Impact of sampling method on identification of proteins differentially expressed in tumors vs. normal tissues.

*<p>randomly selected single tumor samples per case; this analysis was repeated 3 times (tumor samples 1–3).</p><p>+ and − symbols after p-values indicate significant upregulation (+) or downregulation (−) of the respective protein in tumor vs. normal tissues.</p><p>n.s., not significant (>0.05).</p><p><i>Italics</i> indicate the group of proteins identified as differentially expressed by all four approaches.</p

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

Comparison of intratumoral heterogeneity and variation among patients for all proteins, phosphorylated and non-phosphorylated proteins.

<p>CV, coefficient of variation.</p><p>Overall, all proteins combined; Phospho, all phosphorylated proteins combined; Non-phospho, all non-phosphorylated proteins combined.</p><p>Healthy tubes, fallopian tube epithelium from healthy individuals.</p><p>Contralateral tubes, morphologically normal contralateral fallopian tube epithelium from ovarian cancer patients.</p

Variation between 88 individual tumor samples from 13 patients assessed by non-supervised hierarchical clustering based on expression of 36 proteins.

<p>Different patients are color-coded as indicated in the figure legend. High relative expression of proteins is shown in red and low expression in green color. Grey spaces indicate missing data points.</p

Intratumoral heterogeneity and variation among different patients for the expression of 4 exemplary proteins (VEGF, VEGFR, EGFR and p1068EGFR) assessed by reverse phase protein arrays.

<p>Box plots show the median (line within the box), 25th and 75th percentiles, and whiskers are showing 1.5 times the interquartile range.</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

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

Schematic representation of the ICPL-triplex proteomic approach work flow.

<p>The protein samples from week 10 (control and two irradiated) were reduced and alkylated before labeling with ICPL0, ICPl4 and ICPL6. Samples were mixed and further separated using 1D gel electrophoresis and digested as described in Methods. Samples were analyzed by LC-ESI-MS/MS. Quantification of proteins was performed by Proteome Discoverer software using three biological replicates.</p

Growth curve and senescence associated β-gal of HUVECs exposed to chronic low-dose rates.

<p>A) The growth curve is plotted with cumulative population doublings versus time. Growth curves of control (blue), 1.4 mGy/h (orange) and 2.4 mGy/h (green) irradiated HUVECs are shown. Cumulative population doublings from each week are presented as means ± SEM (n = 3). B) Histograms of positively-stained cells for SA-ß-gal (senescence marker) for control (blue), irradiated by 1.4 mGy/h (orange) and irradiated by 2.4 mGy/h (green). Data are presented as means ± SEM (n = 3). (Students t-test; *p<0.05, **p<0.01 and ***p<0.005).</p