Multiplexed imaging reveals heterogeneity of PI3K/MAPK network signaling in breast lesions of known PIK3CA genotype
Activating genetic changes in the phosphatidylinositol-3-kinase (PI3K) signaling pathway are found in over half of invasive breast cancers (IBCs). Previously, we discovered numerous hotspot PIK3CA mutations in proliferative breast lesions. Here, we investigate the spatial nature of PI3K pathway signaling and its relationship with PI3K genotype in breast lesions.
We identified PI3K phosphosignaling network signatures in columnar cell change (CCL), usual ductal hyperplasia (UDH), ductal carcinoma in situ (DCIS), and IBC in 26 lesions of known PIK3CA genotype from 10 human breast specimens using a hyperspectral-based multiplexed tissue imaging platform (MTIP) to simultaneously quantitate PI3K/MAPK pathway targets (pAKT473, pAKT308, pPRAS40, pS6, and pERK) in FFPE tissue, with single-cell resolution.
We found that breast lesional epithelia contained spatially heterogeneous patterns of PI3K pathway phosphoprotein signatures, even within microscopic areas of CCL, UDH, DCIS, and IBC. Most lesions contained 3–12 unique phosphoprotein signatures within the same microscopic field. The dominant phosphoprotein signature for each lesion was not well correlated with lesion genotype or lesion histology, yet samples from the same patient tended to group together. Further, 5 UDH/CCL lesions across different patients had a common phosphosignature at the epithelial–stromal interface (possible myoepithelial cells) that was distinct from both the adjacent lesional epithelium and distinct from adjacent stroma.
We present the first spatial mapping of PI3K phosphoprotein networks in proliferative breast lesions and demonstrate complex PI3K signaling heterogeneity that defies simple correlation between PIK3CA genotype and phosphosignal pattern.
KeywordsPIK3CA Phosphatidylinositol-3-kinase Usual ductal hyperplasia Columnar cell change Hyperspectral imaging
This project was supported by NIH Grant #P30-CA069533 and a OHSU pilot grant from the Knight Cancer Institute and Oregon Center for Spatial Systems Biomedicine. We thank Drs. Esteban Roberts, Karl Garsha, Michael Barnes, and Kandavel Shanmugam (Ventana-Roche) for stimulating discussions and the use of the MTIP platform. We acknowledge the helpful technical assistance of Dominic Mills in dendrogram analysis. A small subset of invasive tumors with PIK3CA E545K mutations was previously reported  with different scope of data analysis and without a comparative group of PIK3CA exon 20-mutated invasive cancers or noninvasive lesions.
Compliance with ethical standards
Conflict of interest
Thomas Jacob, Joe W. Gray, Megan Troxell, and Tania Q. Vu declare that they have no conflict of interest.
Compliance of ethical statement
Tissue specimens were derived from breast precursor lesions, with an IRB-approved protocol.
In these retrospective studies, deidentified materials were used; informed consent was waived by an institutional IRB-approved protocol.
- 18.Roberts E, et al Mapping cancer signaling networks by an integrated multiplexed tissue imaging platform. Technology (in publication) Google Scholar
- 19.Hermanson GT (2008) Bioconjugate techniques, 2nd edn. Academic press, New YorkGoogle Scholar