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Lack of HER2 Overexpression and Amplification in Small Intestinal Adenocarcinoma

Owen T.M. Chan MD, PhD, Zong-Ming E. Chen MD, PhD, Fai Chung, Kevin Kawachi, Dan C. Phan MD, PhD, Eric Himmelfarb MD, Fan Lin MD, PhD, Arie Perry MD, Hanlin L. Wang MD, PhD
DOI: http://dx.doi.org/10.1309/AJCPK6QHNNOEMJIM 880-885 First published online: 1 December 2010

Abstract

HER2 overexpression and amplification have been studied as a therapeutic and prognostic target in a number of human cancers, including esophageal, gastric, and colorectal adenocarcinomas. However, HER2 status has not been well investigated in primary small intestinal adenocarcinoma, probably because of its rarity. In this study, we conducted immunohistochemical analysis and fluorescence in situ hybridization (FISH) for HER2 on 49 primary nonampullar small intestinal adenocarcinomas. The results showed a complete lack of HER2 protein expression in 47 cases (96%) by immunohistochemical analysis. Only 2 cases (4%) showed a 1+ staining pattern. No tumors exhibited 2+ or 3+ HER2 immunoreactivity. By FISH, none of the tumors, including those with 1+ HER2 immunoreactivity, exhibited HER2 gene amplification. These observations demonstrate that HER2 protein overexpression and gene amplification are infrequent events, if they occur at all, in small intestinal adenocarcinoma. Thus, routine immunohistochemical and/or FISH testing for HER2 for potential targeted anti-HER2 therapy may not be beneficial for patients with primary small intestinal adenocarcinoma.

Key Words:
  • Small intestinal adenocarcinoma
  • HER2
  • Immunohistochemistry
  • Fluorescence in situ hybridization
  • Gene amplification

Primary small intestinal adenocarcinoma (SIA) is a rare malignancy with a poor prognosis. The 5-year overall survival rate for patients with SIA is 26% to 40% and has remained unchanged since the 1970s.15 Not surprisingly, survival is related to tumor stage, and the 5-year individual survival rates for stage I, II, III, and IV tumors are 65%, 48%, 35%, and 4%, respectively.6 Primary therapy for SIA is surgical resection, which has not changed for the past 30 years.13,7 Unfortunately, the currently available adjuvant chemotherapies following surgical resection seem to have no significant additional benefit.8

The absence of effective treatment diversity for SIA and the resultant poor patient survival underscore the importance of understanding tumor pathobiology in order to develop new therapies. Studies of HER2 in breast carcinoma serve as a prime example in this regard. HER2 signaling is involved in cell proliferation, differentiation, and survival9; and this oncogene is overexpressed and amplified in 20% to 30% of breast cancers.10 With the development of anti-HER2 targeted therapy, patients with HER2-overexpressing breast carcinoma experienced increased overall survival.11,12 Therefore, knowing the HER2 expression status in breast cancer determines whether a patient would benefit from such targeted therapy.

To extend the potential benefit of anti-HER2 therapy, investigators have examined the HER2 status in a number of nonbreast malignancies.13 HER2 overexpression or amplification has been studied in patients with gastrointestinal tumors such as esophageal adenocarcinoma,14 gastric adenocarcinoma,13,1518 and colorectal adenocarcinoma.13,19,20 Recently, anti-HER2 therapy has been tested in patients with gastric adenocarcinoma. The phase 3 ToGA trial demonstrated that the addition of trastuzumab to standard chemotherapy against HER2+ advanced gastric cancer led to increased median overall survival to 13.5 months in contrast with 11.1 months with standard chemotherapy alone.21 This work suggests that anti-HER2 therapy may have positive therapeutic effects for gastrointestinal tumors.

The aim of the current study was to further determine the prevalence of HER2 overexpression and amplification in SIA because HER2 status has been investigated in only a limited number of cases with conflicting findings.8,22 Our data showed a lack of HER2 protein overexpression and gene amplification in the cases examined in this study. Thus, HER2-targeted therapies may have only limited therapeutic benefit, if any, for these rare but highly aggressive tumors.

Materials and Methods

Study Population

This retrospective study involved 49 patients who underwent surgical resection for SIA at the authors’ institutions. These included 23 cases (47%) from the jejunum, 9 (18%) from the ileum, 8 (16%) from the duodenum (distal from the ampulla of Vater), and 9 (18%) with unspecified locations. Ampullar carcinomas were excluded from the study because of their heterogeneous histogenesis. Cases with a clinical history of adenocarcinoma in other anatomic locations were also excluded. The study was approved by the institutional review boards at Cedars-Sinai Medical Center, Los Angeles, CA, and the coauthors’ institutions. H&E-stained slides were rereviewed to confirm the diagnosis. Formalin-fixed, paraffin-embedded tissue blocks were selected to include adenocarcinoma and normal-appearing, nonneoplastic intestinal mucosa in the same blocks, if possible.

Immunohistochemical Detection of HER2 Protein

Immunohistochemical detection of the HER2 protein was performed on 4-μm tissue sections using prediluted PATHWAY anti–HER2/neu rabbit monoclonal antibody (clone 4B5) obtained from Ventana Medical Systems, Tucson, AZ. The staining was conducted on the Ventana BenchMark XT automated slide stainer using the onboard heat-induced epitope retrieval method in high pH CC1 buffer (Ventana). The staining was visualized using the ultraView DAB Universal Detection Kit (Ventana), which involved a hydrogen peroxide substrate and 3,3′-diaminobenzidine chromogen solution. The slides were subsequently counterstained with hematoxylin. For each batch of staining, there was a positive control sample, composed of a case of ductal carcinoma of the breast known to overexpress HER2 protein, and a negative control sample in which the primary antibody was replaced by non-human-reactive rabbit IgG.

Scoring of the HER2 immunostains was conducted according to published criteria for breast carcinoma, slightly modifying the system based on the DAKO (Carpinteria, CA) HercepTest kit guide23 and the Ventana PATHWAY HER2 package insert. Briefly, a score of 0 denotes no staining observed in tumor cells. A score of 1+ denotes weak, incomplete membrane staining in any percentage of tumor cells or weak, complete membrane staining in fewer than 10% of tumor cells. A score of 2+ denotes weak, complete membrane staining in 10% or more of tumor cells or intense, complete membrane staining in fewer than 30% of tumor cells. A score of 3+ denotes uniform, intense, complete membrane staining in more than 30% of tumor cells, as recommended by the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines.24,25

FISH Detection of HER2 Gene Amplification

FISH tests for HER2 gene amplification were performed on 4-μm tissue sections that were subjected to deparaffinization, air drying, and dehydration in 70%, 85%, and 100% ethanol, followed by proteolytic pretreatment using a commercial kit (paraffin pretreatment reagent kit, Vysis, Downers Grove, IL). After denaturation for 5 minutes at 74°C in 70% formamide–standard saline citrate solution, hybridization was performed overnight at 37°C in a humidified chamber using SpectrumOrange-labeled gene-specific probes (Vysis) for HER2 together with SpectrumGreen-labeled probes (Vysis) for centromere 17 (CEP17) as a reference. Slides were then washed and counterstained with 0.2 mmol/L 4′,6-diamidino-2-phenylindole (DAPI) in an antifade solution. Twenty nuclei were counted for each slide, enumerating the total HER2 and CEP17 signals per cell. The HER2 gene was considered amplified in a tumor if the HER2/CEP17 ratio was more than 2.2. A ratio of 1.8 to 2.2 was considered equivocal, and a ratio of less than 1.8 was considered nonamplified.

Results

The patient demographics and tumor characteristics are summarized in Table 1. The median age of the patients was 68 years (range, 33–85 years), and men constituted 65% (32/49) of the study population (male/female ratio, 1.9:1). Adenocarcinomas occurred in all locations of the small intestine, with most of the tumors found in the jejunum (23 [47%]) and the fewest in the duodenum (8 [16%]). The tumors manifested at varied stages, with most tumors resected at stages II to IV.

Immunohistochemical and FISH Detection of HER2

HER2 protein expression and gene amplification were assayed in our study population by immunohistochemical analysis and FISH Table 2. Only 2 cases (4%) exhibited a 1+ HER2 staining pattern Image 1A, while the remaining 47 specimens (96%) demonstrated no staining Image 1B. There were no specimens showing a 2+ or 3+ staining pattern by immunohistochemical analysis. Positive breast carcinoma control samples with 2+ and 3+ HER2 immunostaining patterns are shown in Image 1C and Image 1D.

Because the HER2 protein may not be consistently assayable in formalin-fixed tissues owing to possible nonstandardized fixation methods,26 we also analyzed HER2 gene amplification by FISH. No amplification of HER2 was detected in any of the SIAs in our study population (Table 2). A representative FISH analysis for HER2 is shown in Image 2.

Discussion

In an effort toward understanding tumor pathobiology and searching for potential therapeutic targets, the current work investigated whether HER2 protein overexpression and gene amplification occurred in SIA. In our study population, none of the 49 SIAs showed greater than 1+ immunoreactivity, which is considered nonamplification.24 These results correlated with the absence of gene amplification as determined by FISH. Thus, our work indicates that HER2 overexpression or amplification is an extremely rare event, if it occurs at all, in SIA.

To date, only 2 studies have investigated HER2 protein expression in SIA. Overman et al8 used monoclonal antibody clone neu Ab8 (Labvision, Fremont, CA) and reported HER2 immunohistochemical expression in only 1 (2%) of 54 small intestinal (nonampullar) adenocarcinomas. In this study, positive HER2 immunostaining was defined as 2+ or 3+ membranous staining in 10% or more of the tumor cells. Our results using monoclonal antibody clone 4B5, which has been validated for determining HER2 status in breast cancer,27 corroborate the study findings by Overman et al8 and indicate that HER2 overexpression or amplification is not a primary oncogenic abnormality in SIA. In contrast, Zhu et al22 examined HER2 protein expression in duodenal (nonampullar) adenocarcinoma and found that 9 (60%) of 15 cases exhibited positive expression by immunohistochemical analysis. While the disparity of our findings and those of Zhu et al22 is unclear, it may be related to differences in immunohistochemical reagents and interpretation criteria used in different studies. For example, monoclonal antibody clone 3B5 (Oncogene Science, Cambridge, MA) was used to detect HER2 protein expression in the study by Zhu et al,22 which may have a different specificity than clone 4B5, which was used in our study. In addition, HER2 immunostaining was graded for staining intensity (ie, ±, +, ++) and percentage of positively stained tumor cells in the study by Zhu et al.22 A tumor was considered to be positive for HER2 expression if more than 5% of the tumor cells exhibited “+” or greater staining intensity.22 In contrast, our study used the ASCO/CAP guideline recommendations to assess the immunostaining.24,25

View this table:
Table 1
View this table:
Table 2

One of the advantages of our study is that the observed lack of HER2 protein overexpression in SIA, as assayed by immunohistochemical analysis, is substantiated by the absence of HER2 gene amplification, as demonstrated by FISH analysis. In contrast, the previous studies on HER2 in SIA used only immunohistochemical analysis,8,22 and their findings were not further confirmed by FISH analysis for gene amplification. Because variable fixation and antigen retrieval methods could introduce staining variability in immunohistochemical analysis,26 the corroborating FISH data lend credence to our study.

Image 1

HER2 protein expression in small intestinal adenocarcinoma by immunohistochemical analysis. A, Only 2 (4%) of 49 tumors exhibited weak, incomplete membrane staining in <10% of the tumor cells (1+). B, The majority of the tumors (47 [96%]) demonstrated no immunoreactivity. No tumor demonstrated a 2+ or 3+ staining pattern as exemplified by positive breast carcinoma controls (C, 2+; D, 3+) (AD, ×400).

Another advantage of our study is that the immunohistochemical and FISH analyses were conducted on whole tissue block sections instead of tissue microarrays, as used in the work by Overman et al.8 The use of whole tissue blocks enables the analysis of much larger tissue areas with greater tumor representation. This issue is significant in light of potential intratumoral heterogeneity for HER2 expression.28 Given that SIA is a rare malignancy, our work considerably increases the number of SIA cases tested for HER2 status among the few existing studies.8,22

Despite morphologic similarity, SIA seems to differ from colorectal adenocarcinoma pathogenetically.29,30 However, the observed frequency of HER2 overexpression or amplification in SIA in our study is similar to that reported in colorectal adenocarcinoma. The available studies have shown that HER2 overexpression or amplification (defined as 3+ immunohistochemical staining or assessed by FISH) is also an infrequent event in colorectal adenocarcinoma, ranging from 0% to 3% in published series.19,20,31,32 This is in marked contrast with gastric and esophageal adenocarcinomas, in which HER2 overexpression or amplification seems to have a greater role in tumorigenesis. The reported frequencies of HER2 overexpression and amplification in gastric and esophageal adenocarcinomas are 8% to 27%15,16,33,34 and 12% to 15%,14,35 respectively. Our findings indicate that HER2 overexpression or amplification is not a primary event in the pathogenesis of SIA.

Image 2

HER2 gene amplification in small intestinal adenocarcinoma by fluorescence in situ hybridization. All tumors in our study population (n = 49) demonstrated no amplification of the HER2 gene. A representative sample is depicted. Green signal, CEP17 probe; orange signal, HER2 gene probe.

While HER2 overexpression or amplification has clinical importance in the treatment of breast and gastric carcinomas,10,36 our study indicates that HER2 does not have the same clinical significance for SIA. The results of this work are noteworthy because they suggest that routine testing for HER2 in SIA is likely unnecessary, and anti-HER2 therapy may not have a major therapeutic role in treating adenocarcinoma of the small intestine.

CME/SAM

Upon completion of this activity you will be able to:

  • discuss the role of HER2 in carcinogenesis.

  • define the role of anti-HER2 immunoglobulin therapy for HER2 protein-expressing tumors.

  • interpret HER2 gene amplification by fluorescence in situ hybridization and protein overexpression by immunohistochemistry.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 1008. Exam is located at www.ascp.org/ajcpcme.

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