Genetic heterogeneity and actionable mutations in HER2-positive primary breast cancers and their brain metastases

Abstract

Brain metastases constitute a challenge in the management of patients with HER2-positive breast cancer treated with anti-HER2 systemic therapies. Here we sought to define the repertoire of mutations private to or enriched for in HER2-positive brain metastases. Massively parallel sequencing targeting all exons of 254 genes frequently mutated in breast cancers and/or related to DNA repair was used to characterize the spatial and temporal heterogeneity of HER2-positive breast cancers and their brain metastases in six patients. Data were analyzed with state-of-the-art bioinformatics algorithms and selected mutations were validated with orthogonal methods. Spatial and temporal inter-lesion genetic heterogeneity was observed in the HER2-positive brain metastases from an index patient subjected to a rapid autopsy. Genetic alterations restricted to the brain metastases included mutations in cancer genes FGFR2, PIK3CA and ATR, homozygous deletion in CDKN2A and amplification in KRAS. Shifts in clonal composition and the acquisition of additional mutations in the progression from primary HER2-positive breast cancer to brain metastases following anti-HER2 therapy were investigated in additional five patients. Likely pathogenic mutations private to or enriched in the brain lesions affected cancer and clinically actionable genes, including ATR, BRAF, FGFR2, MAP2K4, PIK3CA, RAF1 and TP53. Changes in clonal composition and the acquisition of additional mutations in brain metastases may affect potentially actionable genes in HER2-positive breast cancers. Our observations have potential clinical implications, given that treatment decisions for patients with brain metastatic disease are still mainly based on biomarkers assessed in the primary tumor.

Keywords: HER2-positive; actionable genetic alterations; brain metastasis; metastatic breast cancer; personalized medicine.

Figure 1. The somatic genetic alterations in the primary breast tumor and the three synchronous, spatially distinct brain metastatic lesions of the index case

(A) Longitudinal representation of computer tomography scans, showing coronal sections in T1W1 and the presence of 3 main lesions: BM1, left frontal lobe, BM2, basilar ganglion, BM3, right occipital lobe during the timepoint 1 (06.05.2013), timepoint 2 (12.12.2013) and timepoint 3 (17.03.2014). (B) Bar graphs representing the volumes of main brain lesions that were followed up in the clinics. Note that two small lesions in the basilar ganglion (BM2) were followed up in the clinical care of this patient. (C) Heatmap depicting the cancer cell fractions (CCF) of the mutations as defined by ABSOLUTE [11] in the primary breast cancer and the metachronous brain metastases. Color key for CCF is depicted. Red and orange dots indicate likely pathogenic mutations and mutations affecting cancer genes [–14], respectively. Asterisk (^*) indicate hotspot mutation [17]. The presence of loss of heterozygosity is represented by a diagonal bar, and clonal mutations are indicated by an orange box. (D) Genome plots of the primary tumor and the three brain metastatic lesions highlighting ERBB2 gene amplification, KRAS amplification and CDKN2A homozygous deletion. Smoothed Log₂ ratios were plotted on the y-axis according to their genomic positions indicated on the x-axis. (E) Phylogenetic tree constructed from the somatic mutations, amplifications and homozygous deletions highlighting the main genetic alterations for each sample sequenced. The branch lengths are proportional to the number of genetic alterations.

Figure 2. The repertoire of somatic genetic alterations in primary breast cancers and their respective brain metastases

Heatmap depicting the cancer cell fractions (CCF) of the mutations as defined by ABSOLUTE [11] in the primary breast tumors and the metachronous brain metastases. CCFs are depicted according to the color key. Red and orange dots indicate likely pathogenic mutations and mutations affecting cancer genes [–14], respectively. Asterisks (^*) indicate hotspot mutations [17]. The presence of loss of heterozygosity (LOH) of the wild-type allele is represented by a diagonal bar, and mutations considered clonal by ABSOLUTE [11] are indicated by an orange box.

Figure 3. Progression and associated shift in clonal composition of Case 12

(A) Fluorescent in situ hybridization representative micrographs characterize the HER2 status of the primary breast cancer, brain metastasis #1, brain metastasis #2 as HER2-amplified. HER2/CEP17 ratio and HER2 gene copy number are shown for each sample. Scale bar: 5 microns. (B) Schematic shows shifts in clonal composition from the diagnosis of primary breast cancer to the development of two sequential brain metastases over a period of 60.8 months. Genes affected by likely pathogenic mutations are illustrated. P53 expression has a focal pattern in the primary tumor and is enriched in the brain metastasis (more than 80% of the cells) as depicted in the representative micrograph of immunohistochemistry with antibodies against p53 (See Materials and Methods, original magnifications 100X).