PAM50 gene expression subtypes represent a cornerstone in the molecular classification of breast cancer and are included in risk prediction models to guide therapy. We aimed to illustrate the impact of included genes and biological processes on subtyping while considering a tumor’s underlying clinical subgroup defined by ER, PR, and HER2 status. To do this we used a population-representative and clinically annotated early-stage breast tumor cohort of 6233 samples profiled by RNA sequencing and applied a perturbation strategy of excluding co-expressed genes (gene sets). We demonstrate how PAM50 nearest-centroid classification depends on biological processes present across, but also within, ER/PR/HER2 subgroups and PAM50 subtypes themselves. Our analysis highlights several key aspects of PAM50 classification. Firstly, we demonstrate the tight connection between a tumor’s nearest and second-nearest PAM50 centroid. Additionally, we show that the second-best subtype is associated with overall survival in ER-positive, HER2-negative, and node-negative disease. We also note that ERBB2 expression has little impact on PAM50 classification in HER2-positive disease regardless of ER status and that the Basal subtype is highly stable in contrast to the Normal subtype. Improved consciousness of the commonly used PAM50 subtyping scheme will aid in our understanding and interpretation of breast tumors that have seemingly conflicting PAM50 classification when compared to clinical biomarkers. Finally, our study adds further support in challenging the common misconception that PAM50 subtypes are distinct classes by illustrating that PAM50 subtypes in tumors represent a continuum with prognostic implications.

PAM50 基因表达亚型代表了乳腺癌分子分类的基石，并被纳入指导治疗的风险预测模型中。我们的目的是说明所包含的基因和生物过程对亚型的影响，同时考虑由 ER、PR 和 HER2 状态定义的肿瘤的潜在临床亚组。为此，我们使用了具有人群代表性和临床注释的早期乳腺肿瘤队列，该队列包含 6233 个样本，通过 RNA 测序进行分析，并应用排除共表达基因（基因集）的扰动策略。我们展示了 PAM50 最近质心分类如何取决于 ER/PR/HER2 亚组和 PAM50 亚型本身之间以及内部存在的生物过程。我们的分析强调了 PAM50 分类的几个关键方面。首先，我们证明了肿瘤最近的 PAM50 质心和次近的 PAM50 质心之间的紧密联系。此外，我们还发现，第二佳亚型与 ER 阳性、HER2 阴性和淋巴结阴性疾病的总生存率相关。我们还注意到，无论 ER 状态如何， ERBB2表达对 HER2 阳性疾病中的 PAM50 分类几乎没有影响，并且与正常亚型相比，基础亚型高度稳定。提高对常用 PAM50 亚型分型方案的认识将有助于我们理解和解释与临床生物标志物相比具有看似冲突的 PAM50 分类的乳腺肿瘤。最后，我们的研究通过说明肿瘤中的 PAM50 亚型代表具有预后意义的连续体，进一步支持挑战 PAM50 亚型是不同类别的常见误解。