MYC gene amplification is often acquired in lethal distant breast cancer metastases of unamplified primary tumors

Abstract

In breast cancer, amplification of MYC is consistently observed in aggressive forms of disease and correlates with poor prognosis and distant metastases. However, to date, a systematic analysis of MYC amplification in metastatic breast cancers has not been reported. Specifically, whether the MYC amplification status may change in metastases in comparison to the corresponding primary breast tumor, and potential variability among different metastases within the same patient have also not been assessed. We generated single patient tissue microarrays consisting of both primary breast carcinomas and multiple matched systemic metastases from 15 patients through our previously described rapid autopsy program. In total, the 15 tissue microarrays contained 145 primary tumor spots and 778 spots derived from 180 different metastases. In addition, two separate tissue microarrays were constructed composed of 10 matched primary breast cancers and corresponding solitary metastases sampled not at autopsy but rather in routine surgical resections. These two tissue microarrays totaled 50 primary tumor spots and 86 metastatic tumor spots. For each case, hormone receptor status, HER2/neu, EGFR and CK5/6 expression were assessed, and the cases were characterized as luminal, basal-like or HER2 based on published criteria. Both fluorescence in situ hybridization and immunohistochemistry for MYC was performed on all cases. Of the 25 cases, 24 were evaluable. While 4 of 24 primary tumors (16%) demonstrated MYC amplification, an additional 6 (25% of total evaluable cases) acquired MYC amplification in their systemic metastases. Of note, there was remarkably little heterogeneity in MYC copy number among different metastases from the same patient. MYC immunoreactivity was increased in metastases relative to matched primaries in the surgical cohort, although there was no perfect correlation with MYC amplification. In conclusion, amplification of MYC is a frequent event in breast cancer, but occurs more frequently as a diffuse, acquired event in metastatic disease than in the corresponding primary. These observations underscore the importance of MYC in breast cancer progression/metastasis, as well as its relevance as a potential therapeutic target in otherwise incurable metastatic disease.

Figure 1

Representative MYC immunohistochemical staining and corresponding two-color fluorescence in situ hybridization (MYC in red and centromere 8 in green) in matched primary and metastatic breast cancers from rapid autopsies (A–H) and surgical specimens (I–P). The primary in case 9 was MYC positive by immunolabeling and scored as a moderate expressor (A), while the corresponding metastasis was MYC negative (B). This result was inconsistent with fluorescence in situ hybridization, where the metastasis acquired MYC amplification (D), but no abnormalities in MYC copy number were detected in the primary (C). In contrast, the primary in case 11 was MYC negative (E), while the metastasis was MYC positive (F, moderate expressor). By fluorescence in situ hybridization, MYC was duplicated in the primary (G) and amplified in the metastasis (H). In case 25, both the primary (I) and metastasis (J) were MYC positive. However, only half the number of cells stained for MYC in the metastasis as compared to the primary. By fluorescence in situ hybridization, MYC copy number relative to centromere 8 remained unchanged (K, primary and L, metastasis). For case 20, an increase in MYC immunostaining was observed in the metastasis (N, moderate expressor) as compared to the primary (M, low expressor). Further, while MYC was neither duplicated or amplified in the primary (O), MYC was amplified in the metastasis (P). See Tables 2 and 3 for c-myc immunohistochemistry and fluorescence in situ hybridization MYC to centromere 8 ratios for each primary and corresponding metastases.

Figure 2

Comparison of MYC copy number in primary breast cancer, lymph node metastases upon initial diagnosis, and subsequent metastases on autopsy by two-color fluorescence in situ hybridization (MYC in red and centromere 8 in green) for cases 11 (A–D) and 12 (E–H). At the time of diagnosis, both the primary (A, case 11; E, case 12) and axillary lymph node metastases (B, case 11; F, case 12) were characterized by a similar MYC duplication. However, systemic metastases harvested at autopsy for both cases (C and D, case 11; G and H, case 12) acquired a MYC amplification. Metastases for case 11 include axillary lymph node (C) and lung (D), while case 12 include 2 separate brain metastases (G and H). See Table 3 for fluorescence MYC to centromere 8 ratios for each primary and corresponding metastases.