Genomic aberrations in normal tissue adjacent to HER2-amplified breast cancers: field cancerization or contaminating tumor cells?

Abstract

Field cancerization effects as well as isolated tumor cell foci extending well beyond the invasive tumor margin have been described previously to account for local recurrence rates following breast conserving surgery despite adequate surgical margins and breast radiotherapy. To look for evidence of possible tumor cell contamination or field cancerization by genetic effects, a pilot study (Study 1: 12 sample pairs) followed by a verification study (Study 2: 20 sample pairs) were performed on DNA extracted from HER2-positive breast tumors and matching normal adjacent mammary tissue samples excised 1-3 cm beyond the invasive tumor margin. High-resolution molecular inversion probe (MIP) arrays were used to compare genomic copy number variations, including increased HER2 gene copies, between the paired samples; as well, a detailed histologic and immunohistochemical (IHC) re-evaluation of all Study 2 samples was performed blinded to the genomic results to characterize the adjacent normal tissue composition bracketing the DNA-extracted samples. Overall, 14/32 (44 %) sample pairs from both studies produced genome-wide evidence of genetic aberrations including HER2 copy number gains within the adjacent normal tissue samples. The observed single-parental origin of monoallelic HER2 amplicon haplotypes shared by informative tumor-normal pairs, as well as commonly gained loci elsewhere on 17q, suggested the presence of contaminating tumor cells in the genomically aberrant normal samples. Histologic and IHC analyses identified occult 25-200 μm tumor cell clusters overexpressing HER2 scattered in more than half, but not all, of the genomically aberrant normal samples re-evaluated, but in none of the genomically normal samples. These genomic and microscopic findings support the conclusion that tumor cell contamination rather than genetic field cancerization represents the likeliest cause of local clinical recurrence rates following breast conserving surgery, and mandate caution in assuming the genomic normalcy of histologically benign appearing peritumor breast tissue.

Fig. 1

Frequency of significant allelic gain (green) or loss (red) based on 20K MIP array platform analysis of 12 paired Study 1 samples, plotted as a function of genomic location, with HER2-positive tumor frequencies shown above (A) and their adjacent normal breast tissue frequencies shown below (B). The 17q chromosome location of the HER2/ERBB2 amplicon is indicated above each panel. Vertical axis labels indicate fraction of samples with gained (0 to +1.0) or lost (0 to −1.0) allelic probes

Fig. 2

Histologic (H&E) images from representative Study 2 cryosections of a HER2-positive cancer (A) and a normal-appearing cancer-adjacent sample (B). All cancer-adjacent samples were characterized for constituent histologic elements including fat, stroma, and epithelium as illustrated above and summarized in Table 2

Fig. 3

Total and allele-specific copy number variations based on 300K MIP array platform analysis of paired Study 2 samples, plotted as a function of genomic location. A Summary box-plot of log2 copy number variations across all 20 normal samples and their matching 20 tumor samples. Red (increased copy number) and blue (decreased copy number) vertical lines indicate upper and lower quantile variations, colored by their median value; note different copy number scales for normal (−0.4 to +0.6) and tumor (−2 to +3) samples. Black dots indicate median values across all samples. B Genome-wide allele-specific copy number comparisons for the 9 HER2-positive tumor (T)–normal (N) sample pairs in which there was some genomic aberration detectable in the N sample, with 7 of these showing 17q12–21 HER2 copy number gains (# 2, 8, 9, 16, 17, 18, 20). Major (yellow) and minor (blue) allelic changes (scaled from 0 to 8 for all samples) are shown with estimated copy numbers >1.5 considered significant gains

Fig. 4

IGV display comparing Study 2 chromosome 17 gains/losses and HER2/ERBB2 amplicon gains in the 7 T/N sample pairs for which increased 17q and HER2 copy numbers were seen the N samples (top heatmap panel), and the 2 other sample pairs in which the N samples showed normal chromosome 17 copy numbers but aberrant genomes elsewhere (bottom heatmap panel). Heatmaps show segmented overall copy number variations with red intensities indicating regions of copy number gain, blue intensities indicating regions of copy number loss, and white indicating no copy number change. The HER2/ERBB2 locus is indicated by the red rectangle on the ideogram (~35 mb)

Fig. 5

Histologic (H&E) and IHC (+3 HER2 overexpression) images of scattered, occult tumor cell clusters (25–200 μ) identified in four HER2-positive cancer-adjacent normal samples (# 2N, 8N, 18N, 20N)