Comparative analysis of the EGFR, HER2, c-MYC, and MET variations in colorectal cancer determined by three different measures: gene copy number gain, amplification status and the 2013 ASCO/CAP guideline criterion for HER2 testing of breast cancer

Gene copy number (GCN) variation, i.e., copy number difference in a genomic segment, occurs commonly and is one of the main mechanisms in shaping the human genome [1]. GCN variation has been found to play an important role in the stimulation of cell proliferation and decrease of apoptosis as well as in promoting development and progression of various cancers [1, 2]. Recent advancements in molecular genetics have made it possible to detect various GCN changes in human cancers [1, 2]. To date, many studies have reported the prognostic and predictive value of GCN variation in breast, lung, gastric and colorectal cancer (CRC) [3, 4]. Investigation of the molecular profiles of malignant cells may yield valuable prognostic information about each type of cancer. In addition, the sub-classification based on genetic alterations is also essential to select the patients who may benefit from drugs specifically targeted to a particular molecular alteration. Despite these clinical implications, the appropriate and standardized criterion to define positivity of each GCN variation remains uncertain, particularly in CRC. Therefore, further studies are required to develop the adequate criterion for the identification of genetic alterations and determination of reliable biomarkers.

CRC is known to be a heterogeneous disease with diverse molecular alterations including genetic changes in expression of c-MYC and MET, members of the human epidermal growth factor receptor (EGFR) family [5]. Human EGFR is a receptor kinase, which is a part of a complex signaling cascade that regulates cell proliferation and differentiation. EGFR is a representative member of the EGFR family. Dysregulation of EGFR expression is considered to be an important genetic alteration in the targeted treatment of advanced CRC. Several studies have assessed the prognostic value for overall survival of patients and predictive effect of the anti-EGFR treatment in CRC [6, 7]. However, the results of these studies remain unclear. This may be explained by the relatively small number of patients involved and discrepant cut-off values of GCN variation used in these studies.

The prognostic significance of changes in HER2 (human epidermal growth receptor 2), c-MYC and MET is also uncertain. Previous studies of Kavanagh et al. [8] and Marx et al. [9] did not consider HER2 gene amplification as a significant prognostic factor in CRC. In aforementioned studies, they used different cut-off value for determining the HER2 genetic status; the HER2 signal >4.0 [8] and the HER2/CEP17 ratio ≥2.0 [9]. However, there had been some controversy about the cut-off value and the prognostic significance of HER2 gene alterations in CRC [10, 11]. In CRC, a number of studies have addressed the prognostic and predictive value of c-MYC and MET gene alterations applying different criteria, such as the target gene/corresponding CEP signal ratio of ≥2–3 for amplification and the target GCN gain of ≥4 copies [12‐15]. Only few studies have been examined the GCN alteration of these genes with a criterion of the ASCO/CAP 2013 guideline for HER2 testing of breast cancer.

In this study, we analysed GCN variation of the EGFR, HER2, c-MYC, and MET genes in 334 colorectal cancer tissue samples using silver-enhanced in situ hybridization (SISH). In particular, we defined GCN variation according to several criteria and compared them with clinicopathological data and patient outcomes.

Much effort has been focused on improving CRC treatment strategies, and a number of genetic alterations associated with CRC development have been evaluated. Aberrant activation of EGFR, HER2, c-MYC, or MET is considered to be a promising target for specific molecular treatments in various cancers, including CRC [7, 12, 15, 18]. Although many studies suggested a prognostic and predictive impact of such genetic alterations, a standardized criterion for them in CRC has not been established. In this study, we evaluated the genetic status of EGFR, HER2, c-MYC and MET in 334 CRC samples and its association with patients’ prognosis in 334 CRC samples using dual-colour SISH analysis. We evaluated the applicability of the ASCO/CAP HER2 guideline criterion for breast cancer to assess the EGFR, HER2, c-MYC, and MET gene status. We also assessed GCN gain and amplification of these genes in the CRC samples. We found that comparative analysis of the genetic status by using several criteria supported the conclusion that GCN gain and GCN result according to the ASCO/CAP criteria were independent prognostic factors in consecutive CRC patients.

Genetic alterations of HER2 have been studied particularly actively in breast and gastric cancers [17, 19]. Currently, evaluation of the HER2 status in breast cancer is included as part of a routine test according to the ASCO/CAP criteria. However, because many previous studies evaluated the genomic status of these genes in CRC by disparate criteria, the prognostic and predictive impact of the HER2 status has been controversial. Our study is one of the few studies, in which genetic changes in CRC were assessed according to the 2013 ASCO/CAP criterion. When we applied the ASCO/CAP HER2 test criterion to CRC cases, EGFR and c-MYC positivity showed significant association with poor prognosis. In the cumulative analysis of EGFR, HER2, c-MYC, and MET, CRC cases with positive results for any of these genes was a significant predictor of poor overall survival. Although further studies that would evaluate this concordance with fluorescence in situ hybridization and immunohistochemistry methods are needed, our findings suggest that the 2013 ASCO/CAP HER2 testing criterion for breast cancer is applicable for determination of genetic changes in CRC.

We defined GCN gain as the signal equal or higher than the cut-off value of 4.0 for each gene signal per cell. In our results, GCN gain in the EGFR, c-MYC, and MET genes, determined according to this criterion, was significantly associated with patient survival. In the multivariate analysis, application of this cut-off value revealed that GCN gain in any of the four genes analysed was a significant poor prognosis factor in CRC, in line with the results obtained using the ASCO/CAP criterion. In addition, we defined the equivocal result according to the ASCO/CAP guideline, namely when 6> gene copy number ≥4.0 and found that for such CRC cases, the prognosis was worse than for those with negative or positive results. Thus, we demonstrated in our study that the cut-off value of ≥4.0 as well as the ASCO/CAP criterion could be applied as a reliable cut-off point to determine GCN gain as a molecular prognostic marker in CRC.

As has been published previously [6, 13, 14], we observed that the rates of c-MYC and MET amplification were relatively low. Interestingly, approximately 20–30% of genetic alterations in c-MYC and MET were detected using GCN gain and the ASCO/CAP criteria. The discrepancy was also found in the rates of CRC cases with multiple genetic changes. There were only 5 cases of CRC with amplifications of multiple genes. However, there were 35 cases of CRC accompanied by GCN gains and ASCO/CAP positivity in multiple genes. These genetic changes were considered to have clinical significance because they were associated with poor prognosis. Specific drugs targeting EGFR represent one of the main treatment strategies in advanced CRC patients [20, 21], and some studies reported the association between the EGFR GCN and resistance to anti-EGFR treatment [7, 22, 23]. Currently, the association between other genetic changes and the resistance to EGFR inhibitors is a subject of intense scientific scrutiny. Furthermore, attempts to exploit these genetic changes for targeted therapy are currently underway in several preclinical and clinical trials [18, 24, 25]. Based on this knowledge, the ability to detect the presence of these genetic changes in CRC has a great clinical significance. Our findings, therefore, suggest that GCN gain and the ASCO/CAP criterion are more useful to detect genetic alterations accurately.

In present study, we analysed somatic gene copy alteration of TCGA dataset and compare the results from that of Korean CRC cohort. The rate of high-level amplification of each gene using array-based method was lower than amplification rate obtained by SISH method. The proportion of MET gene amplification was highest and that of EGFR gene amplification was lowest among four genes in both cohorts. Correlation between copy number gain and MSI-H status was also observed in both cohorts. However the direct comparison of these results derived by different detection methods would be unsuitable.

Several platforms for detection of GCN alteration are currently available, but it is still unclear whether array-based platform is an ideal detection method. A lack of reproducibility and concordance for array-based method has been shown in previously published study by Pinto et al. [26]. Normal tissue contamination or unknown ploidies of tumor cells is another challenges for array-based platform. In HER2 testing for breast or gastric cancer, in situ hybridization method is the gold standard to assess patient’s further treatment [27]. In this study, the prognostic significance of GCN gain was only confirmed in Korean CRC when the genetic status analysed by GCN gain and ASCO/CAP guideline criterion. From the clinical viewpoint, detection of GCN alteration by classic in situ hybridization method could be more useful.

Despite several genes being proposed as prognostic markers based on the GCN gain or the ASCO/CAP criterion data, our results have some limitations. Some genetic alterations, including changes in EGFR and MET, were observed at relatively low frequencies, and these alterations were detected in less than 5% of cancers analysed in our study. Based on our results, evaluation of these genetic changes in large cohorts of samples using GCN gain or the ASCO/CAP guideline criterion is needed to determine their prognostic impact. Additionally, EGFR, HER2, c-MYC, and MET have been identified to be potential biomarkers that predict the efficacy of the pharmacological treatments targeted against protein products of these genes in various cancers. The determination and exact evaluation of cases with positive copy number changes in these genes may help in developing more effective treatment strategies by selecting patients who may benefit from the targeted treatments directed against the corresponding genetic changes. Further studies are necessary to explore if the GCN gain defined by the cut-off point of 4.0 and by the ASCO/CAP criterion has a predictive impact on the efficacy of treatments targeted to specific genetic changes in CRC. We analysed the genetic status using TMA, which may not be representative of the whole CRC tumour due to the use of only small cores taken from each specimen. Some previous studies also reported that cancers often had genomic heterogeneity [7, 9], so a small tissue core of TMA may not fully represent each cancer at the genetic level. Thus, we need efficient validation of the heterogeneity of these genetic alterations in CRC.

We evaluated EGFR, HER2, c-MYC, and MET gene status by determining the degree of amplification, GCN gain and the 2013 ASCO/CAP HER2 testing guideline criterion for breast cancer. In the cumulative analysis of these genes, we revealed that genetic variation in any of four genes according to the GCN gain defined by the cut-off point of 4.0 or to the ASCO/CAP criterion was an independent prognostic factor in CRC.