Multiplexed imaging reveals heterogeneity of PI3K/MAPK network signaling in breast lesions of known PIK3CA genotype

Abstract

Purpose Activating genetic changes in the phosphatidylinositol-3-kinase (PI3K) signaling pathway are found in over half of invasive breast cancers (IBC). 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. Methods We identified PI3K phosphosignaling network signatures in columnar cell change (CCL), usual ductal hyperplasia (UDH), ductal carcinoma in situ (DCIS) and IBC in 29 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. Results 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. Conclusion 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.

Spa-RQ: an Image Analysis Tool to Visualise and Quantify Spatial Phenotypes Applied to Non-Small Cell Lung Cancer

“…In some clinical samples, clear stromal MAPK activity could be detected, in accordance with the increased stromal component in human disease compared to murine tumours. The detection of signalling heterogeneity in tumours carrying identical driver mutations matches a previous report demonstrating PI3K/MAPK heterogeneity in PIK3CA mutant breast cancer, a rare example of a study applying multiplexed techniques to investigate phosphoprotein networks 28 . An interesting question for future mechanistic exploration is whether such spatial phenotyping of in situ signalling heterogeneities can further refine the predictive stratification of tumours linked to drug response.…”

Solid papillary carcinoma with reverse polarity of the breast harbors specific morphologic, immunohistochemical and molecular profile in comparison with other benign or malignant papillary lesions of the breast: a comparative study of 9 additional cases

“…The phosphatidylinositol-3-kinase (PIK3) cell signaling pathway is often dysregulated and activated in human cancers, and in more than half of breast invasive tumors [44]. We identified PIK3CA or PIK3R1 alterations in all our study groups, regardless of malignancy.…”

Breast cancer intratumour heterogeneity: current status and clinical implications

“…Moreover, the detection of tumour and non‐tumour cells can be used to identify tumour boundaries, which could be useful in supporting pathologists in digital slide workflow for the identification of regions of interest. In addition, spatial heterogeneity in the epithelial–stromal interface PI3K/MAPK signature can be assessed by the use of THRIVE workflows …”

“…In addition, spatial heterogeneity in the epithelial-stromal interface PI3K/MAPK signature can be assessed by the use of THRIVE workflows. 114…”