Common Protein Biomarkers Assessed by Reverse Phase Protein Arrays Show Considerable Intratumoral Heterogeneity in Breast Cancer Tissues

Abstract

Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2–5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22–43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range18–38%) or between different tumor zones (CV 24%, range 17–38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18–34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29–98%) and lymph node metastases (CV 65%, range 40–146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.

Pan-HER, an Antibody Mixture Simultaneously Targeting EGFR, HER2, and HER3, Effectively Overcomes Tumor Heterogeneity and Plasticity

“…Heterogeneous expression patterns have been described for EGFR in glioblastoma (20,21) and for HER2 in breast cancer (22,23), gastric cancer (24), and salivary duct carcinoma (20). In addition, biomarker studies in primary breast cancer have revealed a highly heterogeneous expression pattern for the whole HER family (25). Consequently, biomarker assessment and subsequent selection of an optimal targeted therapy remains challenging even when multiple biopsies are available.…”

Histopathological markers of treatment response and recurrence risk in ovarian cancers and borderline tumors

“…The overall goal of the first research project [5] was to investigate the intratumoral heterogeneity of proteins with clinical relevance to breast cancer by analyzing 35 target proteins including 15 phosphorylated proteins. This included predictive markers for therapies targeting HER2 [50], diagnostic marker E-Cadherin [51], and prognostic markers including HER2, estrogen and progesterone receptors [42], and uPA and PAI-1 [52, 53].…”

“…All 35 proteins studied by reverse phase protein microarrays (RPPA) showed similar heterogeneity with a mean coefficient of variation (CV) of 31% (range 22–43%) within primary breast cancers and 35% (range 10–79%) within different lymph node metastases from the same patient [5]. …”

“…Interestingly, the significance of this correlation was lost when only one sample was randomly chosen for each primary tumor. Similarly, a significant correlation between higher expression of phosphorylated pGSK3β, uPA, PAI-1, and HER4 in ER positive compared to negative tumors was only observed when using mean expression values of all samples for each primary tumor and lost significance when using only single samples [5]. Significant correlations between protein expression and clinico-pathologic parameters were also observed for tumor stage (pERK) and lobular versus ductal subtype (PGSK3β, p 727 STAT3, uPA, E-Cadherin), all of which lost significance when using only single samples for each primary tumor.…”

Receptor-targeted Drug delivery: Current Perspective and Challenges

“…Despite these tests, one may not always know what fraction of the tissue expresses receptors to be targeted, the distribution pattern of receptors in tissue, and what is the expression at cellular level (density of receptors on cell surface), as these, in turn, vary depending on how the sample for biopsy has been taken [42]. This can also be ascribed to the heterogeneity of receptor expression at cellular level that a diseased tissue possesses [42]. For example, for tumors, cells at the periphery and those at the core of the tumor experience different microenviroments and blood supply, which leads to differences in expression at cellular level.…”