RAS/RAF mutations and microsatellite instability status in primary colorectal cancers according to HER2 amplification

Abstract

HER2 amplification-associated molecular alterations and clinicopathologic features in colorectal cancers (CRCs) have not been well established. In this study, we assessed the prevalence of HER2 amplification and microsatellite instability (MSI) status of 992 patients with primary CRC. In addition, molecular alterations of HER2 amplified and unamplified CRCs were examined and compared by next-generation sequencing. HER2 amplifications were found in 41 (4.1%) of 992 primary CRCs. HER2 amplification was identified in 1.0% of the right colonic tumors, 5.1% of the left colonic tumors, and 4.8% of the rectal tumors. Approximately 95% of HER2 amplification was observed in the left colon and rectum. Seven (87.5%) of eight metastatic tumors showed HER2 amplification. Most clinicopathologic features were unrelated to HER2 amplification except tumor size and MSI status. All 41 HER2 amplified CRCs were microsatellite stable. In a molecular analysis of frequently identified somatic mutations in CRCs, HER2 amplified CRCs showed a lower rate of KRAS mutations (24.4%) but a higher rate of TP53 mutations (83%) than unamplified CRCs. No BRAF and NRAS mutations were identified in HER2 amplified CRCs. Our study suggests that HER2 amplified CRCs are mutually exclusive of MSI and harbor less frequent KRAS/NRAS/BRAF mutations but frequent T53 mutations.

Figure 1

A flow chart showing each step of detecting HER2 expressed and amplified tumors in 992 invasive CRCs using immunohistochemistry and DISH. Among 992 unselected CRCs, there were 149 (15%) tumors with 1 + labeling, 50 (5%) tumors with 2 + labeling, and 33 (3.3%) tumors with 3 + labeling by immunohistochemistry. HER2 DISH was performed on tumors with all 1 + , 2 + , and 3 + labeling. Finally, 41(4.1%) tumors were found to be HER2 amplified.

Figure 2

Representative micrographs of HER2 immunohistochemistry and DISH on the primary and metastatic CRCs are shown. (A) The primary CRC reveals a diffuse and strong staining pattern (3 +) of HER2 immunostaining at low magnification (4×). (B) The corresponding primary tumor shows diffusely located individuals and clusters of black dots within neoplastic cells on DISH. (C) At high magnification (40×), neoplastic cells exhibit intense membranous staining (3 +) of HER2 (left) and highly clustered black dots (right) representing amplified HER2 gene signals. (D) Seven of eight metastatic CRCs harbor HER2 amplification on DISH, representing 87.5% of the concordance rate between the primary and metastatic CRCs. The metastatic CRC in the liver displays a diffuse and strong staining pattern (3 +) of HER2 at low magnification (4×). (E) The corresponding metastatic tumor shows diffuse and strong black dots on DISH. (F) At high magnification (20×), neoplastic cells within the liver parenchyma reveal a strong membranous staining pattern (3 +) of HER2 (left) and highly clustered black dots (right) representing amplified HER2 gene signals.

Figure 3

This illustration shows the anastomotic site where HER2 amplified tumors occur in the colorectum. The most common primary tumor site was the left colon (48.8%), followed by the rectum (46.3%) and the right colon (4.9%). In HER2 amplified left colonic tumors, seventeen (85%) were observed in the sigmoid colon. Moreover, in HER2 amplified rectal tumors, eleven (57.9%) occurred in the upper rectum, a median of 11 cm (range, 7–15 cm) located above the anal verge. All two (4.9%) HER2 amplified tumors were found in the ascending colon.

Figure 4

In order to compare molecular differences between HER2 amplified and unamplified CRCs, previous molecular data for 314 HER2 unamplified CRCs were available. As there are known differences in the frequencies and distributions of mutations in key oncogenes and tumor suppressors between right and left-sided CRCs, a control group of HER2 unamplified CRCs was selected after matching tumor sites with 41 HER2 amplified CRCs. The most common HER2 amplified tumor site was the left colon (48.8%), followed by the rectum (46.3%) and the right colon (4.9%). A flow chart outlines the selection process of 200 CRCs as a control group after matching tumor sites. The 200 CRCs selected consisted of 5% right-sided colon cancer, 50% left-sided colon cancer, and 45% rectal cancers, with varying frequencies of KRAS/NRAS/BRAF mutations.

Figure 5

(A) Somatic mutational profile and copy number variations of HER2 in HER2 amplified CRCs are illustrated. The most frequently identified somatic mutations were found in the TP53 gene, followed by the APC gene. Predominantly, missense mutations were found in KRAS and TP53 mutations. In contrast, mostly truncating and inframe mutations were observed in APC mutations. Copy numbers of HER2 ranged from 5 to 43, with a mean copy number of 13. (B) Comparative analysis of frequently identified somatic mutations between HER2 amplified and unamplified CRCs. No BRAF and NRAS mutations were identified in HER2 amplified CRCs. HER2 amplified CRCs harbored KRAS mutations less frequently than unamplified CRCs (24% vs 59%; p < 0.0001). In contrast, TP53 mutations were more frequently identified in HER2 amplified CRC than unamplified ones (83% vs 62%; p = 0.0014). There were no differences in frequencies of other gene mutations. All HER2 amplified CRCs were MSS phenotypes.

Figure 6

Kaplan–Meier cumulative survival curves. (A) Survival according to T category (p < 0.001); (B) Survival according to pathologic TNM stage (p < 0.0001); (C) Survival according to HER2 amplified and unamplified tumors (p = 0.827); (D) Survival according to MSI-H and MSS/MSI-L tumors (p = 0.065).