT baseline group; the TCH regimen had approximately the same cardiac toxicity as the ACT group; patient age limited to 70 years FinHer Trial completed; 2005 1,010 Adjuvant trial comparing RFS OS, time to NA NA 96% alive at 3 NA NA 89% disease-free [283] trial based in the addition of distant yrs (HR, 0.41 at 3 yrs (HR, Finland; HER-2 trastuzumab to 2 CT recurrence 0.16–1.08 ; 0.42 0.21–0.83 ; positive defined as regimens (docetaxel p  .007) p  .01) gene amplification followed by detected by CISH cyclophosphamide, in approved epirubicin, and 5- reference labs fluorouracil or vinorelbine followed by cyclophosphamide, epirubicin and 5- fluorouracil) (continued) Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 343 Table 5. (Continued) Median Trial status and Enrollment n of duration of FDA HER-2 testing completion patients Trial design and Primary Secondary ORR (HR; response, Median OS, Median DFS, approval Trial name entry criteria date enrolled agents used endpoint endpoint p-value) mos mos TTP, mos TTF, mos mos pCR, % impact Reference Lapatinib Phase III; ALTTO, 2010 8,000 ALTTO is sponsored by OS TTP, RR, NA NA NA NA NA NA [335] trials ALTTO BIG 2–06/N063D; (estimated) the NCI, part of the U.S. safety HER-2 positive in NIH, and local labs (FISH, GlaxoSmithKline, the CISH, or IHC) maker of lapatinib, and defined by ASCO– is being coordinated by CAP guidelines; The Breast Cancer HER-2 equivocal Intergroup of North local lab results America (TBCI), based sent to central lab in the U.S., and BIG in Brussels, Belgium TEACH Phase III; lapatinib 2012 3,000 The objective of the DFS OS, RFS, NA NA NA NA NA NA [336] for 1 year versus TEACH trial is to toxicity, placebo; HER-2 determine whether quality of testing (FISH adjuvant therapy with life positive or IHC lapatinib for 1 year will 3) by central lab improve DFS in women with early-stage ErbB- 2–overexpressing breast cancer; eligible women must have completed adjuvant CT, be free of disease, and have either a new diagnosis and be unable or unwilling to receive trastuzumab or have a remote diagnosis of ErbB-2–overexpressing breast cancer and not have received prior trastuzumab Neoadjuvant treatment of breast cancer Trastuzumab Phase III; HER-2 Invasive operable breast pCR Control T3FEC Control T3FEC [294] trials Buzdar positive defined as cancer stages IIa to III; (HR, 94.7 85.2– (HR, 26.3 9.0– et al. 3 positive by IHC Ni inflammatory breast 100.0 ,1yr); 51.0 ); or FISH positive cancer allowed; patients TH3FECH TH3FECH treated with paclitaxel (HR, 100.0 (HR, 65.2 43.0– followed by FEC (T3 85.2–100.0 ,1 84.0 ; p  .016) FEC) compared with yr); p  .041) patients who received the same CT with 24 wks of simultaneous trastuzumab (TH3 FECH) (continued) Downloaded from by DeepDyve user on 01 February 2022 344 HER-2 in Breast Cancer Table 5. (Continued) Median Trial status and Enrollment n of duration of FDA HER-2 testing completion patients Trial design and Primary Secondary ORR (HR; response, Median OS, Median DFS, approval Trial name entry criteria date enrolled agents used endpoint endpoint p-value) mos mos TTP, mos TTF, mos mos pCR, % impact Reference Chang et al. Phase II; FISH T3 or T4, any N, M0; pCR Control (no [432] positive docetaxel and carboplatin trastuzumab), both preoperatively and 9%; trastuzumab, postoperatively; patients 36% were randomized to receive either postoperative trastuzumab only or both preoperative (concurrent with docetaxel and carboplatin) and postoperative trastuzumab GETN(A)-1 Phase II; IHC 3 2006 70 Patients with HER-2– pCR Trastuzumab  [292] or FISH positive positive, stage II/III, docetaxel noninflammatory, carboplatin, 43% operable breast cancer requiring a mastectomy (but who wanted to conserve the breast) received trastuzumab (4 mg/kg on day 1), followed by 2 mg/kg trastuzumab weekly, plus docetaxel (75 mg/m every 3 weeks) and carboplatin (area under curve, 6) for six cycles before surgery NOAH Phase III; HER-2 2006 228 Phase III trial of EFS pCR, RR, Control (no [296] positive defined as neoadjuvant safety trastuzumab), 3 positive by IHC trastuzumab in 23%; or FISH positive combination with CT in trastuzumab, patients with HER-2– 43%; p  .002 positive locally advanced breast cancer Lapatinib Phase III; adjuvant 2019 450 Neo-ALTTO is a 3-arm, pCR at safety and [337] trials and neoadjuvant randomized, multicenter, 18 wks tolerability, Trastuzumab lapatinib and/or open-label, phase III study ORR, % plus lapatinib trastuzumab of neoadjuvant lapatinib, patients trials ALTTO treatment neoadjuvant trastuzumab, with node- and Neo- optimization trials and a combination of negative ALTTO neoadjuvant lapatinib and disease at trastuzumab in women surgery, with ErbB-2–positive conversion primary breast cancer; to breast after treatment for 6 wks, conservation, the same targeted therapy DFS, OS, will be repeated for 12 RR, PET/ wks with the addition of CT, CTCs paclitaxel (continued) Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 345 achieved when the test was performed exactly according to the manufacturer’s instructions. Concern over IHC accu- racy using standard formalin-fixed, paraffin-embedded tis- sue sections has encouraged the use of the FISH assay for its ability to predict trastuzumab response rates. Reports that FISH could outperform IHC in predicting trastuzumab re- sponse [271] and well-documented lower response rates of 2 IHC staining versus 3 IHC staining tumors [272] have resulted in an approach that either uses IHC as a primary screen with FISH testing of all 2 cases or primary FISH- based testing. In a recently published study in which trastu- zumab was used as a single agent, the response rate in 111 assessable patients with 3 IHC staining was 35%, and the response rate for 2 cases was 0%; the response rates in patients with and without HER-2 gene amplification de- tected by FISH were 34% and 7%, respectively [272]. In another study of breast cancer treated with trastuzumab plus paclitaxel, in patients with HER-2– overexpressing tumors, the overall response rates were in the range of 67%– 81%, compared with 41%– 46% in patients with normal expres- sion of HER-2 [272]. The CB11 and TAB250 antibodies for IHC and FISH featured the strongest significance [273]. In- terestingly, in a recently published review from New York and Italy, it was noted that, although FISH-based testing is more expensive and not as widely available as IHC, the data suggested that FISH was actually the most cost-effective option [274]. In summary, although the superiority of one method over the other continues to be controversial [162, 275–277], approximately 80% of laboratories in the U.S. are screening all new cases with IHC and triaging selected, usually 2, cases for FISH testing, whereas 20% of testing uses FISH as the only method. It remains to be seen whether the newly approved CISH method will gain market share in the near and long term. Adjuvant Setting Table 5 outlines a series of clinical trials demonstrating the efficacy of trastuzumab-based regimens in the adjuvant set- ting [185, 278, 279]. The major phase III trastuzumab- based adjuvant trials (the NSABP B-31, North Central Cancer Treatment Group N9831, Herceptin Adjuvant [HERA], and Breast Cancer International Research Group [BCIRG] 006 trials) used a variety of cytotoxic agents in various combinations, doses, and orders of administration [280 –283]. When a 12-month course of trastuzumab was added to adjuvant chemotherapy, the DFS time was 33%– 52% greater and the OS time was 34%– 41% greater [280 – 283]. The improvements in DFS were independent of age, nodal status, hormonal status, or tumor size in all trials. As in the metastatic disease trials, cardiac toxicity was the most significant adverse event, occurring in 0%– 0.9% of pa- Table 5. (Continued) Median Trial status and Enrollment n of duration of FDA HER-2 testing completion patients Trial design and Primary Secondary ORR (HR; response, Median OS, Median DFS, approval Trial name entry criteria date enrolled agents used endpoint endpoint p-value) mos mos TTP, mos TTF, mos mos pCR, % impact Reference CHERLOB Phase IIb 2011 120 Randomized trial of pCR ORR, [338, 339] preoperative CT plus safety, gene trastuzumab, lapatinib, expression or the combination in profile HER-2–positive operable breast cancer 2cm Abbreviations: ALTTO, Adjuvant Lapatinib and/or Trastuzumab Treatment Optimization; ASCO–CAP, American Society of Clinical Oncology–College of American Pathologists; BCIRG, Breast Cancer International Research Group; BIG, Breast International Group; CHERLOB, Preoperative Chemotherapy Plus Trastuzumab, Lapatinib or Both in HER-2–Positive Operable Breast Cancer; CT, chemotherapy; CTA, clinical trial assay; CTCs, circulation tumor cells; DFS, disease-free survival; EFS, event-free survival; FDA, U.S. Food and Drug Administration; FEC, 5-fluorouracil, epirubicin, cyclophosphamide; FinHer, Finnish Herceptin; FISH, fluorescence in situ hybridization; HERA, Herceptin Adjuvant; HR, hazard ratio; IHC, immunohistochemistry; M, metastasis stage; MBC, metastatic breast cancer; N, node stage; NA, not applicable; NCCTG, North Central Cancer Treatment Group; NCI, National Cancer Institute; NIH, National Institutes of Health; NOAH, Neoadjuvant Trastuzumab in Locally Advanced Breast Cancer; NS, not significant; NSABP, National Surgical Adjuvant Breast and Bowel Project; ORR, overall response rate; OS overall survival; pCR, pathologic complete response; PET/CT, positron emission tomography/computed tomography; PFS, progression-free survival; RFS, relapse-free survival; T, tumor stage; TEACH, Tykerb Evaluation After Chemotherapy; TTF, time to treatment failure; TTP, time to progression. Downloaded from by DeepDyve user on 01 February 2022 346 HER-2 in Breast Cancer tients in the control arms and in 0%–3.8% of patients in the temic clearance, (b) a low volume (4 l) of distribution, and trastuzumab-containing arms, a level considered to be be- (c) a very long, 28-day half-life [286]. Whether trastu- low the safety cutoff points set by the individual studies’ zumab is used in the adjuvant setting or for the treatment of independent data monitoring committees [279]. These ad- metastatic disease, the recommended dosage is the same. juvant trastuzumab trials have achieved these notable clin- The clinical relevance of trastuzumab kinetic variability ical results despite lacking a standardized approach to and elimination routes is unknown [286]. Drug– drug inter- HER-2 testing. Of note in the adjuvant treatment trials was actions have not been reported. After a loading dose, tras- the impact of single-agent trastuzumab, which was featured tuzumab is typically given by i.v. perfusion at a dose based in the treatment of node-negative patients in the HERA trial on body weight, in weekly (adjuvant, neoadjuvant, and [281]. In addition, in an early study, trastuzumab mono- metastatic disease protocols) or every-3-week (adjuvant therapy achieved a 30% overall response rate for IHC 3 protocols) regimens. For metastatic disease, trastuzumab or FISH-positive tumors in the metastatic setting [272]. In treatment is typically continued until the time of disease current clinical practice, trastuzumab monotherapy is used, progression. A short-course regimen of trastuzumab (9 on occasion, for patients judged to be at risk for serious ad- weeks) is under investigation and appears promising in verse events if exposed to a combination with cytotoxic terms of activity, tolerance, and cost. agents. The strategy for using trastuzumab monotherapy, with or without endocrine therapy, for tumors judged to be Trastuzumab Benefit in HER-2–Negative Tumors. In low risk by routine clinicopathologic or molecular assess- the NSABP B-31 clinical trial of adjuvant trastuzumab plus ment is controversial. Some of the trials have included ei- chemotherapy [280], it was noted that a significant number ther IHC 3 or FISH-positive tumors as entry criteria, of patients originally considered HER-2 positive by the lo- whereas others, such as the Finnish Herceptin trial, have cal laboratory, and who appeared to benefit from the addi- used the CISH method. In addition, some trials have re- tion of trastuzumab, were ultimately considered HER-2 quired central HER-2 testing confirmation before entry into negative by the ratio-based FISH method performed at the the trial, whereas others have performed centralized labo- NSABP central laboratory [287]. There are a number of po- ratory testing after trial results were completed. Finally, the tential technical explanations for this observation includ- adjuvant trials were started before the publication of the ing: (a) because of a variety of factors (wrong tissue block, ASCO-CAP HER-2 testing guidelines [212] were pub- DNA degradation in specimen shipping, loss of the HER- lished and thus frequently used the 2.0 ratio cutoff for a 2–positive focus on deeper sectioning, etc.), the central lab- positive FISH result rather than the recommended 2.2 cut- oratory negative result may actually be incorrect; (b) some off recommended by the expert task force. tumors feature chromosome 17 polysomy and overexpres- sion of HER-2 protein that would not be detected when only Trastuzumab Combinations. Since the FDA approval in ratio-based FISH results were evaluated centrally; and (c) 1998 of two trastuzumab plus chemotherapy combinations, because HER-2 status change occurs most commonly for a number of additional approaches have gained favor in the borderline positive (or negative) cases, it is possible that the clinical practice community. The National Comprehensive current threshold for HER-2 amplification (i.e., doubling of Cancer Network (NCCN) Clinical Practice Guidelines DNA copy number) is not the optimal threshold and tumors [284] currently recommend the following regimens for the with lesser average amplification (i.e., ratio 1.0) may also first-line treatment of HER-2–positive MBC: trastuzumab benefit. It is also possible that other biologic pathways that plus single agents— either paclitaxel (every 3 weeks or are not linked to HER-2 gene amplification are inhibited by weekly), docetaxel (every 3 weeks or weekly), or vinorel- trastuzumab. In the early stages of trastuzumab clinical de- bine (weekly). For combination therapies, the NCCN rec- velopment in MBC, it was noted that HER-2–negative tu- ommends trastuzumab plus paclitaxel and carboplatin mors by IHC rarely responded to the antibody, and that, if (every 3 weeks) or docetaxel plus carboplatin. Recently, all patients were treated, the low response rate in HER-2– carboplatin-based trastuzumab combinations have gained negative cases would significantly dilute the enhanced re- interest as a result of both the apparent boost in efficacy as sponse rate in HER-2–positive cases and mask the overall measured by a higher overall response rate and longer pro- clinical benefit of the novel therapeutic. One intriguing pos- gression-free survival time and the cardioprotective bene- sible predictor of trastuzumab benefit in HER-2–negative fits of avoiding an anthracycline-containing regimen [285]. breast cancer is the preliminary observation that HER-2– negative tumors that overexpress neuregulin, an activating Trastuzumab Administration and Pharmacokinetics. ligand for HER-4, are inhibited by HER-2–targeted therapy The pharmacokinetics of trastuzumab feature (a) low sys- [288]. Alternatively, it is also possible (although unprece- Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 347 dented) that a drug may have no significant anticancer ac- consensus definition of resistance in both the adjuvant tivity in a particular disease subset in the metastatic stage and metastatic settings. In the neoadjuvant setting, resis- (i.e., HER-2–normal cancers), but may have antitumor ac- tance has been defined as a failure to achieve a pCR or tivity against the same subset in the micrometastatic stage. near pCR. A number of biomarkers proposed as predic- Immunological mechanisms could possibly underlie such tors of trastuzumab resistance are listed in Table 6. It an effect. should also be noted that trastuzumab is typically com- bined with one or more cytotoxic agents and attempts to Neoadjuvant Setting determine individual biomarkers predictive of trastu- The results of trastuzumab-based neoadjuvant studies zumab resistance will be significantly impacted by the (Table 5) have received significant recent interest in the biologic pathways related to the resistance or sensitivity oncology community [289]. Virtually all completed and of the tumor cells to the companion agents as well. in progress clinical trials have demonstrated a significant enhancement in the rate of pathologic complete response HER-2 Gene Copy Number. It has been reported that tu- (pCR), the primary endpoint in these studies, in cases of mors with higher HER-2 gene copy numbers (e.g., 10 patients with HER-2–positive breast cancer that received HER-2 copies per nucleus) are more sensitive to trastu- trastuzumab in the neoadjuvant setting [290 –297]. This zumab [301, 302]. Despite this evidence, all patients with benefit of the addition of trastuzumab in the neoadjuvant gene copy numbers 6.0 per nucleus or gene ratios of setting appears to be independent of, if not enhanced by, HER-2/CEP17 2.2 are equally considered HER-2 posi- the coexistence of ER positivity [297]. Among the poten- tive by the ASCO-CAP task force, and HER-2 gene copy tial explanations for the apparent greater chemosensitiv- number is not currently used to determine the intensity or ity of HER-2–positive tumors cotreated with duration of trastuzumab therapy. trastuzumab in the neoadjuvant setting is the concept that HER-2 gene amplification is in some way related to the Shedding of HER-2 Protein. Early in the time line of tras- growth and survival of breast cancer stem cells [298, tuzumab development there was concern that significant 299]. The higher pCR rates in HER-2–positive breast shedding of the HER-2 surface receptor (p185neu) protein, cancers treated with neoadjuvant trastuzumab may con- as evidenced by a high serum HER-2 protein ELISA test, ceivably reflect the inhibition of both stem cell and pro- would be associated with resistance of the tumor as a result genitor cell proliferation and invasion by removing or of the neutralization of the infused antibody [303]. How- downregulating HER-2–mediated growth signals [299]. ever, follow-up studies have not confirmed that a high se- Also of interest in the neoadjuvant trial results is the pos- rum HER-2 level at the outset of trastuzumab therapy is sible observation that HER-2–targeted therapy can con- predictive of clinical tumor resistance. vert a HER-2–positive breast cancer into a HER-2– negative tumor [300]. In a recent report, nearly one third Dimerization Status. Previous studies have suggested that of the patients with HER-2–amplified breast cancer HER-2 dimerization status (HER-2:HER-2 homodimers treated with a taxane and anthracycline-based chemo- versus HER-2:HER-3 and HER-2:HER-4 heterodimers) therapy with concomitant trastuzumab in the neoadju- can predict response to trastuzumab-based therapy in MBC vant setting that failed to achieve pCR were found to [221, 222, 304]. Validation of these initial observations has have converted to HER-2–negative disease [300]. Fur- not been presented in large cohorts of patients and prospec- ther validation of these findings awaits additional pro- tive testing of dimerization status as a predictor of trastu- spective studies. zumab resistance has not been published to date. Biomarkers of Trastuzumab Resistance Fc Receptor Status and Antibody-Dependent Cellular Since trastuzumab was introduced for the treatment of Cytotoxicity Response. Antibody-dependent cellular cy- MBC in 1998, there has been growing interest in the dis- totoxicity (ADCC) is considered a major aspect of the covery and potential clinical utility of biomarkers de- mechanism of action of trastuzumab [305–309]. Interac- signed to predict resistance to the drug. Current tions with the Fc receptor may be a critical step in the acti- approaches to HER-2 testing provide a negative predic- vation of natural killer lymphocytes and ADCC response. tor of drug response: the test does not predict which pa- Preliminary studies have linked both germline polymor- tients will respond to trastuzumab, it predicts which phisms and post-translational modifications (glycosylation patients are unlikely to benefit. The study of resistance and fucosylation) of the Fc receptor with the impaired biomarkers has been limited to a degree by the lack of a ADCC response associated with monoclonal antibody ther- Downloaded from by DeepDyve user on 01 February 2022 348 HER-2 in Breast Cancer Table 6. Biomarkers of trastuzumab resistance Proposed impact on trastuzumab Biomarker response Proposed mechanism Validation status References High HER-2 gene copy Resistance Higher HER-2 gene Majority of reports [298, 299] number copy number predicts favor, but large greater dependency of scale validation is tumor cells on the lacking HER-2 pathway Shedding of HER-2 protein Resistance Circulating shed HER-2 Not validated [300] protein would bind infused trastuzumab and reduce therapeutic targeting Dimerization status Resistance HER-2 homo- and Not validated [220, 221, 301] heterodimerization status would predict response Fc receptor status and Resistance Polymorphisms and Not validated [302–306] ADCC response other dysfunction of Fc receptor would reduce the ADCC response to infused trastuzumab PTEN deficiency/PI3K Resistance Loss of PTEN Most highly [307–311] pathway activation expression and validated activation of PI3K trastuzumab pathway creates resistance marker resistance to drugs targeting HER-2 tyrosine kinase signaling c-MYC amplification Sensitivity Coamplification of c- Observed in [312–314] MYC and HER-2 may adjuvant and promote apoptosis in neoadjuvant trials, tumor cells exposed to but not, to date, in combination regimens metastatic disease of trastuzumab and trials; further cytotoxic agents validation required IGF-1R overexpression Resistance Activation of IGF Not fully [314–318] signaling pathway validated; overcomes inhibition of conflicting studies HER-2 signaling published pathway MUC4 overexpression Resistance MUC4 expression Not validated [319] induces steric hindrance of trastuzumab binding to p185neu (HER-2) receptor p95HER-2 expression Resistance p95HER-2 expression Not validated [320, 321] enables constitutive signaling of HER-2 tyrosine kinase even when p185HER-2 receptor’s extracellular domain is bound by trastuzumab (continued) apeutics such as trastuzumab [309]. The clinical develop- will require validation of these retrospective observations ment of Fc receptor assays to predict trastuzumab resistance in prospective trials. Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 349 Table 6. (Continued) Proposed impact on trastuzumab Biomarker response Proposed mechanism Validation status References Phosphorylated HER-2 Sensitivity Phosphorylated HER-2 Not validated [322] receptor receptor was identified in a subset of HER-2– positive breast cancers and associated with an higher response rate to trastuzumab monotherapy and combination therapy with cytotoxic agents Topoisomerase IIa Sensitivity Topoisomerase IIa Not validated [323] amplification amplification is associated with anthracycline benefit in HER-2–positive tumors in some, but not all, studies Basal phenotype Resistance Basal-like phenotype of Not validated [324] breast cancer is associated with IGF-1R overexpression and resistance to inhibition of trastuzumab- mediated blockade of HER-2 tyrosine kinase signaling CD44 overexpression Resistance CD44 binding at the Not validated [325] cell surface may reduce ADCC for trastuzumab Vascular endothelial growth Resistance Growth in tumor Not validated [326] factor overexpression neovasculature may reduce antitumor effects of trastuzumab; this finding is the basis for the trastuzumab plus bevacizumab combination trials miRNA Resistance miRNA signatures have Not validated [327] not, to date, been linked to HER-2 status; nonetheless, the association of expression of specific miRNAs with response to hormonal and cytotoxic therapy suggests that miRNA biomarkers of trastuzumab may soon be uncovered Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; HER-2, human epidermal growth factor receptor 2; IGF- 1R, insulin-like growth factor 1 receptor; miRNA, micro-RNA; MUC4, mucin 4; PI3K, phosphatidylinositol 3 kinase; PTEN, phosphatase and tensin homologue deleted on chromosome ten. Phosphatase and Tensin Homologue Deleted on mor suppressor gene expression and activation of the PI3K Chromosome Ten Deficiency/PI3K Pathway Activation. A pathway with resistance to trastuzumab-based therapy number of studies have linked the loss of phosphatase and [310 –314]. Although the potential for PTEN status to pre- tensin homologue deleted on chromosome ten (PTEN) tu- dict trastuzumab response appears quite promising, the as- Downloaded from by DeepDyve user on 01 February 2022 350 HER-2 in Breast Cancer sociations have not been uniformly observed by all phorylation status and trastuzumab response have not been investigators and additional validation in prospective stud- performed to date. ies is clearly required before this biomarker can achieve Amplification. Although topoisomer- widespread clinical adoption. Topoisomerase II ase II amplification has been linked to the benefit of an- thracycline chemotherapy in HER-2–positive breast EGFR Expression. Early studies suggested that amplifi- cancers, specific association of topoisomerase II status cation of EGFR and overexpression of EGFR would confer with response to trastuzumab-based regimens in either the clinical resistance to trastuzumab [315]. This observation adjuvant or metastatic disease settings has not been con- has not been validated in large-scale follow-up studies. firmed [326]. In the BCIRG 006 trastuzumab adjuvant trial, topoisomerase II gene copy number detected by FISH was c-MYC Amplification. Based on data from the NSABP studied as a predictive biomarker, although interim reports B-31 trastuzumab adjuvant trial, tumors that feature coam- have not confirmed its utility [282]. plification of the c-MYC oncogene and HER-2 benefited by the addition of trastuzumab to chemotherapy in terms of Basal Phenotype. The basal-like phenotype of breast can- both recurrence-free survival and OS, compared with pa- cer is associated with IGF-1R overexpression and resis- tients whose tumors lacked the c-MYC amplification [316]. tance to inhibition of trastuzumab-mediated blockade of In a neoadjuvant study, PgR-negative status and c-MYC HER-2 tyrosine kinase signaling [327]. In the basal-like amplification were both associated with higher pCR rates phenotype, HER-2–positive status is quite infrequent, oc- after the addition of trastuzumab to chemotherapy [317]. curring in 10% of cases [327]. Thus c-MYC gene copy status may be a biomarker of tras- tuzumab response in the adjuvant or neoadjuvant settings, CD44 Tumor Cell Overexpression. It has been postulated although this requires large-scale studies for confirmation. that CD44 binding at the cell surface may reduce ADCC for trastuzumab [328]. Insulin-Like Growth Factor 1 Receptor Status. The overexpression of the insulin-like growth factor 1 receptor High Tumor/Serum Vascular Endothelial Growth (IGF-1R) has been associated with resistance to trastu- Factor Levels. Overexpression of vascular endothelial zumab in some studies [318, 319], but not in others [318 – growth factor (VEGF) in breast cancers and high serum lev- 321]. Experimental models favor the idea that activation of els of VEGF have been postulated as a cause of trastuzumab IGF-1R confers resistance to trastuzumab [322]. Trials ex- resistance [329]. amining the potential synergism between trastuzumab and novel anti–IGF-1R therapeutics have been initiated. MicroRNA. MicroRNA (miRNA) signatures have not, to date, been linked to HER-2 status. Nonetheless, the associ- Mucin 4. The mucin 4 glycopeptide may be secreted by ation of expression of specific miRNAs with response to some breast cancers and interfere with trastuzumab binding hormonal and cytotoxic therapy suggests that miRNA bio- to the HER-2 receptor [323]. markers of trastuzumab may soon be uncovered [330]. p95HER-2. The accumulation of truncated forms of the Lapatinib: HER-2 Testing and the Prediction of HER-2 receptor (p185HER-2) that lack the extracellular Response to Lapatinib Therapy trastuzumab-binding domain of the intact receptor have Lapatinib (Tykerb; Glaxo Smith Kline, Research Triangle been associated with resistance to trastuzumab in preclini- Park, NC) is an orally available small-molecule dual inhib- cal studies [324]. Amino terminally truncated carboxyl ter- itor of the EGFR and HER-2 tyrosine kinases [331]. minal fragments of HER-2 are termed p95HER-2. In one published clinical study involving 46 patients, breast tu- Metastatic Disease Setting mors that expressed p95HER-2 showed a lesser or absent Lapatinib was approved by the FDA in 2007 for use in com- response to trastuzumab-based regimens in a retrospective bination with capecitabine for the treatment of HER-2– analysis [324]. positive MBC that has progressed with standard treatment [332]. In the phase III registration trial involving 399 pa- Phospho–HER-2. Preliminary studies have linked HER-2 tients (Table 5), the addition of lapatinib to capecitabine receptor phosphorylation status to response to trastuzumab- produced a longer median time to progression by 8.5 weeks (27.1 weeks, versus 18.6 weeks for capecitabine alone) based regimens [325]. Large-scale studies of HER-2 phos- Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 351 with an HR of 0.57 (p  .00013) [331]. The response rates treatment of visceral disease in MBC appears to have un- were 23.7% and 13.9%, respectively, with an HR of 1.9 masked the clinical problem of progressive CNS disease in (p  .017) [331]. Patients were eligible for the trial based on HER-2–positive patients, a clinical syndrome not fre- either a HER-2 IHC score of 3 or a FISH ratio 2.0. In an quently encountered in the pretrastuzumab era. In a report updated efficacy and biomarker report, OS in the lapatinib of 39 patients heavily pretreated with trastuzumab and tax- plus capecitabine treated group trended toward a longer sur- anes who had progressed despite radiation, two patients vival duration [333]. In a lapatinib monotherapy trial for pa- achieved a partial response based on the Response Evalua- tients who had progressed on a trastuzumab regimen, the tion Criteria In Solid Tumors, and five additional patients response rate in the HER-2–positive group was 4.3%, indi- were found to have experienced at least a 30% volumetric cating modest clinical activity of the drug as a single agent reduction in their CNS lesions [340]. The potential efficacy [334]. of lapatinib in trastuzumab-resistant brain metastases awaits further documentation in larger case cohorts. Adjuvant Setting The Adjuvant Lapatinib and/or Trastuzumab Treatment Inflammatory Breast Cancer Optimization (ALTTO) and Tykerb Evaluation After In a phase II trial, lapatinib treatment has shown early Chemotherapy (TEACH) trials are two of the major phase promise in the treatment of HER-2–positive inflammatory III trials that are currently evaluating lapatinib in the adju- breast cancer [331]. vant setting (Table 5) [335, 336]. The ALTTO trial plans to enroll 8,000 patients and is sponsored by the National Can- Lapatinib Administration and Pharmacokinetics cer Institute, part of the U.S. National Institutes of Health, With oral administration of the FDA-recommended daily and GlaxoSmithKline, and is being coordinated by The dose of 1,250 mg/day, the time of maximum plasma con- Breast Cancer Intergroup of North America in the U.S. and centration of lapatinib is 3– 4 hours [331]. Lapatinib is me- the Breast International Group in Brussels, Belgium [335]. tabolized primarily by the cytochrome P450 system via the The TEACH trial is designed to determine whether adju- 3A4 isozyme, resulting in a single metabolite active against vant therapy with lapatinib for 1 year will improve DFS in EGFR but not HER-2. With continuous dosing, the half-life women with early-stage HER-2–positive breast cancer. of lapatinib is 24 hours [331]. This trial plans to enroll 3,000 patients [333]. Efficacy data from adjuvant trials featuring lapatinib in combination with Biomarkers of Lapatinib Resistance cytotoxic agents are not available at this time. In that lapatinib was approved 9 years after trastuzumab, considerably less information has been published concern- Neoadjuvant Setting ing markers of efficacy or resistance to the drug [331, 341– The Neo-ALTTO trial is a randomized, open-label, multi- 343]. center, phase III study comparing the efficacy of neoadju- vant lapatinib plus paclitaxel with that of trastuzumab plus Serum HER-2 ECD Status. In the lapatinib plus capecit- paclitaxel and with concomitant lapatinib and trastuzumab abine versus capecitabine trial, preliminary study of plus paclitaxel given as neoadjuvant treatment in HER-2– biomarkers failed to identify tissue EGFR or HER-2 bio- positive primary breast cancer [337]. The CHERLOB trial markers predictive of lapatinib resistance [333]. However, is a randomized trial of preoperative chemotherapy plus a significant reduction in lapatinib response was associated trastuzumab and lapatinib or the combination of trastu- with cases in which the starting serum HER-2 ECD levels zumab and lapatinib in HER-2–positive operable breast were high [329]. In another study, high serum HER-2 ECD cancer featuring a tumor diameter 2 cm [338, 339]. pCR levels did not predict benefit from lapatinib-based combi- is the endpoint for both of these lapatinib neoadjuvant trials nation therapy [344]. that compare lapatinib plus cytotoxic agents with lapatinib plus trastuzumab plus cytotoxic agents. Efficacy data have Tissue HER-2 Status. All completed and in-progress clin- not been published on these trials to date. ical trials employing lapatinib have required that entering patients have tumors that are HER-2 positive by FISH or HER-2–Positive CNS Metastases IHC. A number of studies have confirmed that HER-2 pos- A major goal for the development of lapatinib has been the itivity is required for lapatinib clinical benefit [341]. In one potential efficacy in cases of CNS involvement in patients recent report, on a clinical trial that originally found limited with HER-2–positive MBC progressing on trastuzumab- lapatinib benefit in cases of HER-2– unamplified tumors based regimens. The relative success of trastuzumab in the tested by FISH, lapatinib efficacy was found to be limited to Downloaded from by DeepDyve user on 01 February 2022 352 HER-2 in Breast Cancer HER-2–positive cases when tumors were retested by an ac- MBC patients who progressed on trastuzumab-based regi- ademic central laboratory and scored by a pathologist rather mens, significant synergy, as measured by the progression- than a technician [345]. free survival duration, was shown [351]. Tissue EGFR Status. Although lapatinib’s mechanism of Trastuzumab Plus Bevacizumab action includes the inhibition of the tyrosine kinase activity The BEvacizumab and Trastuzumab Adjuvant Therapy in of both HER-1 (EGFR) and HER-2, a number of studies HER-2-Positive Breast Cancer trial is a multicenter phase have failed to link amplification of EGFR or overexpres- III randomized adjuvant trial comparing chemotherapy plus sion of EGFR with the efficacy of lapatinib-based therapies trastuzumab with chemotherapy plus trastuzumab and the [346]. In one study, there was no correlation between EGFR anti-VEGF ligand bevacizumab [352]. No efficacy data are expression (IHC or mRNA) and responsiveness to lapatinib available at this time. In an ongoing neoadjuvant trastuzumab– regardless of HER-2 status [346]. bevacizumab trial, to date, the addition of bevacizumab has not resulted in a higher rate of pCR [353]. However, in a Chromosome 17 Polysomy. Although extra copies of recent interim report of the combination in a trial of locally chromosome 17 have been linked to the efficacy of trastu- advanced disease treated in the neoadjuvant setting, early zumab in patients whose HER-2 FISH ratio test is negative evidence of synergistic efficacy was noted [354]. for HER amplification, in one recent study, polysomy of chromosome 17 was not associated with lapatinib benefit in Trastuzumab Plus Everolimus HER-2–negative tumors [347]. As documented in a preclinical study, one of the strategies for overcoming the resistance of PTEN-deficient breast IGF-1R. In a preclinical study, lapatinib inhibited IGF-1R cancers to trastuzumab is the targeting of the Akt pathway signaling and growth-promoting effects in parental and re- using a mammalian target of rapamycin (mTOR) inhibitor sistant cells [348]. The studies indicating that IGF-1R sig- [355]. RAD001 (everolimus) is an inhibitor of mTOR cur- naling can cause trastuzumab resistance have encouraged rently in clinical trials for the treatment of HER-2–positive the concept that lapatinib will prove efficacious in breast breast cancer in combination with trastuzumab. In an ongo- cancers that have progressed on trastuzumab. ing clinical trial, early efficacy data suggest the possibility of significant synergism from the addition of everolimus to ER Signaling. In one preclinical/clinical study, it was pos- a trastuzumab and taxane regimen in the metastatic disease tulated that signaling through the ER pathways was a setting [356]. significant mechanism of resistance to lapatinib [349]. Trastuzumab Plus Heat Shock Protein 90 Inhibitors PTEN. Although data are limited in comparison with tras- Inhibition of the chaperone protein heat shock protein 90 tuzumab [317–321], in one study, in contrast to the rela- (HSP90) results in increased degradation of HER-2 ECD tively strong supporting data for trastuzumab, loss of PTEN [357, 358]. Two anti-HSP90 agents that have been com- expression was not associated with lapatinib resistance in bined with trastuzumab in early-stage clinical trials are either cell lines or clinical specimens [350]. geldanamycin and tenespimycin (17-AAG; Kosan Bio- sciences, Hayword, CA). Reports of a trial of tenespimycin combined with trastuzumab in advanced pretreated MBC Trastuzumab–Lapatinib Combinations and have shown good safety and tolerability [359] and early in- Other Targeted Therapies dications of significant clinical activity in HER-2–positive Trastuzumab Plus Lapatinib disease [360]. A number of clinical trials are examining the potential syn- ergy of using both trastuzumab and lapatinib for HER-2– Duration of Anti–HER-2 Targeted Therapy positive breast cancer in the neoadjuvant, adjuvant, and A number of recent reviews have summarized the lack of metastatic disease settings. Data from the ALTTO, Neo- standardization of the duration of treatment with anti– ALTTO, and CHERLOB trials are not yet mature, and ef- HER-2 targeting agents in HER-2–positive breast cancer ficacy data are currently not available to assess the impact [361–364]. Although the current recommended duration of of combining HER-2–targeted agents [336, 338, 339]. In a trastuzumab treatment is 1 year in the adjuvant setting, dif- recent interim report, testing the efficacy of the combina- ferent treatment durations, from 9 weeks to 2 years, have tion of trastuzumab and lapatinib compared with lapatinib been studied with, to date, no optimal duration of treatment alone in a heavily pretreated population of HER-2–positive achieving consensus among investigators [265]. In the Downloaded from by DeepDyve user on 01 February 2022 Ross, Slodkowska, Symmans et al. 353 lapatinib plus capecitabine registration trial, oral lapatinib evaluating pertuzumab efficacy in MBC have not been suc- therapy was maintained until the time of disease progres- cessful to date [372, 373]. The observation that pertuzumab sion or based on adverse events [331, 332]. In a recent can elicit a metabolic response detected by position emis- study, a higher efficacy but similar toxicity were found sion tomography scanning in HER-2–negative MBC has when trastuzumab was continued beyond progression and fueled continued interest in the development of the anti- second-line chemotherapy with capecitabine was initiated body in subsets of breast cancer patients [374]. In a more [365]. However, there is increasing evidence that continu- recent phase II study of trastuzumab and pertuzumab com- ation of anti–HER-2 therapy after progression on trastu- bination therapy in HER-2–positive metastatic disease, a zumab confers clinical benefit. In a recent review by the 40% clinical benefit rate with multiple complete and partial NCCN, it was noted that 74% of patients with MBC who responses was described [375]. had progressed after first-line trastuzumab-based therapy continued to receive trastuzumab in a second-line protocol Ertumaxomab [366]. Currently, no specific biomarkers appear to be capa- Ertumaxomab (Fresenius Biotech, Hamburg, Germany) is a ble of preselecting an individual patient for a short-term or trifunctional bispecific antibody targeting HER-2 on tumor long-term treatment regimen. A variety of markers, includ- cells and CD3 on T cells that has the capability to redirect T ing serum-based assays and imaging studies, have been cells, macrophages, dendritic cells, and natural killer cells proposed to guide the cessation or continuance of treatment to the sites of tum