HER2 protein expression and HER2 gene amplification are infrequent in small intestinal carcinomas
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- Gu, M.J., Hong, S., Jung, S.J. et al. Virchows Arch (2013) 462: 603. doi:10.1007/s00428-013-1425-1
Human epidermal growth factor receptor 2 (HER2/neu) gene amplification and HER2 protein overexpression have been associated with clinicopathological parameters and clinical outcome in many carcinomas. The aim of this study was to evaluate the frequency and prognostic impact of HER2 protein overexpression and gene amplification in small intestinal carcinoma (SIC). We performed immunohistochemistry (IHC) for HER2 protein and silver in situ hybridization for the HER2 gene in a total of 194 SICs. A total of 184 cases (94.8 %) were IHC 0 and 6 cases (3.1 %) were IHC 1+ with no gene amplification. HER2 protein overexpression (IHC 3+) with concordant gene amplification was detected in four cases (2.1 %), using the American Society of Clinical Oncology–College of American Pathologists guidelines for breast cancer. HER2 gene amplification was observed in an equivocal (IHC 2+) metastatic tumor in lymph node. No significant correlation was observed between HER2 status and clinicopathological parameters. Although HER2 protein overexpression and amplification were rare and did not correlate with clinicopathological parameters, further studies will be necessary to answer the question as to whether adjuvant therapy targeting the HER2 receptor might improve outcome in patients with a SIC with HER2 gene amplification and protein overexpression.
KeywordsSmall intestineCarcinomaHuman epidermal growth factor receptor 2ImmunohistochemistrySilver in situ hybridization
Overexpression of human epidermal growth factor receptor 2 (HER2) protein and amplification of the HER2 gene have been observed in many human carcinomas, including breast, gastric, colon, lung, and urothelial carcinomas [1–3]. HER2 gene amplification and/or overexpression in tumors is associated with poor prognosis and constitutes an important predictor of response to HER2-targeted therapy [3–5]. Small intestinal carcinoma is a very rare tumor with poor overall outcome. The 5-year overall survival rate for patients with small intestinal carcinoma (SIC) is 26 to 40 % . Research efforts have focused on the identification of better molecular targets for adjuvant chemotherapy following surgical resection; however, due to its rarity, the molecular carcinogenesis of SIC is still poorly understood and there has been little improvement in survival over the last decade. We sought to (1) determine the exact frequency of HER2 protein expression, (2) to clarify the relationship between HER2 protein expression and gene amplification, and (3) to determine correlation between HER2 status and clinicopathological parameters in a large series of SICs.
Materials and methods
A total of 194 SIC cases were collected from 20 institutions in Korea, and tissue microarrays (TMAs) were made. Primary carcinomas arising from the duodenum, jejunum, and ileum were included in this study. Primary ampulla of Vater carcinoma or extension of other gastrointestinal carcinoma was excluded. The histology from all tumor specimens was initially evaluated by gastrointestinal pathologists from each institution and finally reviewed by M.J. Gu. Reviews of medical records and pathology reports were used to identify patient’s age, sex, most recent follow-up, and survival status and presence or absence of disease related to occurrence of SIC. The study was approved by the Human Ethics Review Board of each institution that participated in the Korean Small Intestinal Cancer Study Group.
Four or five 1-mm cores were obtained from the most representative tumor area of each paraffin block and arrayed in a new recipient block: two or three cores from the SIC, one from a concurrently excised metastatic lymph node, and one from normal intestinal mucosa. In total, seven TMA blocks were made containing 194 SICs. The controls included normal liver, spleen, kidney, placenta, and breast carcinoma.
After deparaffinization and rehydration, 4-μm sections were immunostained for HER2 (clone 4B5, PATHWAY HER2/neu rabbit monoclonal antibody; Ventana Medical Systems, USA). The staining was performed on a Ventana BenchMark® platform automated slide stainer (Ventana Medical Systems, Tucson, AZ, USA) using the onboard heat-induced epitope retrieval method in high pH CC1 buffer (99 °C, 1 h). The staining was visualized using the iVIEWTM DAB Detection Kit (Automated BenchMark®, Ventana), which included a hydrogen peroxide substrate and a 3,3′-diaminobenzidine chromogen solution. The slides were subsequently counterstained with hematoxylin. For scoring of the HER2 immunoreactivity, the American Society of Clinical Oncology–College of American Pathologists (ASCO-CAP) guidelines for breast cancer and gastric cancer scoring criteria were assessed.
Silver in situ hybridization
Silver in situ hybridization (SISH) was performed using a Ventana BenchMark® platform automated slide stainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturers’ protocol. The INFORM HER2 DNA probe and INFROM Chromosome 17 probe were detected using the ultraView SISH Detection Kit. The slides were briefly counterstained with Ventana Hematoxylin II. The protocol for SISH staining has been described previously. Following the manufacturer’s guidelines, scoring of SISH was carried out assuming that a single signal is counted as 1 gene copy, a small cluster as 8 gene copies, and a large cluster as 16 gene copies. In accordance with the ASCO-CAP guidelines, we defined amplification as a HER2/CEP17 ratio of more than 2.2. A ratio of 1.8 to 2.2 was considered equivocal, and a ratio of less than 1.8 was defined as nonamplified.
Statistical comparisons were performed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). The chi-square test and Fisher’s exact test were performed for examination of associations between clinicopathological factors and HER2 expression. Overall patients’ survival was defined as the time from surgical resection of SICs to death of patients or last follow-up of the patients. The Kaplan–Meier method was used for calculation of survival rate. A comparison of survival rate with regard to clinicopathological parameters was performed using the log-rank test. A p value of less than 0.05 was considered statistically significant.
Characteristics of patients
A total of 121 male and 73 female patients with a median age of 59 years (range 23–86 years) were included in this study. Tumors were located in the duodenum in 105 cases (54.1 %), the jejunum in 59 cases (30.4 %), and the ileum in 30 cases (15.5 %). Regarding growth features, there were 35 cases of polypoid (18.8 %), 12 cases of nodular (6.5 %), and 149 cases of infiltrative (74.7 %). Tumor size ranged from 1 to 16 cm (mean 4.4 cm).
Regarding histological subtypes, there were 176 adenocarcinomas (90.7 %), 9 mucinous carcinomas (4.6 %), 4 signet ring adenocarcinomas (2.1 %), and 5 undifferentiated carcinomas (2.6 %). Vascular and lymphatic invasion was observed in 51 (26.3 %) and 95 (49.0 %) cases, respectively. Pancreatic invasion was found in 68 cases (35.1 %). Eighty-nine (50.9 %) cases showed metastasis to regional lymph nodes. Retroperitoneal tumor seeding was observed in 14 cases. Regarding pTNM staging, pTis was observed in 4 cases, pT1 in 7 cases, pT2 in 9 cases, pT3 in 63 cases, and pT4 in 111 cases. A pN0 was observed in 86 cases, pN1 in 59 cases, and pN2 in 29 cases. No patient showed distant metastasis. There were 2 cases in stage 0, 13 cases in stage I, 71 cases in stage II, and 89 cases in stage III.
Results of immunohistochemical and silver in situ hybridization
Comparison of number of cases for HER2 protein expression between breast and gastric cancer scoring criteria
Breast cancer criteria
Gastric cancer criteria
184 (94.8 %)
184 (94.8 %)
6 (3.1 %)
4 (2.1 %)
0 (0 %)
2 (1.0 %)
4 (2.1 %)
4 (2.1 %)
Clinicopathological characteristics of HER2-positive cases
Using gastric cancer scoring criteria, the results of HER2 protein expression for IHC 0 and IHC 3+ were identical to those obtained using breast cancer scoring criteria. Of the IHC 1+ cases by breast cancer scoring criteria, two cases were classified as IHC 2+ (1.0 %) but without accompanying HER2 gene amplification (Fig. 1e, f). For case 184, HER2 scoring results were identical to those obtained with the breast cancer scoring criteria.
HER2 IHC was repeated to assess tumor heterogeneity on full tumor blocks (all IHC 1+ to IHC 3+ cases and 15 cases of randomly selected IHC 0 cases). The immunohistochemical results for IHC 0 to IHC 3+ were identical to those obtained on the corresponding TMA cores: all IHC 0 cases were completely negative. HER2 protein was expressed in 70 % or more tumor cells in IHC 1+ cases. The IHC 2+ metastatic tumor cells and IHC 3+ cases exhibited HER2 expression more than 90 % of tumor cells. HER2 overexpression or gene amplification was not associated with any specific clinicopathological parameters.
HER2, an important member of the HER family, is encoded by a gene located on chromosome 17q21 and acts as a proto-oncogene in modulating proliferation, invasion, and apoptosis . HER2 protein overexpression and/or gene amplification has been observed in many carcinomas, including breast, colon, urothelial, lung, gastric, and gastroesophageal junction adenocarcinoma . HER2 protein overexpression has been reported in up to 8.2 to 53.4 % of gastric carcinomas and 0 to 83 % of colorectal carcinomas [1, 7–9]. This variation in the reported frequency may be due to sample size, different antibodies, differences in technical methods, and differences in interpretation methods.
To the best of our knowledge, three studies of HER2 protein expression in SIC have been reported with discrepancies in the observed frequency of HER2 protein overexpression by immunohistochemistry. Overman et al.  reported HER2 protein expression in only 1 of 54 cases of small intestinal adenocarcinoma. Zhu et al.  observed HER2 protein expression in 9 of 15 cases. Chan et al.  reported that 47 of 49 cases (95.9 %) completely lacked HER2 protein expression with the remaining two IHC 1+ cases without amplification by fluorescence in situ hybridization (FISH). These discrepancies were probably due to the different antibodies used, the different processing techniques, or the use of different interpretation methods. Our study was performed using the same antibody, the same immunohistochemical method using the Ventana BenchMark® platform automated slide stainer, and the same interpretation criteria (ASCO-CAP guideline recommendations) as those used in the Chan study . In our study, 4 cases showed HER2 protein overexpression (IHC 3+, 2.1 %), 1 case showed equivocal staining in metastatic tumor (IHC 2+), 6 cases (3.1 %) were IHC 1+, and the remaining 184 cases (94.8 %) showed lack of expression. By SISH, all tumors with HER2 protein overexpression (IHC 3+) exhibited HER2 gene amplification and IHC 2+ metastatic cancer cells exhibited HER2 gene amplification in spite of the absence of amplification in the primary tumor. No HER2 gene amplification was observed in any tumor with IHC 1+ or IHC 0. We regard our results reliable as our laboratory subscribes to an IHC quality control program. In the Chan study, IHC and FISH were performed on whole tissue block sections but neither HER2 protein overexpression nor gene amplification was observed. Sample size might be responsible for the lower number of positive cases in the Chan study. We did not find intratumoral heterogeneity for HER2 expression in SIC; tumor cells were evenly stained for HER2 protein in at least two or three of the cores and at least 70 % or more of tumor cells in a full tissue block. Incomplete membrane and basolateral staining was observed, but focal staining as observed in gastric cancer was uncommon in SIC. We also found HER2 gene amplification more often IHC 3+ using gastric cancer scoring criteria, with cases that would have scored IHC 2+ using breast cancer scoring criteria.
Trastuzumab (HerceptinTM), which has been used extensively in the treatment of breast carcinoma, has been approved as the standard treatment for patients with advanced and with recurrent locally inoperable and metastatic gastric cancers and has resulted in improved survival [12, 13]. Due to the small number of tumors with HER2 overexpression/amplification, we were unable to correlate HER2 status with overall outcome or other clinicopathological parameters. Chan et al.  suggested that routine testing for HER2 in small intestinal adenocarcinoma is likely unnecessary and that anti-HER2 therapy may not have a major therapeutic role in the treatment of SIC. Although no survival difference for HER2 status was observed in our study, complete concordance was observed between HER2 protein overexpression and gene amplification in primary tumors and their metastases. A study on a larger number of cases of SIC will be necessary to determine whether adjuvant therapy targeting the HER2 receptor might play a role in the improvement of patient outcome with HER2 gene amplification and to answer the question as to whether testing for HER2 should be routinely included in the diagnostic work-up for patients with SIC.
In conclusion, HER2 protein overexpression/gene amplification was observed in 2.1 % of SIC. This finding indicates that HER2 overexpression or amplification does not play a major role in carcinogenesis in SIC. However, further studies on patients with HER2 overexpression/amplification will be necessary in order to determine whether adjuvant therapy targeting HER2 receptor may improve the outcome of patients with HER2 overexpression/amplification.
Conflict of interest
The authors declare that they have no competing interests.