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

<div><p>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.</p> </div

Reproducibility of protein extraction and reverse-phase-protein-arrays.

<p>Bivariate correlations of HER2, pHER2, uPA and PAI-1 expression (A) from three independent protein extractions and (B) from two independent RPPA analyses. Graphs are showing the Spearman’s rho for each pairwise correlation.</p

Intratumoral heterogeneity and variation amongst different patients in the expression of 4 exemplary proteins (E-cadherin, EGFR, ER, and HER2) assessed by reverse-phase-protein-arrays.

<p>Box plots are showing the median (line within the box), 25^th and 75^th percentiles, and whiskers are showing 1.5 times the interquartile range; the small box sign is showing the mean, and small cross signs show the maximum and minimum values. <b><i>Italics (bold)</i></b>, all cases containing lymph node metastases; * invasive lobular cancers; (L), two largest primary tumors (7.9 and 8.0 cm diameter).</p

Heterogeneity in the expression of 35 proteins assessed by reverse-phase-protein-arrays.

<p>There was considerable intratumoral heterogeneity in the expression of all 35 proteins analyzed, and even more pronounced variation amongst different patients.</p><p>Intratumoral, differences between individual samples of the same primary tumor or between individual lymph node metastases from the same patient.</p><p>CV, coefficient of variation given as percentage; Primary tumor, whole tumor including central, peripheral, and intermediate zone; LN, lymph node; Total, total case including all primary tumor and lymph node samples.</p><p>Phospho, all phosphorylated proteins combined; Non-phospho, all non-phosphorylated proteins combined;</p><p>Overall, all 35 proteins combined.</p