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Technical Articles

HER2 Testing in Gastric and Esophageal Adenocarcinoma: Emerging Therapeutic Options and Diagnostic Challenges

Christa L. Whitney-Miller, MD David G. Hicks, MD

Department of Pathology and Laboratory Medicine University of Rochester Medical Center Rochester, New York, USA

History of Gastric and Esophageal Adenocarcinoma Gastric carcinoma is a substantial health care problem worldwide, with over 900,000 new diagnoses and 700,000 deaths worldwide annually, making it the second leading cause of cancer mortality in the world (1, 2). In the United States, approximately 21,000 people will be diagnosed with gastric carcinoma each year, including almost 13,000 men and over 8,000 women. This will result in over 10,000 deaths, including 6,000 men and 4,000 women. The average age at the time of diagnosis is 71. The case fatality rate of gastric carcinoma is 0.75, significantly higher than most other common malignancies (1). Median survival is just 8-10 months, and 5-year survival only 7% (3). Currently, gastric carcinoma is the 13th most common cause of cancer death in the United States; however, until the 1930s, it was the leading cause of cancer death. The basis

for this drop is not wholly recognized. Some postulate the availability of refrigeration and hence consumption of more fresh and less salted and smoked food, while others propose that this may be related to the use of antibiotics to treat Helicobacter pylori infections (3). Approximately 16,500 cases of esophageal carcinoma are diagnosed annually in the United States, resulting in 14,500 deaths. Esophageal carcinoma is more common in men, about 3-4x. Overall, the incidence is about the same among blacks and whites, though blacks are more likely to get squamous cell carcinoma, while whites are more likely to get adenocarcinoma. Five-year survival is 18% in whites and 11% in blacks (3). The rate of esophageal adenocarcinoma is increasing in white men from Western countries at approximately 1% a year, while it is stable in white women and decreasing in blacks (3).

Surgical resection is the mainstay of therapy for both gastric and esophageal carcinoma; however, in the United States, most patients are diagnosed at an unresectable stage, particularly those with gastric carcinoma. For these patients, chemotherapy may prolong life, but survival rates remain low. Given the poor prognosis and the fact that most gastric and esophageal cancers are diagnosed at a more advanced or unresectable stage, new therapeutic strategies, treatment options and novel therapeutic targets are desperately needed.

New AJCC Definition for Adenocarcinoma of the Esophagus, GE Junction and Stomach The seventh edition of the AJCC1 Cancer Staging Manual (4) redefines esophageal cancer as any tumor whose epicenter is in the lower esophagus, gastroesophageal junction or proximal 5cm of the stomach that extends

AJCC: American Joint Committee on Cancer

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into the gastroesophageal junction (GEJ) or esophagus. Tumors with an epicenter in the stomach greater than 5cm distal to the GEJ, or those within 5cm of the GEJ but not extending into the GEJ or esophagus, are stage grouped using the gastric cancer staging system.

general, HER2 over-expression is related to In worse prognosis in esophageal adenocarcinoma. Different studies have found HER2 over-expression in esophageal adenocarcinoma to be associated with increasing depth of invasion, lymph node and distant metastases and poor survival.

HER2 in Breast Cancer HER2 Biology HER2 is a member of the HER-family of growth factor receptors, which includes HER1, HER3, and HER4, all of which are involved in the complex regulation of cell growth, proliferation and survival. The HER2 gene is located on chromosome 17 and is constitutively expressed in many different types of normal epithelium. This gene is translated into a 185-kD membrane growth factor receptor protein, which transmits signals regulating normal cell growth, development and survival. The binding of several high affinity ligands to HER receptor-family members leads to receptor dimerization and activation of intracellular signaling through receptor tyrosine kinases. The over-expression of the HER2 protein increases the likelihood of receptor dimerization and activation of these signaling pathways (5). In a subset of 15-20% of early stage breast cancer, there is a genomic alteration involving the HER2 gene locus leading to amplification of the gene on the order of 2 to 20 fold relative to the number of copies of chromosome 17. HER2 gene amplification is an early event in tumor development for this subset of breast cancers and drives HER2 gene and protein expression, resulting in a marked increase in the number of HER2 receptor protein molecules on the surface of each tumor cell (5). The ensuing signaling cascade and signal transduction drives cellular proliferation, migration, enhances cell survival pathways and promotes angiogenesis, contributing to

an aggressive biology and clinical behavior for tumors with this molecular alteration (6). Like HER2, other members of this receptor family have been implicated in the development of cancer. HER1, also known as EGFR, has been implicated in cancers of the lung, colon and head and neck. In addition to breast cancer, HER2 over-expression and/or gene amplification have also been reported in carcinomas of the colon, bladder, ovary, endometrium, lung, head and neck, esophagus and stomach.

to be effective at inhibiting the growth of HER2 over-expressing breast cancer cells. In numerous clinical trials, trastuzumab 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 (8). Recent clinical trials data have demonstrated that adjuvant trastuzumab in combination with or following chemotherapy can reduce the relative risk of recurrence by up to 50% in early stage HER2-positive breast cancer (8). The clinical efficacy of trastuzumab in selective patients who over-express HER2 provides the rationale for testing all newly diagnosed breast cancer patients for the HER2 alteration. Clinical assays to assess the HER2 status include immunohistochemistry (IHC) which detects protein over-expression or fluorescence in situ hybridization (FISH) which detects gene amplification (7, 9). Both assays have been clinically validated to predict a clinical response and patient benefit from trastuzumab treatment. Data from clinical trials suggest that only those breast cancer patients whose tumors demonstrate protein over-expression and/or gene amplification by the above assays are likely to benefit from therapy with trastuzumab (8).

HER2 as a Target for Therapy in Breast Cancer HER2 over-expression in breast cancer represents an ideal target for therapy, given the location of the receptor on the surface of tumor cells and its role in driving the biology and the clinical course of the disease (7). 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 (5). Trastuzumab demonstrates a high affinity and specificity for the HER2 receptor and in preclinical studies was shown

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HER2 Expression in Gastric and Esophageal Adenocarcinoma A number of recent studies have reported the over-expression of HER2 in a subset of 20-30% of patients with gastric and esophageal adenocarcinoma; however, the full clinical significance of this finding remains to be clearly defined. In some studies, HER2 appears to be an important prognostic factor in gastric cancer; however, the literature is conflicting in this respect, and not all studies have shown a clear association between HER2 over-expression and poor prognosis. It is clear that HER2 protein over-expression and gene amplification are much more heterogenous in gastric cancer compared to breast cancer. This has implications for the clinical testing of biopsy specimens (Table 1), as well as for the use of small specimens such as tissue micro-arrays in the research setting. Also evident from early studies is that there is less correlation between IHC and FISH results; this is also discussed in more detail, as follows. But in general, HER2 over-expression is related to worse prognosis in esophageal adenocarcinoma. Different studies have found HER2 overexpression in esophageal adenocarcinoma to be associated with increasing depth of invasion, lymph node and distant metastases and poor survival (10).

HER2 Expression, Histologic Type and Primary Tumor Site The exact numbers are variable, but proximal gastric carcinomas with intestinal phenotype are generally found to have a higher prevalence of HER2-positivity (range 8-34%) than distal diffuse gastric carcinomas (range 1-7%). A sample of study results including the region represented is summarized in Table 1. The rate of HER2-positivity in esophageal adenocarcinoma is also variable (range 0-73%), but may be slightly higher than gastric carcinomas, with one meta-analysis showing approximately 25% of esophageal adenocarcinomas over-expressing HER2 (10).

(16 were from the gastroesophageal junction and 3 were esophageal) with both IHC (Dako HercepTest) and FISH (Dako HER2 FISH pharmDx) kits (Fig. 1). When the breast cancer scoring criteria were applied, 11 cases were found to be FISH positive, but IHC negative or equivocal. The remaining 157 (93.5%) were concordant. Further analysis of these 11 cases (FISH + cases in Table 2) revealed three reasons for inconsistent results. The first group (n=3) consisted of cases with intense immunoreactivity in <10% of cells, which is scored as negative (IHC score=0) by HercepTest criteria. However, these IHC 3+ clones were FISH+. A second group (n=2) exhibited moderately intense incomplete basolateral reactivity in >10% of cells. According to breast cancer criteria, incomplete membranous reactivity is IHC = 0 or 1. However, of the seven cases with moderately intense incomplete membranous reactivity, two were FISH+. The third group (n=5) consisted of samples exhibiting moderately intense complete membranous reactivity, scored as 2+ by HercepTest criteria. Of the 14 cases that exhibited moderately intense complete membranous reactivity, 5 were FISH+. Given these results, a consensus panel made the

Summary of Hoffmanns Assessment and Validation of HER2 Scoring System in Gastric Cancer Accurate determination of HER2 status is critical to ascertain which patients might benefit from trastuzumab. A validated algorithm exists for HER2 assessment in breast cancer; however, prior to a recent publication from Hoffman, et al. (16), a validated scoring system did not exist for HER2 assessment in gastric cancer. Hoffman, et al. (16) analyzed 168 resection specimens, most (n=149) from the stomach

Author

Population

n Intestinal (%+)

Histologic Type Diffuse (%+) 2 7 6 6 1 Mixed/unknown (%+) 5 14 20 GEJ 24 25 32

Location Stomach 12 9.5 18

Tanner (11) Gravalos (12) Lordick (13) Matsubara (14) Park (15)

Findland Spain International Japan Korea

231 166 1527 87 161

21.5 16 34 32.5 8

Table 1. Proximal gastric carcinomas with intestinal phenotype are found to have a higher prevalence of HER2 over-expression than distal diffuse gastric cancer in
published studies. Abbreviations: GEJ: Gastroesophageal junction

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Case # 3 2 5 5 9 1

IHC Result Strong complete staining in <10% Moderately intense incomplete basolateral staining in >10% Moderately intense incomplete basolateral staining in >10% Moderately intense complete staining in >10% Moderately intense complete staining in >10% Faint complete staining in <10%

IHC Score by Breast Criteria 0 1+ 1+ 2+ 2+ 0

FISH Result + + + +

Table 2. Breakdown of the discordant cases between IHC and FISH from the Hoffman (16) validation study of HER2 over-expression using breast cancer
scoring criterion.

Pattern No reactivity or membranous reactivity in <10% of cells Faint/barely perceptible membranous reactivity in >10% of cells; cells are reactive only in part of their membrane Weak to moderate complete or basolateral membranous reativity in >10% of cells Moderate to strong complete or basolateral membranous reativity in >10% of cells Biopsy samples with cohesive IHC3+ or FISH+ clones, irrespective of size (even if <10%)

Score/Classification 0/negative 1+/negative 2+/equivocal 3+/positive 3+/positive

Table 3. Consensus panel recommendation for HER2 evaluation of gastric cancer from the Hoffman validation study (16). Cases with equivocal results by immunohistochemistry should undergo FISH analysis.

following recommendations for HER2 scoring in gastric cancer, as seen in Table 3 (16).

ToGA Trial The results from the first successful phase III clinical trial of anti-HER2 therapy in combination with chemotherapy in advanced HER2-positive gastric cancer represents a major advancement in systemic therapy for this deadly disease. Only 20% of the patients screened and subsequently enrolled for this study were found to be HER2-positive when utilizing both immunohistochemistry and FISH. The ToGA study (trastuzumab with chemotherapy in HER2-positive advanced Gastric Cancer) looked at the combination of trastuzumab with cisplatin/capecitabine (or FU) compared with cisplatin/capecitabine chemotherapy alone in a multicenter multinational random assignment phase III trial. In HER2-positive gastric tumors, the

addition of trastuzumab to chemotherapy was associated with a significant improvement in overall survival compared with chemotherapy alone (13.8 months (trastuzumab + cis/capecitabine) versus 11.1 months (cis/capecitabine alone), p = 0.0002). This landmark study was the first to show a survival advantage with a targeted therapy in a prospective randomized trial and has established a new treatment option for patients with metastatic HER2-positive gastric cancer (17, 18). Several small Phase I studies have looked at trastuzumab in esophageal carcinoma and found acceptable toxicity profiles.

A subset of these carcinomas over-express HER2, a growth factor receptor more commonly associated with breast cancer. A monoclonal antibody to HER2 is commercially available and has been shown to improve overall survival in gastric cancer patients with HER2 over-expressing tumors. Studies of trastuzumab in esophageal carcinoma are in preliminary stages. HER2 testing in gastric cancer is generally similar to HER2 analysis in breast cancer, with some slight interpretative differences recommended regarding basolateral staining and sub-clones. With the availability of ever-more targeted antibody therapies to treat tumors of different organ systems, pathologists need to keep abreast of testing methodologies so they can provide the information oncologists and surgeons need to treat their patients.

Conclusions Esophageal and gastric carcinomas are substantial health concerns worldwide and both have traditionally had dismal prognoses.

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Figure 1. Gastric adenocarcinoma. Upper left panels shows faint incomplete membranous staining (IHC 0/negative). Upper right panel shows moderate basolateral staining in >10% of cells (IHC 2+/equivocal). Lower left panel shows strong complete staining in most cells (IHC 3+/positive). Dako HercepTest.

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15. Park DI, Yun JW, Park JH, et al. HER2/neu amplification is an independent prognostic factor in gastric cancer. Dig Dis Sci 2006; 51:1371-1379. 16. Hofmann M, Stoss O, Shi D, Bttner R, van de Vijver M, Kim W, Ochiai A, Rschoff J, Henkel T. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology 2008; 52:797-805. Epub 2008 Apr 18. 17. Van Cutsem E, Kang K, Chung H, Shen L, Sawaki A, Lordick F, et al. Efficacy results from the ToGA trial: a phase III study of trastuzumab added to standard chemotherapy (CT) in first-line human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer (GC). In: Annual proceedings. Orlando, FL, USA: American Society of Clinical Oncology; May 29-June 2, 2009 [abstract #LBA 4509]. 18. Bang Y, Chung H, Xu J, Lordick F, Sawaki A, Lipatov O, et al. Pathological features of advanced gastric cancer (GC): relationship to human epidermal growth factor receptor 2 (HER2) positivity in the global screening programme of the ToGA trial. In: Annual proceedings. Orlando, FL: American Society of Clinical Oncology; May 29-June 2, 2009 [abstract #4556].

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