Somatic mutations and copy number variations in breast cancers with heterogeneous HER2 amplification

Abstract

Intratumour heterogeneity fuels carcinogenesis and allows circumventing specific targeted therapies. HER2 gene amplification is associated with poor outcome in invasive breast cancer. Heterogeneous HER2 amplification has been described in 5-41% of breast cancers. Here, we investigated the genetic differences between HER2-positive and HER2-negative admixed breast cancer components. We performed an in-depth analysis to explore the potential heterogeneity in the somatic mutational landscape of each individual tumour component. Formalin-fixed, paraffin-embedded breast cancer tissue of ten patients with at least one HER2-negative and at least one HER2-positive component was microdissected. Targeted next-generation sequencing was performed using a customized 53-gene panel. Somatic mutations and copy number variations were analysed. Overall, the tumours showed a heterogeneous distribution of 12 deletions, 9 insertions, 32 missense variants and 7 nonsense variants in 26 different genes, which are (likely) pathogenic. Three splice site alterations were identified. One patient had an EGFR copy number gain restricted to a HER2-negative in situ component, resulting in EGFR protein overexpression. Two patients had FGFR1 copy number gains in at least one tumour component. Two patients had an 8q24 gain in at least one tumour component, resulting in a copy number increase in MYC and PVT1. One patient had a CCND1 copy number gain restricted to a HER2-negative tumour component. No common alternative drivers were identified in the HER2-negative tumour components. This series of 10 breast cancers with heterogeneous HER2 gene amplification illustrates that HER2 positivity is not an unconditional prerequisite for the maintenance of tumour growth. Many other molecular aberrations are likely to act as alternative or collaborative drivers. This study demonstrates that breast carcinogenesis is a dynamically evolving process characterized by a versatile somatic mutational profile, of which some genetic aberrations will be crucial for cancer progression, and others will be mere 'passenger' molecular anomalies.

Keywords: HER2 amplification; breast cancer; copy number variations; intratumour heterogeneity; next-generation sequencing; somatic mutation.

Fig. 1

Heterogeneous CNVs and heterogeneous HER2 and EGFR expression in the tumour of patient #1. The scatter plot illustrates the presence of a HER2 copy number gain in one DCIS component and in the MC, as well as an EGFR copy number gain in the HER2‐negative DCIS component (A). Immunohistochemistry for HER2, with an overview of breast cancer #1 (B; original magnification 12.5× – scale bar size = 2.5 mm), and detailed microphotographs of the HER2‐positive DCIS (C), the HER2‐positive MC (D) and the HER2‐negative DCIS (E; original magnification 100× – scale bar size = 250 µm). Immunohistochemistry for EGFR, which was positive in the HER2‐negative DCIS component (F) and negative in the HER2‐positive DCIS (G) and in the HER2‐positive MC (H; original magnification 100× – scale bar size 300 µm).

Fig. 2

Heterogeneous HER2 overexpression and CNVs in the breast cancer of patient #3. Immunohistochemistry for E‐cadherin (A) illustrates the absence of expression in all tumour components (original magnification 50× – scale bar size = 500 µm). Immunohistochemistry for HER2 (B) demonstrates a positive 3+ score in the HER2‐amplified LCIS component (orange squares) and an equivocal 2+ score in the HER2 nonamplified LCIS component (blue diamond) and the ILC of classic type (grey triangle; original magnification 50x – scale bar size = 500 µm). The scatter plot confirms the HER2 copy number gain in the HER2‐positive LCIS (indicated by orange squares) and its absence in the HER2‐negative components (C). Additionally, the presence of an FGFR1 copy number gain in the ILC is noted (indicated by grey triangles).

Fig. 3

Overview of detected pathogenic and likely pathogenic variants in 26 genes per patient. Green and blue squares indicate missense and nonsense mutations, respectively. Red and orange squares indicate deletions and insertions, respectively. Splice site mutations are indicated by purple squares.