Human epidermal growth factor receptor 2 (HER2) is an oncogenic driver, and a well-established therapeutic target in breast and gastric cancers. While the role of HER2 as a prognostic biomarker in colorectal adenocarcinomas (CRCs) remains uncertain, its relevance as a therapeutic target has been established. We undertook the present study to evaluate the frequency of HER2 expression in CRC and to correlate it with various clinicopathological variables.

To correlate HER2 protein expression and HER2 gene amplification with clinicopathological features and survival in surgically resected CRC.

About 1195 consecutive surgically resected CRCs were analyzed by immunohistochemical staining (IHC) to assess HER2 protein expression, and 141 selected tumors were further evaluated by fluorescence in situ hybridization (FISH) to assess HER2 gene amplification. Follow-up information was available for 1058 patients, and using this information we investigated the prevalence of HER2 protein overexpression and gene amplification in a large series of surgically resected CRCs, and evaluated the relationship between overexpression and clinicopathological parameters and prognosis.

HER2 IHC scores of 3+, 2+, 1+, and 0 were seen in 31 (2.6%), 105 (8.8%), 475 (39.7%), and 584 (48.9%) tumors, respectively. HER2 gene amplification was seen in 24/29 tumors with an IHC score of 3+ (82.8%; unreadable in 2/31), 12/102 tumors with an IHC score of 2+ (11.8%; unreadable in 2/104), and 0 tumors with IHC score of 1+ (0/10). HER2 gene amplification was seen in 36/1191 tumors (3.0%; unreadable in 4/1195). Among the tumors with HER2 IHC scores of 3+ and 2+, the mean percentage of tumor cells with positive IHC staining was 90% (median 100%, range 40%-100%) and 67% (median 75%, range 5%-95%), respectively (P < 0.05). Among tumors with IHC scores of 2+, those with HER2 gene amplification had a higher number of tumors cells with positive IHC staining (n = 12, mean 93%, median 95%, range 90%-95%) than those without (n = 90, mean 70%, median 50%, range 5%-95%) (P < 0.05). HER2 gene status was significantly associated with distant tumor metastasis and stage (P = 0.028 and 0.025). HER2 protein overexpression as measured by IHC or HER2 gene amplification as measured by FISH was not associated with overall survival (OS) or disease-specific survival for the overall group of 1058 patients. However, further stratification revealed that among patients with tubular adenocarcinomas who were 65 years old or younger (n = 601), those exhibiting HER2 gene amplification had a shorter OS than those without (mean: 47.9 mo vs 65.1 mo, P = 0.04). Among those patients with moderately to poorly differentiated tubular adenocarcinomas, those with positive HER2 tumor IHC scores (2+, 3+) had a shorter mean OS than those with negative HER2 IHC scores (0, 1+) (47.2 mo vs 64.8 mo, P = 0.033). Moreover, among patients with T2 to T4 stage tumors, those with positive HER2 IHC scores also had a shorter mean OS than those with negative HER2 IHC scores (47.1 mo vs 64.8 mo, P = 0.031).

HER2 protein levels are correlated with clinical outcomes, and positive HER2 expression as measured by IHC confers a worse prognosis in those patients 65 years old or younger with tubular adenocarcinomas.

Colorectal adenocarcinoma (CRC) is one of the most common malignancies and remains one of the leading causes of cancer-related death in the world[1]. The vast majority of colorectal cancers are adenocarcinomas. Until recently, the role of chemotherapy in treating CRC has been fairly limited, and as such there is a need to develop more effective therapeutic regimes for CRC.

Human epidermal growth factor receptor 2 (HER2) represents a promising therapeutic target. The HER2 gene is a proto-oncogene located on chromosome 17q21 encoding HER2 (ErbB-2), which is involved in cell proliferation and differentiation[2]. The HER2 protein is a transmembrane receptor tyrosine kinase and a member of the family of the epidermal growth factor receptors[3]. Oncogenic activation of HER2 commonly occurs through gene amplification, which results in protein overexpression on the cell membrane that is involved in signal transduction to regulate cell growth[4]. HER2 overexpression/amplification is linked to trastuzumab response in breast/gastric cancers. For breast cancer in particular, HER2 serves as both a prognostic factor and also a therapeutic target, with targeted therapy against HER2 using the monoclonal antibody Trastuzumab (Herceptin) having become the standard of care for patients whose breast carcinomas exhibit HER2 gene amplification[5-7]. A recent randomized phase III trial (ToGA) also revealed that the addition of trastuzumab to chemotherapy improved response rates, median progression-free survival, and overall survival in patients with advanced gastric or gastroesophageal junction cancer with HER2 overexpression[8].

Compared to breast and gastric cancers, the data regarding HER2 protein overexpression and gene amplification in CRCs are limited[9-18]. The numbers of cases in these previous studies were relatively small, and their analyses were primarily reliant upon tissue microarrays[9-18]. In addition, the reported rates of HER2 protein overexpression ranged widely from 3% to 47.4% across studies, indicating that the clinical significance of HER2 in CRC has not yet been fully clarified. In this study, we assessed HER2 overexpression in 1195 CRC patients via immunohistochemical staining (IHC) in 1195 patients and analyzed HER2 gene status via fluorescence in situ hybridization (FISH) in a subset of these patients (all tumors with IHC scores of 2+ and 3+, and some a score of 1+). We then correlated HER2 protein overexpression and HER2 gene status with clinicopathologic features and prognosis to gain insight into the importance of this marker in the context of CRC.

In this study we assessed HER2 protein overexpression and HER2 gene amplification in a large series of 1195 colorectal adenocarcinomas. 11.4% (136/1195) of these colorectal adenocarcinomas were positive for HER2 staining by IHC (scores of 2+ and 3+). HER2 gene amplification was identified in 11.8% (12/102) of tumors with HER2 IHC scores of 2+ and in 82.8% (24/29) of tumors with HER2 IHC scores of 3+. No tumors with HER2 IHC scores of 1+ showed evidence of HER2 gene amplification by FISH. Our findings indicate that HER2 protein overexpression occurs in a small percentage of colorectal adenocarcinomas, similarly to what has been reported in previous studies. There are only 9 prior studies regarding HER2 status in colorectal adenocarcinomas in the English literature[9-17]. Three of these nine studies used tissue microarrays (104, 518, and 1851 patients, respectively) and the remaining 6 used full tissue blocks (ranging from 138 to 317 patients)[9-17]. Our study is the largest to date to employ full tumor tissue blocks for analysis (1195 patients). The reported rates of HER2 protein overexpression in the literature ranged from 2.7% to 15.5%, with the exception of one study in which the authors reported 47.4% positive HER2 staining in 137 colorectal adenocarcinomas[9-17]. One explanation for this dramatic range in HER2 overexpression positivity across studies may be that antibodies used for staining varied among research groups. The HER2 antibody used in the study with highest positive HER2 staining (47.4%) was a polyclonal rabbit antibody. Even though those authors demonstrated a high HER2 overexpresssion rate, the percentage of cases exhibiting HER2 gene amplification was only 8% in those with 3+ HER2 staining, and 0% in 38 cases with 2+ HER2 staining, raising questions regarding the validity of the IHC scoring. The positive rates of HER2 protein staining in studies using the DAKO HER2 antibody were 2.7% to 15.5%, while it was 8% in the study using Ventana pathway antibody by Kavanagh et al[14] and was 11.4% in our study using this same Ventana pathway.

Our study also indicates that there is a high concordance between HER2 IHC 3+ staining and HER2 gene amplification in colorectal adenocarcinomas. In our study 83% of tumors (24/29) with 3+ HER2 staining exhibited HER2 gene amplification. There were 6 other studies in the literature on this topic, however the number of cases analyzed in 5 of these 6 studies was small (less than 10). Marx et al[15] found that 100% of tumors (27/27) with 3+ HER2 staining (as measured with the DAKO antibody) harbored HER2 gene amplifications. In the study by Park et al[10], only 2 of 27 tumors with 3+ HER2 staining showed evidence of HER2 gene amplification, however, as described above, the IHC staining in that study was performed using a polyclonal antibody of uncertain reliability. In 3 other smaller studies (2-6 cases with 3+ HER2 staining), this concordance rate with gene amplification was high (100%). Variations in the antibodies used likely led to differential staining and scoring, contributing to the disagreement in HER2 gene amplification in tumors with 3+ HER2 scores. Nevertheless, these studies, including ours, have shown that HER2 IHC 3+ staining is highly predictive of HER2 gene amplification in colorectal adenocarcinomas. In contrast, the percentage of tumors with HER2 IHC 2+ staining showing evidence of HER2 gene amplification was highly variable. In our study, such amplification was only evident in 20% of tumors, similar to what has been reported by Kavanagh et al[14] (2/9 or 22%, using the Ventana Pathway antibody) and Nathanson et al[9] (1/3 or 33%, using the DAKO antibody). In contrast, Marx et al[15] observed HER2 gene amplification in 75% of tumors with HER2 scores of 2+ (using the DAKO antibody). In the study by Park et al[10], using the Zymed antibody, no gene amplification events were observed in such tumors. In the study by Ooi et al[11], 2/2 tumors with 2+ HER2 staining showed evidence of HER2 gene amplification (using an antibody from Nicheri, Japan). Similarly, variations in the antibodies used likely led to differential staining and scoring, contributing to the observed high levels of reported variation in HER2 gene amplification in tumors with 2+ HER2 scores.

The high concordance between HER2 IHC 3+ staining and HER2 gene amplification indicates that gene amplification is the major factor accounting for HER2 protein overexpression in colorectal adenocarcinomas with HER2 IHC scores of 3+. However, HER2 gene amplification was seen in only 12/102 (11.8%) tumors with HER2 IHC scores of 2+, and among these tumors, those with evidence of HER2 gene amplification had a higher number of tumor cells with positive IHC staining (n = 12, mean 93%) than those without (n = 90, mean 70%) (P < 0.05). Our study thus confirmed reported results regarding colorectal cancer, and these results were similar to those observed in breast cancer where IHC 3+ HER2 overexpression is highly correlated with HER2 gene amplification, whereas 2+ staining is equivocal for gene amplification[21-24].

In our present study, HER2 gene amplification was higher in patients with a more advanced disease stage or distant metastases. This suggests that HER2 may play some role in tumor progression and would thus be a valuable prognostic factor for certain CRC patients. In our study, the significance of HER2 gene amplification was best observed in the subpopulation of patients who were 65 years or younger with tubular adenocarcinomas. However, we did not observe a significant difference in prognosis for all colorectal carcinoma patients, regardless of HER2 amplification or HER2 expression status.

Although we found a statistically significant association between HER2 gene amplification and tumor depth of invasion, advanced stage, and distant metastasis, several other studies have failed to show such associations[15,19,25,26]. Those studies, however, reported a correlation between HER2 gene amplification and high grade histology, higher tumor stage, and positive nodal status[27,28]. The role of HER2 as a prognostic factor in CRCs is still controversial, as some of the earlier studies have failed to identify any specific association with prognosis[28,29] while others reported a correlation between HER2 gene amplification and poorer survival[30]. Similar observations have been made for gastric cancer, although a larger number of studies have supported the association between HER2 overexpression and poorer survival[31]. Possible explanations for such discrepancies include: Antibody resources, detection methods, scoring systems, and patient populations (different ethnic groups with different genetic backgrounds). It has been clearly demonstrated that HER2 gene amplification differs significantly between right/left-sided and rectal carcinomas. We were not able to produce similar results, potentially due to differences in groups analyzed, HER2 testing methods, and tumor biological characteristics[32,33].

In summary, we investigated HER2 protein status and HER2 gene status in a large series of colorectal adenocarcinomas. Our results show that HER2 protein overexpression is evident in 11.4% of colorectal adenocarcinomas. HER2 IHC scores of 3+ are highly correlated with HER2 gene amplification, while this correlation is weaker in tumors with IHC 2+ staining. We also observed an association between HER2 gene amplification and prognosis in patients with tubular adenocarcinomas who were 65 years or younger. Our study indicates that a certain population of patients with colorectal adenocarcinomas may benefit with HER2-targeted therapy.