Quantification of intrinsic subtype ambiguity in Luminal A breast cancer and its relationship to clinical outcomes.

Background: PAM50 gene profiling assigns each cancer to a single intrinsic subtype. However, individual cancers vary in their adherence to a prototype, and due to bulk tissue sampling, some may exhibit expression patterns that indicate intra-tumor admixture of multiple subtypes. Our objective was to develop admixture metrics from PAM50 gene expression profiles in order to stratify Luminal A (LumA) cases according to their degree of subtype admixture, and then relate such admixture to clinical and molecular variables.

Methods: We re-constructed scaled, normalized PAM50 profiles for 1980 cases (674 LumA) in the METABRIC cohort and for each case computed its Mahalanobis (M-) distance from its assigned centroid and M-distance from all other centroids. We used t-SNE plots to visualize overlaps in subtype clustering. With Normal-like cases excluded, we developed two metrics: Median Distance Criteria (MDC) classified pure cases as those located within the 50th percentile of the LumA centroid and > =50th percentile from any other centroid. Distance Ratio Criteria (DRC) was computed as the ratio of M-distances from the LumA centroid to the nearest non-assigned centroid. Pure and admixed LumA cases were compared on clinical/molecular traits. TCGA LumA cases (n = 509) provided independent validation.

Results: Compared to pure cases in METABRIC, admixed ones had older age at diagnosis, larger tumor size, and higher grade and stage. These associations were stronger for the DRC metric compared to MDC. Admixed cases were associated with HER2 gain, high proliferation, higher PAM50 recurrence scores, more frequent TP53 mutation, and less frequent PIK3CA mutation. Similar results were observed in the TCGA validation cohort, which also showed a positive association between admixture and number of clonal populations estimated by PyClone. LumA-LumB confusion predominated, but other combinations were also present. Degree of admixture was associated with overall survival in both cohorts, as was disease-free survival in TCGA, independent of age, grade and stage (HR = 2.85, Tertile 3 vs.1).

Conclusions: Luminal A breast cancers subgrouped based on PAM50 subtype purity support the hypothesis that admixed cases have worse clinical features and survival. Future analyses will explore more extensive genomic metrics for admixture and their spatial significance within a single tumor.