40P A peculiar chromosome 17 copy number profile stratifies HER2-low breast carcinomas

About half of all breast cancers (BCs) harbor HER2-low expression, which can be targeted with the anti-HER2 antibody-drug conjugate trastuzumab deruxtecan (T-DXd), leading to a relevant survival benefit in about 50% of advanced BC patients. Whether these carcinomas constitute a discrete BC entity remains debated. Recently, we and others have provided evidence of genomic heterogeneity within the HER2-low spectrum of BC. Of note, chromosome 17 (chr17) often displays copy number (CN) alterations in BC and HER2-low BCs often show increased HER2 and CEP17 CN in diagnostic in situ hybridization tests. We performed shallow whole-genome sequencing (CUTseq method) on 84 HER2-low carcinomas [28 cases with HER2 immunohistochemistry (IHC) score 1+(1), 15 HER2 not-amplified cases with score 2+ (2N), 41 2+ cases with HER2 copy number in the equivocal range (2E)]. A control group of 40 HER2-negative (score 0) and 30 HER2-positive (score 3+ or score 2+ harboring HER2 amplification) was also analysed. To further dissect HER2-low heterogeneity, we also subjected four 2E samples to single-cell DNA sequencing (single-cell CUTseq). By applying k-means clustering on either genome-wide or chr17 (mapping site of the HER2 locus) CN profiles, 3 distinct CN clusters emerged. The clusters were partially independent from IHC classes and HER2-low BCs were split between different clusters. Among HER2-low cases, a cluster enriched in 2E samples (71%) and characterized by a conserved unbalancing of chr17 (loss of p-arm and gain of the whole q-arm) was associated with a significantly shorter progression-free survival (PFS) compared to the other HER2-low subgroups subjected to comparable adjuvant treatment. Single-cell analysis from 3,200 cells isolated from four 2E samples recapitulated bulk genome-wide copy number profiles, corroborating the chr17 CN unbalance as a clonalnrearrangement of a subset of 2E lesions. HER2-low BCs harbour heterogenous copy number landscapes. Notably, a characteristic chr17 CN profile identifies a specific subgroup of HER2-low carcinomas with dismal prognosis, suggesting this might be used as a novel risk biomarker in this subset of breast neoplasms.