Uncovering the genomic heterogeneity of multifocal breast cancer

Abstract

Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as 'oncogenic' in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole-genome rearrangement screen was further conducted in 8/36 patients. Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome-wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers.

Keywords: breast cancer; genomic heterogeneity; multicentric; multifocal; oncogenic mutations; targeted sequencing.

Figure 1

Distribution of non‐silent substitutions and indels in the ‘homogeneous’ MFBC group. Heat maps representing the non‐silent exonic substitutions and indels of the homogeneous multifocal lesions (L), which have all the mutations in common between all the lesions. Orange indicates the presence of an oncogenic mutation; grey the presence of a mutation of unknown significance; and white the absence of the mutation. When a sample name is grey, it means that it was not initially sequenced, due to a lack of sufficient DNA, but was included in the validation phase.

Figure 2

Distribution of non‐silent substitutions and indels in the ‘intermediate’ MFBC group. Heat maps representing the non‐silent exonic substitutions and indels of the intermediate multifocal lesions, presenting both common and private mutations. The colour code, abbreviations, and meaning of the grey colour for sample names are the same as in Figure 1. When numbers are present after the gene symbol, it means that different mutations were observed in the same gene.

Figure 3

Distribution of non‐silent substitutions and indels in the ‘heterogeneous’ MFBC group. (A) Heat maps representing the non‐silent exonic substitutions and indels of the heterogeneous multifocal lesions, which have no mutation in common between the lesions. The colour code and abbreviations are the same as in Figure 1. (B) Immunohistochemical staining illustrating the loss of PTEN at the protein level only in the lesion of patient PD13774 carrying the PTEN mutation.

Figure 4

Genome‐wide alterations. Bar plots showing the distribution and the type of somatic rearrangements (A) and the percentage of private and common (present in all investigated lesions from that patient) rearrangements (B). (C) Genome‐wide Circos plots of somatic rearrangements of the three patients classified in the ‘heterogeneous’ group. The chromosomes are represented in the outer ring. Within the inner ring, each blue line represents a common rearrangement, whereas each green or red line represents a rearrangement private to lesion 1 or 2, respectively. Bar plots showing the distribution (D) and the percentage of private and common (present in all investigated lesions from that patient) SCNAs (E). (F, G) Log₂‐based estimate (log₂ ratio) of SCNAs, represented in light red, of two of the three patients classified as ‘heterogeneous’.