HER 2 Amplification or Overexpression in Upper GI Tract and Breast Cancer with Clinical Diagnosis and Treatment

© 2013 Zhou and Hicks, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HER2 Amplification or Overexpression in Upper GI Tract and Breast Cancer with Clinical Diagnosis and Treatment


Introduction
EGFR and HER2 family with signal pathway and carcinogenesis: The human epidermal growth factor receptors (HER-2) gene is localized to chromosome 17q and encodes a transmembrane tyrosine kinase receptor protein. Numerous studies were done from basic mechanism of HER family for cell proliferation and oncogenesis, HER2 overexpression or amplification in various solid tumors to clinical treatment of breast cancer, gastroesophageal cancer by trastuzumab in many recent reviews [1][2][3][4][5][6][7][8].
HER2 belongs to a family including epidermal growth factor receptor (EGFR), HER2, HER3 and HER4, which are a group of transmembrane glycoproteins, collectively named receptor tyrosine kinases (RTKs), whose cytoplasmic domains harbor an enzymatic activity, namely tyrosine-specific phosphorylation [9]. The family of epidermal growth factor molecules, which comprises different ligands sharing a 50-60 amino acid receptor-binding domain, bind with subtype RTKs. Each receptor consists of an extracellular ligand-binding domain, a transmembrane domain, and a tyrosine kinase portion [10]. Upon ligand binding, the otherwise inactive monomeric receptors form active homodimers or heterodimers, thereby leading to receptor phosphorylation and signaling via various biochemical pathways (Fig.1), such as the mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3-kinase (PI3K), phospholipase C-γ, and transcription factors like the signal transducers and activators of transcription (STATs) or SMAD proteins [1]. These modules of cellular activation and the respective growth factors (GFs)s are co-opted in several phases of tumor progression.
HER-2 gene amplification in breast cancer has been associated with increased cell proliferation, cell motility, tumor invasiveness, progressive regional and distant metastases, accelerated angiogenesis, and reduced apoptosis [11]. Overexpression of HER2 in human Since breast cancer showed better prognosis with trastuzumab treatment for HER2 positive breast cancer patients and similar HER2 positive cancers were identified in gastric and gastro-esophageal cancer, clinical trial ToGA was performed in gastric carcinoma. ToGA (Trastuzumab for Gastric Cancer) was an open-label, international, phase 3, randomized controlled trial undertaken in 122 centers in 24 countries [22]. Clinical trial ToGA used trastuzumab combined with standard chemotherapy for HER2 positive gastric cancer and gastro-esophageal junction cancer which demonstrated a significant improvement of gastric cancer survival. Now, trastuzumab is approved for treatment of gastric cancer in European, United States, Japan and other multiple countries. Figure 2. Clinically approved and experimental therapeutic strategies targeting ErbB-2/HER2 in carcinomas. Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of HER2, is approved for the treatment of HER2-overexpressing breast cancer. The antibody recruits immune effector mechanisms and can induce apoptosis, block angiogenesis and inhibit tumor cell proliferation. Similarly, Pertuzumab is able to prevent heterodimerization of HER2 with other family members. Unlike the ultimate specificity of Trastuzumab and Pertuzumab to HER2, tyrosine kinase inhibitors like the reversible inhibitor Lapatinib (approved for treatment of breast cancer) and the irreversible inhibitor Neratinib variably inhibit a broad range of tyrosine kinases. The drug has completed phase II clinical trials. HSP90 is a molecular chaperone required for proper folding of protein kinases like HER2. Hence, HSP90 inhibitors, such as 17-AAG, which block the ATP/ADP binding pocket of HSP90 and target HER2 for proteasomal degradation are in clinical trials. A naturally occurring truncated form of HER2, p95-HER2, has been implicated as a mechanism conferring resistance to Trastuzumab. Its formation is mediated by processing of the membrane bound HER2 by matrix metalloproteinases (MMPs) of the ADAM (a disintegrin and metalloproteinase) family. INCB3619 and INCB7839 are potent inhibitors of ADAM10 and ADAM17. ADAM10 is the principle sheddase for different molecules associated with tumor cell proliferation, whereas ADAM17 is the main sheddase for the EGFR ligands TGF-alpha, AR, NRGs, and HB-EGF. These similar inhibitors may effectively block truncation of HER2 and onset of patient resistance to Trastuzumab, but clinical testing has not been completed. ( The clinical efficacy of Trastuzumab likely entails a combination of immunological and nonimmunological mechanisms [1]. The ability of Trastuzumab to elicit antibody-dependent cellular cytotoxicity critically influences the efficacy of Trastuzumab-based therapies. Nonimmunological mechanisms of Trastuzumab action include the inhibition of HER2 activation and downstream signaling. Alternatively, Trastuzumab may act by removing HER2 from the cell surface. Because it binds to an epitope near the cleavage site of HER2's extracellular domain, Trastuzumab inhibits HER2 activation by metalloproteinase-mediated shedding of the extracellular domain. The resulting interference with HER2-mediated downstream signaling processes shuts down cell proliferation, angiogenesis, invasive growth, resistance to apoptosis, and DNA repair, thus sensitizing tumor cells to conventional therapeutic modalities such as chemotherapy, endocrine treatment and radiotherapy. Lapatinib, small molecule kinase inhibitor: Lapatinib, binding either reversibly or irreversibly to the nucleotide-binding cleft of their target kinases, is a highly specific, reversible inhibitor that blocks the catalytic action of both HER2 and EGFR 23 . Experiments in vitro and xenograft models, established the ability of Lapatinib to inhibit both the intact form of HER2 and the truncated intracellular form (p95-HER2), which is not recognized by Trastuzumab.
Similar to Trastuzumab, Lapatinib combined with chemotherapy was found to be better effect than capecitabine alone in HER2-positive women with advanced breast cancer that progressed after treatment with regimens that included Trastuzumab, an anthracycline and a taxane [24]. In addition, Lapatinib demonstrated clinical activity and was well tolerated as first-line monotherapy in HER2-amplified, locally advanced or metastatic breast cancer [25,26]. Recently, laptinib showed a synergistic effect with trastuzumab in vitro and in vivo to inhibit HER2 amplified human gastric cancer cells and animal model [23]. Clinical phase II trial of lapatinib as first line therapy in patients with advanced or metastatic cancer showed well tolerated, which will be another potential drug to target HER2 receptors.

HER2 in gastric adenocarcinoma
Gastric cancer is the fourth most common cancer worldwide and the second most common cause of cancer-related death in the world [31,32]. The incidence of gastric cancer varies substantially worldwide, with the highest rates (>20 per 100,000) occurring in Japan, China, Eastern Europe, and South America, but the lowest rates (<10 per 100,000) finding in North America, southern Asia, North and East Africa, Australia, and New Zealand. In addition, it is more common in men than in women (10.9 vas 5.5 per 100,000). Although the survival of gastric cancer is improved in recently years in Western countries the 5 year survival is still around 5-20%. The multimodality treatments including surgery and neoadjuvent chemotherapy have a limited effect on the overall survival. In breast cancer, HER2 overexpression and amplification were reported around 25% and associate with poorer prognosis [2]. Trastuzumab treatment of HER2 positive breast cancer patient improved survival. HER2 overexpression and amplification were reported in gastric and gastroesophageal junction (GEJ) tumors from 6-43%. In addition, trastuzumab were found to inhibit tumor growth in gastric carcinoma cell lines, animal model and xenograft models [33][34][35]. Recently international large scale phase III clinical trial called ToGA showed that trastuzumab added to standard chemotherapy significantly improved the response rate, median progression-free survival, and overall survival of gastric adenocarcinoma [22]. Trastuzumab combined with standard chemical therapy (such as capecitabine or 5fluorouracil and cisplatin) now is approved by European Medicines Agency, United States and Japan etc. for the treatment of patients with HER2 overexpression or amplification. Thus clinical tests for HER2 overexpression and amplification in gastric adenocarcinoma patients become a key to recruit eligible patients for clinical treatment and evaluation of treatment effect.
IHC studies on HER2 overexpression: HER 2 overexpression was reported from 7-34% by many studies [3]. For clinical trial and treatment, it is very important to develop a standard HER2 test to recruit eligible patients for trastuzumab treatment. Before clinical trial ToGA, Hofmann and colleagues (2008) [36] first set up an IHC criteria based on HER2 IHC test on 168 gastric and GEJ resection patients (see Table 2). Based on the standard HER2 test on the breast cancer, they further proposed that strong incompletely membranous stain with basolateral "U" shape in gastric cancer was positive for HER2 overexpression. In addition, the HER2 expression showed higher heterogeneity about 4.8% in gastric samples than about 1.4% in breast cancer. They modified breast criteria in several points including incomplete membranous stain pattern and percentage of cells (≥ 10% cut off), which improved the concordance level between IHC and FISH tests to 93.5%. For ToGA clinical trial, Bang et al [22] reported that HER2 positive rate was a 22.1%. In addition, they found that HER2-  Table 3; Ruschoff 2012). In their practical procedure for gastric cancer HER2 test, the surgical specimen cutoff is complete, basolateral, or lateral membranous reactivity in ≥10% of cells; the biopsy specimen cutoff is complete, basolateral, or lateral membranous reactivity in ≥5 clustered cells; the borderline cutoff is immunohistochemistry 1+/immunohistochemistry 2+ or focal staining in <10% cells which recommend for FISH or SISH tests. This new score system further improved Hoffmann's score system, but it still need further proved in future HER2 tests, especially the results mostly based on European laboratories. The large scale HER2 studies in Asia are need to build up an optimal HER2 test system in gastric cancer since the incidence of gastric cancer is much higher in Asian countries.

a. Immunohistochemistry
Testing recommendations  Representative surgical samples or an adequate number of viable biopsy specimens (ideally six to eight) are required  If few biopsies are available, all viable specimens should be tested  Immunohistochemistry should be the initial HER2 testing methodology for gastric cancer and bright-field methodologies are preferred wherever possible HER2-positive per European Medicines Agency license: immunohistochemistry 3+ or immunohistochemistry 2+/fluorescence in situ hybridization-positive or immunohistochemistry 2+/silver in situ hybridization-positive Borderline immunohistochemistry 1+/immunohistochemistry 2+ cases and samples with focal and intense membranous reactivity in <10% cells may also be retested with fluorescence in situ hybridization or silver in situ hybridization (scores for both assays should be indicated separately on the report)  Validated immunohistochemistry HER2 assays should be used Scoring recommendations  Due to the tumor heterogeneity (focal areas of positivity) and incomplete membrane staining commonly seen in gastric cancer, the gastric cancer-specific scoring criteria should be adhered to:  Surgical specimen cutoff: complete, basolateral, or lateral membranous reactivity in ≥10% of cells  Biopsy specimen cutoff: complete, basolateral, or lateral membranous reactivity in ≥5 clustered cells  The 'magnification rule' should be used in conjunction with the scoring criteria  Borderline cases (immunohistochemistry 1+/immunohistochemistry 2+ or focal staining in <10% cells) that score fluorescence in situ hybridization-positive or silver in situ hybridization-positive may be considered HER2-positive (scores for both assays should be indicated separately on the report)

b. In situ hybridization
Testing recommendations  Tumor samples classified as immunohistochemistry 2+ should be retested by fluorescence in situ hybridization or silver in situ hybridization to assess HER2 status  Silver in situ hybridization is a more suitable methodology than fluorescence in situ hybridization for assessing HER2 status in gastric tumor samples as it is a bright-field methodology and thus allows for rapid identification of HER2-positive tumor foci within a heterogeneous sample Validated in situ hybridization HER2 assays should be used Scoring recommendations  The definition of fluorescence in situ hybridization or silver in situ hybridization positivity in gastric or gastro-esophageal junction cancer is a HER2:chromosome 17 ratio of ≥2.0  The entire case should be screened for amplified regions (particularly important for fluorescence in situ hybridization samples where a bright-field image is not available)  At least 20 evaluable, non-overlapping cells in the invasive component should be counted initially  In borderline amplification cases, ~20 additional cells should be recounted or scoring should be performed in an alternative area of tissue The overall HER2 gene count is important:  >6 HER2 gene copies using single probe: considered positive  Four to six HER2 gene copies: dual probe test advised and the ratio should be recalculated by counting an additional 20 cells Ensuring quality and timely HER2 testing results  The use of validated immunohistochemistry and in situ hybridization tests is strongly recommended and appropriate controls should be included in each run  Turnaround time from initial diagnosis to reporting of results should ideally not exceed 5 working days and a multidisciplinary approach is required  Centralized testing is recommended wherever possible and all laboratories should participate in validated quality assurance programs  [41] found that HER2 positive rate are very low with two antibodies: HecepTEST, 5.9% and 4B5, 6.4%. The similar result also was presented in TMA data which were classified as IHC2+ (1.6%) or IHC3+ (3.2%) if breast cancer scoring was applied [42]. As the same group also tested gastric cancer TMAs using gastric cancer specific scoring [36]the corresponding rates were 4% IHC2+ and 13% for IHC3+, demonstrating an about fourfold increase of HER2 positivity rate [42]. Therefore, Rushcoff concluded that it is supposed that application of breast cancer scoring to gastric cancer may produce an up to 50% false-negative rate if IHC is used as the primary test platform as favored by EMEA [37].
FISH, CISH and SISH studies on HER2 amplification: HER2 amplification was first reported in gastric cancer in 1986 43 . Since then, HER2 amplification in gastric cancer was extensively studied (see Table 3). Kimura et al. 44  HER2 amplification or overexpression correlating with patient survival and clinicopathological features: In breast cancer, HER2 amplification or overexpression is clearly associated with poorer prognosis and aggressive disease. However, the prognosis of HER2 amplification or overexpression in gastric cancer is controversial. In addition, the association of HER2 positive gastric cancer with clinicopathological features are also not consistent.
Yonemura et al [49] (1991) first reported HER2 overexpression in 260 primary gastric cancer. Patients with erbB-2 protein-positive tumors had 5-fold greater relative risk of death, as compared with those with erbB-2 protein-negative tumors. erbB-2 protein expression was associated with serosal invasion, lymph node metastasis, and lymphatic invasion. Later, their results were confirmed by Nakajima et al (1999). Nakajima et al. [50](1999) also reported HER2 overexpression in 16.4% of gastric cancer, which was associated with significantly poorer survival. However, Kim et al. [51] (1994) studied the HER2 overexpression in 152 Korea gastric carcinoma patients. They reported that the survival analysis of 104 patients with stage III gastric carcinoma revealed no significant association between c-erbB-2 staining status and survival duration. The 5-year survival rates of the c-erbB-2 positive group and its negative group were 21% and 28%, respectively. In addition, there was little association between staining of c-erbB-2 protein and clinicopathological findings such as age, sex, location, histology, gross type, lymph node status, depth of invasion, and stage. However, other Korea studies found HER2 positive gastric cancer had a poor prognosis [41,52]. Park et al [41] reported that HER-2/neu overexpression and amplification in 182 gastric cancer Korea patients was examined with IHC. Twenty-nine of 182 patients expressed the HER-2/neu protein by IHC. Tumors with HER-2/neu amplification were associated with poor mean survival rates (922 vs 3243 days) and 5-year survival rates (21.4% vs 63.0%; P < 0.05). Age, TNM stage, and amplification of HER-2/neu were found to be independently related to survival by multivariate analysis. In another Korea study with 1,414 cases and 595 tissue microarray cases, HER2-positivity was detected in 12.3% of whole-tissue sections and 17% of TMAs [53]. They found that HER2-positivity was correlated with age, histological type, lymphovascular invasion, and lymph node metastasis. Multivariate analyses of the differentiated gastric carcinoma subgroup revealed that HER2-positivity was an independent poor prognostic.
Zhang et al (2009)  Recently, Jorgensen and Hersom [56] (2012) reviewed previous studies with more than 100 patients and analysis of association between the HER2 status and survival or relevant clinicopathological characteristics. Forty-two publications with a total of 12,749 patients fulfilled the two criteria and were reviewed in detail. The majority of the publications (71%) showed that a HER2-postive status measured either by IHC or ISH was associated with poor survival and/or clinicopathological characteristics, such as serosal invasion, lymph node metastases, disease stage, or distant metastases. Based on the current analysis a clear trend towards a potential role for HER2 as a negative prognostics factor in gastric cancer was shown, suggesting that HER2 overexpression and/or amplification is a molecular abnormality that might be linked to the development of gastric cancer Trastuzumab or other HER2 related medication on treatment of HER2 amplification gastric adenocarcinoma: Trastuzumab, a monoclonal antibody that targets HER2, induces antibody-dependent cellular cytotoxicity, inhibits HER2-mediated signaling, and prevents cleavage of the extracellular domain of HER2 [12]. Trastuzumab were found to inhibit tumor growth in gastric carcinoma cell lines, animal model and xenograft models [23,33,57,58]. Fujimoto-Ouchi (2007) used trastuzumab as a single agent inhibited the tumor growth in both of the HER2-overexpressing models but not in the HER2-negative models, GXF97 and MKN-45. In any combination with capecitabine, cisplatin, irinotecan, docetaxel, or paclitaxel, trastuzumab showed more potent antitumor activity than the anticancer agents alone. A three-drug combination of capecitabine, cisplatin, and trastuzumab showed remarkable tumor growth inhibition. Since breast cancer showed better prognosis with trastuzumab treatment for HER2 positive breast cancer patients, clinical trial was also performed in gastric carcinoma. ToGA (Trastuzumab for Gastric Cancer) was an open-label, international, phase 3, randomised controlled trial undertaken in 122 centers in 24 countries [22]. Patients with gastric or gastro-esophageal junction cancer were eligible for inclusion if their tumors showed overexpression of HER2 protein by immunohistochemistry or gene amplification by fluorescence in-situ hybridization. Participants were randomly assigned in a 1:1 ratio to receive a chemotherapy regimen consisting of capecitabine plus cisplatin or fluorouracil plus cisplatin given every 3 weeks for six cycles or chemotherapy in combination with intravenous trastuzumab. 594 patients were randomly assigned to study treatment (trastuzumab plus chemotherapy, n=298; chemotherapy alone, n=296). Median follow-up was 18·6 months in the trastuzumab plus chemotherapy group and 17·1 months in the chemotherapy alone group. Median overall survival was 13·8 months in those assigned to trastuzumab plus chemotherapy compared with 11·1 months in those assigned to chemotherapy alone (hazard ratio 0·74).
Although the survival improvement about 3 months, it is a great breakthrough for gastric carcinoma treatment since the survival of these cancer has not change for a decade. After ToGA clinical trial, trastuzumab combined with standard chemical therapy (such as capecitabine or 5-fluorouracil and cisplatin) now is approved by European Medicines Agency, United States and Japan etc. for the treatment of patients with HER2 overexpression or amplification. In addition, laptinib showed a synergistic effect with trastuzumab in vitro and in vivo to inhibit HER2 amplified human gastric cancer cells and animal model [23]. Clinical phase II trial of lapatinib as first line therapy in patients with advanced or metastatic cancer showed well tolerated, which will be another potential drug to target HER2 receptors [59].

HER2 in esophageal adenocarcinoma
EAC incidence has increased 6 folds in United States and Western countries in the last three decades and the prognosis is usually very poor with 5-year survival rates ranging from 14-22% [60][61][62][63]. While surgical treatment of EAC can offer cure, many patients first present as a disseminated disease and require systemic therapy. Current chemotherapy regimens provide only minimal survival benefit, predominantly when used in combination with surgery or radiation. Recently clinical trial (ToGA) in Asian and European countries showed that anti-HER2 monoclonal antibody trastuzumab treatment significantly improved the survival of patients with gastric adenocarcinoma and HER2 overexpression and amplification. The clinical trial of trastuzumab to treat esophageal adenocarcinoma patients are approved in United States and European countries. Here is a comprehensive review of HER2 overexpression and amplification in esophageal adenocarcinoma.
IHC studies on HER2 overexpression: In esophageal adenocarcinoma, HER2 overexpression and amplification recently has been reported at frequencies similar to those observed in breast cancer. Based on most reports from English literature, the frequency of HER-2 immunohistochemistry shows an average of 12%. The current problems for IHC test for HER2 overexpression is the standard score criteria of the intensity of IHC stain. Recently, Zhou and his colleagues (2011) set up a new score criteria which is modified from Hoffman's gastric adenocarcinoma score system (  [64] found that HER2 amplification was 10.5% in high-level amplification (≥ 6.0 signals) and 60% in low-level copy number change ( ≥ 2.5-4.0 signals). However, in thin slides (4 µm, n=123), HER2 amplification was found in 9 % in high-level amplification (≥ 6.0 signals) and 6 % in lowlevel copy number change ( ≥ 2.5-4.0 signals). However, there is a huge difference between traditional FISH in thin section (6%) and three-dimensional FISH in thick section (60%) to detect the low-level HER2 amplification. They considered that the tumor cell nuclei were truncated due to standardized thin tissue sectioning. Therefore, three dimension FISH need to be further evaluated to help better understand any prognostic significance. In our study, we found that HER2 amplification was 18% (21/116) detected by CISH and 16.4% (19/116) by high definition microarray in cases of esophageal adenocarcinoma. In addition we found no evidence of HER2 amplification in low grad dysplasia, Barrett's esophagus, columnar cell metaplasia or normal esophageal squamous epithelium. Thus, the frequency of HER2 amplification in esophageal adenocarcinoma appears to be consistent between studies with a range of 15-19% and this event appears not to occur prior to the development of high grade dysplasia. Radu et al 65

HER2 amplification or overexpression correlating with patient survival and clinicopathological factors:
In esophageal adenocarcinoma, the relationship between HER2 amplification and prognosis is limited and controversial [66,67]. Brien et al. [66] found that patients with HER2 amplification (n=11) had shorter survival durations than did patients without amplification (n=43). In contrast, Reichelt et al. 67 found no survival difference between the HER2 amplification (n=16) and no HER2 amplification groups (n=90)(p=0.953).
In addition, Rauser et al. 28 found that HER2 gene amplification was associated with increased disease-specific mortality on 3-dimensional fluorescence in situ hybridization (FISH) analysis in thick slides (16 µm), but not on FISH and immunohistochemical analyses in thin (4 µm) sections. Our results 68 indicate no association of HER2 amplification with patient survival in a large cohort studies (total 232 patients) by both CISH and high density DNA microarrays methods although HER2 amplification group shows better prognosis (23 months vs 25 months). However, Yoon et al 69 (2012) found that HER2 amplification significantly associated with improved overall survival (n=713) with 35% of HER2 positive patients alive at 5 years as compared with 26% of HER2 negative patients. It is interesting that they divided the HER2 positive EAC into two groups: EAC with and without adjacent BE. They found that HER2 positive EAC with BE significantly associated with disease specific survival and overall survival, but HER2 positive EAC without BE was not significantly associated with disease-specific-survival and overall survival. The prognosis of HER2 positive EAC patients still cannot be concluded. At present, we can say HER2 positive EAC patients do not show worse prognosis.
The association between HER2 amplification and these clinicopathological factors were controversial. First, Brien [66] 2000 reported that HER2 amplification was not significantly associated with any clinicopathological features such as depth of tumor invasion, lymph node metastasis, differentiation and pathological stage. Reichelt et al [67] (2007) found that HER2 amplification was not associated with pathological staging (TNM) and grade. In our study [68] [69](2012) study supported our finding that HER2 amplification cases were significantly associated with better differentiation, but HER2 amplification cases were not associated with age and gender. However, they also showed that HER2 amplification was associated with lower depth of tumor invasion (T stage), fewer malignant nodes, and absence of signet ring cells.
In summary, the association of HER2 amplification with survival and clinicopathological features is not very clear. At least HER2 amplification was not associated with worse prognosis in most large cohort studies. In addition, the HER2 amplification may be associated better differentiation, but not associated with age and sex. The large, multiinstitute study is needed to confirm current studies.

HER2 in breast cancers
Among new breast cancer patients, 15% to 20% will develop tumors that harbor a genomic alteration involving the HER2 gene locus. This alteration results in amplification of an amplicon on chromosome 17 that contains the HER2 proto-oncogene [73,74]. Gene amplification is the primary mechanism that drives HER2 receptor protein over-expression in this important subset of breast cancers. HER2 over-expression resulting from gene amplification dramatically increases the likelihood of receptor activation and signaling, contributing to a more aggressive tumor biology and is associated with worse clinical outcome including higher rates of early, predominantly visceral and central nervous system recurrence and mortality. [75,76] In addition to the prognostic impact, HER2 overexpression in breast cancer is highly correlated with a younger age at presentation, higher tumor grade as well as a higher tumor burden compared with HER2 negative disease [77]. HER2 over-expression in breast cancer was recognized early on as being an ideal target for therapy, given the location of the receptor on the surface of tumor cells and its role in driving the clinical course of disease for the subset of patients with the HER2 alteration [78], [79]. The drug Trastuzumab was developed as a targeted biologic therapeutic against the HER2 receptor protein. Trastuzumab is a humanized monoclonal antibody that combines the mouse recognition sequence of a monoclonal antibody (clone 4D5) against an extracellular epitope of the receptors with a human IgG1 [74]. Trastuzumab demonstrates a high affinity and specificity for the HER2 receptor and in preclinical studies was shown to be effective at inhibiting the growth of HER2 over-expressing breast cancer cells. [80] In numerous clinical trials, targeting HER2 has been shown to be remarkably effective against HER2 positive breast cancer in both the metastatic and the adjuvant settings, particularly in combination with cytotoxic chemotherapy. Treatment with the drug Trastuzumab has been shown to improve response rates, time to progression, and even survival when used alone [18] or added to chemotherapy in metastatic setting . [78] The success of therapeutically targeting HER2 in the metastatic setting led to several international, prospective randomized trials that have demonstrated that adjuvant trastuzumab reduces the relative risk of recurrence by half and mortality by one third in early-stage breast cancer. [81][82][83][84] The data from these clinical trials highlights the importance of accurate HER2 testing for every newly diagnosed breast cancer patient in order to help select those patients who will be the most suitable candidates for HER2 targeted therapy. [85] Clinical assays to assess the HER2 status include IHC, which detects protein overexpression, or FISH, which detects gene amplification. [85][86][87] Both assays have been clinically validated in the above mentioned prospective randomized clinical trials and have received FDA approval for predicting a clinical response and patient benefit from HER2targeted treatment. Published data from these clinical trials suggest that only those patients whose breast cancer demonstrates protein over-expression and/or gene amplification by the above assays are likely to benefit from therapy with Trastuzumab. [88] Since the results of HER2 assays stand alone in determining which breast cancer patients will be the most appropriate for HER2-targeted therapy, accurate, reliable and reproducible results are a high priority for ensuring optimal patient treatment.
The ASCO/CAP task force, has published recommendations for HER2 testing, in which the panel has concluded that both tests were equally efficient in identifying patients who are candidates for HER2-targeted therapy, as long as the assays have been properly validated and all aspects of the testing is performed in a highly standardized fashion with a rigorous quality assurance program. [89] This task force also recognized the importance of standardizing pre-analytical variables including tissue handling and fixation to improve the quality of clinical samples for predictive factor analysis. [90] The IHC and FISH methodologies for evaluating the HER2 status in breast cancer are complementary in nature. [91] These tests examine different aspects of the biology that underlies HER2 driven breast tumors. FISH evaluates the status of the HER2 gene in the nucleus and is a surrogate for protein expression, while IHC directly evaluates over-expression of the receptor protein at the surface of the cell. In the majority of HER2-positive cancers, HER2 protein overexpression is the result of gene amplification, thus HER2 gene/protein status should be highly correlated in most cases. Consequently, HER2 gene/protein discordant results in the majority of cases are related to technical issues. However, unusual HER2 genotypes such as polysomy for chromosome 17 and genomic heterogeneity can lead to discrepant noncorrelating cases that may be clinically important. [92,93] For such cases, the assessment of both the gene and the protein may be necessary in order to sort out the most appropriate HER2 status for the purpose of determining therapy.
Despite the remarkable clinical efficacy of HER2 targeted therapy, not all patients respond and de novo as well as acquired resistance remains an important clinical issue. Currently there are no clinically validated factors that can be used to predict resistance to HER2 targeted therapy in breast cancer. Preclinical data and more recent clinical studies have suggested a number of potential mechanisms of resistance including reduction of antibody affinity and binding due to steric hindrance from MUC4 over-expression, constitutively active downstream signaling involving p27 Kip1, PTEN, PI3K, mTOR, and Akt as well as cross-talk with other signaling pathways including EGFR and IGFR-1, that can by-pass HER2-blockade. [