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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, gastro-esophageal cancer by trastuzumab in many recent reviews [1-8].
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 . 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 . 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 . 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 . Overexpression of HER2 in human
mammary epithelial cells induces proliferative advantage, transformed characteristics, tumorigenic growth, and induces proliferative and anti-apoptotic changes that mimic early stages of epithelial cell transformation . HER2 amplification is also seen in early in situ ductal carcinomas without any evidence of invasive disease [13, 14]. HER2 status is maintained during progression to invasive disease, nodal metastasis and distant metastasis [14, 15]. HER2 overexpression has been shown to activate multiple signaling complexes, which results in a striking dysregulation of the global transcriptome .
Clinical treatment targeting on HER2 receptor:It took a long journey to develop monoantibody to target HER2. Murine origin of mAb to HER2 limits their clinical application since immunoglobulin molecules are immunogenic. When injected into humans, it shortens their half-lives in circulation. Winter and colleagues (1988) generated a mouse–human chimeric antibody . Later transgenic mice whose immunoglobulin loc have been genetically inactive, was used to produce the first fully human antibody, Panitumumab, an antibody to EGFR. Then, trastuzumab which carry all human immunoglobulin genes, a monoclonal antibody to HER2, was approved for clinical use in lymphoma and in breast cancer . So far, only two drugs that target HER2, Trastuzumab and a kinase inhibitor called Lapatinib/Tykerb, are approved for clinical application in breast cancer, but several novel drugs are in development (see figure 2).
Trastuzumab, monoclone antibody on HER2: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 . Based on multi-centers and countries clinical trial for HER2 positive breast cancer, [18,19,20] trastuzumab was significantly improve the prognosis of breast cancer. Therefore, it was initially approved for treatment of patients with HER2 overexpressing metastatic breast cancer. Because Trastuzumab also enhances the efficacy of adjuvant chemotherapy in operable or locally advanced HER2-positive tumors , the antibody currently represents the standard of care for patients with early or advanced stages of HER2-overexpressing breast cancer.
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 . 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.
The clinical efficacy of Trastuzumab likely entails a combination of immunological and non-immunological mechanisms . The ability of Trastuzumab to elicit antibody-dependent cellular cytotoxicity critically influences the efficacy of Trastuzumab-based therapies. Non-immunological 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 EGFR23. 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 . 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 . 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.
Lapatinib response correlated with EGFR and HER2 expression levels in patients’ tumors, and associated with increased pre-treatment expression of phosphorylated-HER2 (p-HER2). Lapatinib is able to induce apoptosis of Trastuzumab-resistant breast cancer cells via alteration of IGF-1 signaling, [28, 29] and also block NRG-induced p95-HER2/HER3 heterodimers formation .
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 . Trastuzumab treatment of HER2 positive breast cancer patient improved survival. HER2 overexpression and amplification were reported in gastric and gastro-esophageal 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 [33-35]. 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. 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. 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 . 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) 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  reported that HER2 positive rate was a 22.1%. In addition, they found that HER2-
IHC score criteria
No reactivity or membranous reactivity in <10% of cells; Biopsy specimens < 5 Cells
No reactivity or membranous reactivity in <10% of cells
Faint/barely perceptible membranous reactivity in ≥10% of cells; biopsy specimens≥5 Cells
Faint membranous reactivity in "/>10% of cells;
Weak to moderate complete or basolateral membranous activity in ≥10% of tumor cells; biopsy specimens≥5 Cells
Weak to moderate complete membrane staining in "/>10% of tumor cells
Moderate to strong complete or basal/lateral membranous activity in ≥10% of resection tumor cells; biopsy specimens≥5 Cells
Strong complete membrane staining in "/>10% of tumor cells
FISH HER2/CEP 17
≥ 2At least 20 evaluable, non-overlapping cells in the invasive component
"/> 2.2At least 20 evaluable, non-overlapping cells in the invasive component
<2At least 20 evaluable, non-overlapping cells in the invasive component
<1.8At least 20 evaluable, non-overlapping cells in the invasive component
Consensus panel recommendations on HER2 scoring for gastric cancer
positive rate were higher in GEJ cancer than in gastric cancer (33% vs 21%) and in intestinal than diffuse or mixed cancer (32.2% vs 6.1% vs 20.4%). The concordance between IHC and FISH was 87.5%. Ruschoff and colleaguesl37, 38(2010, 2012) further validate the HER2 test procedure to determine whether pathologists from different sits were able to reproduce the method of gastric cancer HER2 status evaluation as it was used by Ruschoff within the ToGA study. They validated the HER2 status testing procedure in terms of inter-laboratory and inter-observe consensus for IHC scoring a series of 547 gastric cancer tissue samples on a tissue microarray. They published a practical approach of HER2 test in gastric carcinoma. Based on multiple laboratories and 8 pathologists HER2 test results, they further confirmed the HER2 positive rate of 22.8% which is close to 22.1% from Hoffman’s score system. In addition, they compared Daco (HecepTest) and Ventana (Pathway HER2 antibody, 4B5). They found that HercepTest had a higher inter-laboratory discordance than 4B5. Furthermore, Ruschoff and a group of international pathologist reviewed previous HER2 studies; they built up new detailed criteria for gastric and gastro-esophageal HER2 tests (see 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.
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 situhybridization-positive or immunohistochemistry 2+/silver in situhybridization-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 situhybridization or silver in situhybridization (scores for both assays should be indicated separately on the report)
Validated immunohistochemistry HER2 assays should be used
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 situhybridization-positive or silver in situhybridization-positive may be considered HER2-positive (scores for both assays should be indicated separately on the report)
In situ hybridization
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 situhybridization is a more suitable methodology than fluorescence in situhybridization 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 situhybridization HER2 assays should be used
The definition of fluorescence in situhybridization or silver in situhybridization 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 situhybridization 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 HER2gene count is important:
"/>6 HER2gene copies using single probe: considered positive
Four to six HER2gene 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 situhybridization 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
Human epidermal growth factor receptor 2 (HER2) testing recommendations in gastric cancer, (a) immunohistochemistry and (b) in situhybridization (From Ruschoff J, Hanna W, Bilous M, et al. HER2 testing in gastric cancer: a practical approach. Mod Pathol 2012)
HER2 immunohistochemistry features
No reactivity or very faint membranous stain in <10% of cells; biopsy specimens<5 Cells
Faint membranous stain in "/>10% of cells; biopsy specimens≥5 Cells
Weak to moderate complete or baso/lateral membranous stain in "/>10% of tumor cells; biopsy specimens≥5 Cells
Strong complete or basal/lateral membranous stain in "/>10% of tumor cells; biopsy specimens≥5 Cells
HER2 FISH/chromogenic in situ hybridization test
Ratio of average HER2/CEP17 ≥2.0
Ratio of average HER2/CEP17 <2.0
Modified score criteria of HER2 immunohistochemical stain and FISH/chromogenic in situ hybridization for esophageal adenocarcinoma
Recently, in Asia, several IHC HER2 tests focused on comparing the HER2 antibodies from various companies. Cho and colleagues  used four different HER2 antibodies compared to standard FISH test. They found the various positive rates with HercepTest (14%), A0485 (16%), 4B5 (14%), and CB11 (9%). The sensitivity and specificity of IHC compared to FISH was 78.9%/96% for HercepTest, 86.5%/94.4% for A0485, 76.3%/95.6% for 4B5 and 60.5%/98.4% for CB11. Compare to FISH, there was no significantly differences in the sensitivity and specificity among the four IHC tests. However, CB11 had a highest specificity (98%), but a lowest sensitivity (61%). Park et al  (2012) compared HercepTest with 4B5, only 41 cases showed discrepancies, yielding a 96.1% concordance rate. However, HER2 positive rate with both methods are very low: HecepTEST, 5.9% and 4B5, 6.4%.
In addition, the standard breast HER2 test was compared with modified gastric carcinoma HER2 test (Table2). Sever studies used breast cancer score rule [40-42]. Barros-Silva et al.  found 3.9% as IHC2+ and 5.4% as IHC3+ from resection 463 gastric adenocarcinomas using the breast cancer scoring rules. Using breast cancer scoring, Park et al. (2012) 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. As the same group also tested gastric cancer TMAs using gastric cancer specific scoring the corresponding rates were 4% IHC2+ and 13% for IHC3+, demonstrating an about fourfold increase of HER2 positivity rate. 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.
FISH, CISH and SISH studies on HER2 amplification:HER2 amplification was first reported in gastric cancer in 198643. Since then, HER2 amplification in gastric cancer was extensively studied (see Table 3). Kimura et al. 44 first set criteria of FISH test as HER2/CEP17 ≥ 2.0 which is modified from breast standard HER2 FISH test with 83% of concordance between IHC 2+ and 3+ samples. Hoffman et al.36 proved that these FISH criteria for gastric cancer showed a higher concordance (93%) between HER2 amplification and overexpression in gastric cancer. Ruschoff et al. 38, 45 (2010, 2012) further validate the HER2 test procedure to determine whether pathologists from different sits were able to reproduce the method of gastric cancer HER2 status evaluation as it was used by Ruschoff within the ToGA study. HER2 amplification was determined by FISH assays, using either HER2 FISH pharmDX™ (Dako Denmark A/S) or PathVysion® (Abbott Laboratories, Des Plaines, IL, USA). Automated brightfield dual-color silver in situ hybridization (SISH) assay (BDISH; Inform™, Ventana Medical Systems SA) was used to determine gene amplification at three of the participating sites. Based on their experience and previous studies, a new practical procedure for HER2 FISH, CISH or SISH tests were established. The positivity of HER2 FISH, CISH or SISH tests in gastric or gastro-esophageal junction cancer is a HER2/Chromosome 17 ratio ≥ 2.0 and > 6 HER2gene copies using single probe. At least 20 evaluable, non-overlapping cells in the invasive component should be counted initially. If the results are borderline (four to six HER2gene copies or HER2/Chromosome 17 ratio 1.8-2.2),  additional cells should be recounted or scoring should be performed in an alternative area of tissue. However, they also concluded that silver in situhybridization is a more suitable methodology than fluorescence in situhybridization 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.
Comparing FISH and SISH methods for HER2 test in gastric cancer was also reported by several studies. Park et al  (2012) compared both SISH and FISH HER2 tests in Korea gastric adenocarcinoma 588 cases. They found only 9 cases with discrepancy, yielding a 98.3% concordance rate. Garcia-Garcia et al  (2011) compared both SISH and FISH HER2 tests in Spanish gastric adenocarcinoma in 166 cases. They found 96% concordance rate. Long et al  (2011) compared both SISH and FISH HER2 tests in China gastric adenocarcinoma 80 cases. They found only one case with discrepancy, yielding a 99% concordance rate. From above studies, FISH and SISH showed similar positive rates. The only difference between two methods is that SISH is much easier to count the HER2 signals.
HER2 amplification or overexpression in primary tumor vs metastatic tumor was also reported. Bozzetti et al  (2011) tested HER2 status with both FISH and IHC. The found that concordance of HER2 status between primary and metastatic tumor is 98.2% by FISH and 94.9% by IHC. They concluded that HER2 status is maintained in most cases unchanged during the metastatic process.
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  (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. (1999) also reported HER2 overexpression in 16.4% of gastric cancer, which was associated with significantly poorer survival. However, Kim et al.  (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  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 . 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) studied the HER2 and HER3 overexpression in Chinese gastric cancer with 102 cases. Overexpression of HER2 and HER3 was detected around 18.6% and 13.7%. HER2 and HER3 overexpression was correlated with a significantly worse survival (p = 0.046 and 0.024, respectively). The overexpression rates of HER2 and HER3 in phase III-IV (TNM stage) disease were significantly higher than that in phase I-II disease (24.0% vs. 7.7%, p < 0.05 and 22.0% vs. 5.8%, p < 0.05, respectively). They proposed that HER3 may become another molecular target.
In European and United States, Tanner et al  (2005) found that HER2 amplification was present in 12.2% of the 131 gastric cancer and 24% of the 100 GEJ adenocarcinomas in Finland which was associated with poor carcinoma-specific survival. In contrast, Kunz (2012) reported that twelve of 99 (12%) gastric carcinomas were positive for HER2 and seven of 70 (10%) gastroesophageal junction carcinomas were positive for HER2. HER2 status or primary tumor site did not correlate with patient survival.
Recently, Jorgensen and Hersom  (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. 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. 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 . 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.
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%[60-63]. 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, HER2overexpression 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 (Table 3). In our modified score criteria, IHC 2+ will be counted as positive HER2 overexpression since all IHC 2+ case had HER2 amplification with CISH test. However, the recent Mayo Clinic study reported that only 15% of IHC2+ cases showed HER2 amplification with FISH tests with breast HER2 criteria. It is difficult to compare their criteria since there are no pictures in their reports.
FISH and CISH studies on HER2 amplification:In esophageal adenocarcinoma, HER2 amplification recently has been extensively studies. Reichelt et al. found that 15% (16/110) of tumors had HER2gene amplification with FISH. Similarly, Brien et al. showed that 19% (12/63) of esophageal adenocarcinomas had HER2gene amplification. In addition, with 3-dimentional FISH method in thick slides (16 µm, n=124), Rauser et al.  found that HER2amplification 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), HER2amplification was found in 9 % in high-level amplification (≥ 6.0 signals) and 6 % in low-level 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 HER2amplification. 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 HER2amplification 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 HER2amplification in low grad dysplasia, Barrett’s esophagus, columnar cell metaplasia or normal esophageal squamous epithelium. Thus, the frequency of HER2amplification 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 al65 (2012) compared HER2 antibodies with FISH tests. They used CAP definition of HER2 amplification to evaluate the FISH results. They found that the very high HER2 amplification rate (30/103, 29%) with HER2/CEP17 ≥ 2.2 and (32/102, 31%) with HER2/CEP17 ≥ 2.0. From their slides, they used 5 µm instead of 3-4 µm routine section. Actually the similar phenomenon was reported in esophageal adenocarcinoma cases 64. Using 16 µm vs4 µm sections for HER2 FISH tests, they found that 16 µm sections showed higher HER2 amplification than 4 µm. Higher HER2 amplification from Radu may be caused by thicker section.
HER2 amplification or overexpression correlating with patient survival and clinicopathological factors:In esophageal adenocarcinoma, the relationship between HER2amplification and prognosis is limited and controversial [66, 67]. Brien et al.  found that patients with HER2amplification (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 HER2amplification (n=16) and no HER2amplification groups (n=90)(p=0.953). In addition, Rauser et al.28found that HER2gene 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 HER2amplification 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 al69 (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 HER2amplification and these clinicopathological factors were controversial. First, Brien  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  (2007) found that HER2 amplification was not associated with pathological staging (TNM) and grade. In our study , 21 of 116 EAC patients had HER2amplification. Nineteen were male, and 2 female (M:F ratio, 10:1), with a mean age of 63 years (range, 51 to 74 years). The remaining patients (85 males and 10 females [M:F ratio, 9:1]; mean age, 65 years [34 to 85 years]) had no amplification. A Fisher’s exact test shows that there is no significant association between HER2and gender (p=1.0), age (p=0.188,), the stage (p=0.325), and the number of metastatic lymph nodes (p=0.234). However, the frequency of HER2 amplification was found to be significantly higher (p=0.004) in moderately differentiated tumors (13/22) compared with poor or well differentiated tumors (1/6 and 7/61 respectively). Yoon (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, multi-institute study is needed to confirm current studies.
Trastuzumab or other HER2 related medication on treatment of HER2 amplification esophageal adenocarcinoma:Safran et al [70, 71] (2004, 2007) first reported clinical trial with trastuzumab, paclitaxel, cisplatin and radiation for locally advanced esophageal adenocarcinoma patients with HER2 overexpression. They took patients with histologically documented EAC with T3, T4 or lymph nodal disease. They used IHC 2+ and 3+ with more than 10% cells as HER2 positive overexpression and set FISH ratio greater than 2 as HER2 amplification. The median survival for all 19 patients is 24 months, which is similar to prior studies. Esophagitis, nausea, dehydration, and neutropenia were the most common toxicity. However, toxicity was modest with only 2 patients (10%) having grade 3-4 esophagitis. Therefore, trastuzumab does not increase toxicities when added to chemoradiation for patients with esophageal cancer.
ToGA clinical trials in patients with gastric adenocarcinoma (trial vscontrol: 236 vs243 patients) and gastroesophageal junction adenocarcinoma (trial vscontrol: 58 vs48 patients) have shown a significant survival benefit for patients treated with a combination of trastuzumab and standard chemotherapy.[22,72] Now Safran and colleagues started Phase III clinical trial to study Radiotherapy, Paclitaxel, and Carboplatin with versus without Trastuzumab in patients with HER2-overexpressing esophageal adenocarcinoma (RTOG-1010, and NCI web site: http://www.cancer.gov/ncicancerbulletin/062811/page6). Their primary goal is to determine whether trastuzumab increases disease-free survival when combined with radiotherapy, paclitaxel, and carboplatin followed by surgery in patients with HER2-overexpressing esophageal adenocarcinoma. It is interesting to follow up their results.
Among new breast cancer patients, 15% to 20% will develop tumors that harbor a genomic alteration involving the HER2gene 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 over-expression 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 . 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 , . 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. 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.
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 or added to chemotherapy in metastatic setting. 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.[81-84] 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. 
Clinical assays to assess the HER2 status include IHC, which detects protein over-expression, or FISH, which detects gene amplification. [85-87] 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 HER2-targeted 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.  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.  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.  The IHC and FISH methodologies for evaluating the HER2 status in breast cancer are complementary in nature.  These tests examine different aspects of the biology that underlies HER2 driven breast tumors. FISH evaluates the status of the HER2gene 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 over-expression is the result of gene amplification, thus HER2gene/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 HER2genotypes such as polysomy for chromosome 17 and genomic heterogeneity can lead to discrepant non-correlating 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. [94, 95]
Lymph node METASTASIS
Median survival (months)
Association of HER2amplified group and non-HER2amplified group with multiple clinical factors (From Hu Y, Bandla S, Godfrey TE, et al. HER2 amplification, overexpression and score criteria in esophageal adenocarcinoma. Mod Pathol 2011;24:899-907)
Zhongren Zhou and David G. Hick (January 24th 2013). HER2 Amplification or Overexpression in Upper GI Tract and Breast Cancer with Clinical Diagnosis and Treatment, Oncogene and Cancer - From Bench to Clinic, Yahwardiah Siregar, IntechOpen, DOI: 10.5772/53921. Available from:
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