3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways

Abstract

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A "seesaw" relationship between ER/PR and Her2 pathways was observed in ER-PR-Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER-PR-Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER-PR space. Conclusions: The unique distribution of specimens in ER-PR-Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

Keywords: FFPE; QDB; biomarker; high throughput; protein quantitation.

Figure 1.

Distribution of Her2, estrogen receptor (ER), progesterone receptor (PR), and Ki67 levels as absolute and continuous variables among 852 FFPE specimens (a-d), and their correlations with IHC scores provided by local hospital (e-h). a-d: The absolute and quantitative levels of Her2, ER, PR, and Ki67 were measured with the quantitative dot blot (QDB) method using lysates prepared from 2 × 15 μm FFPE slices. The average levels of these biomarkers were expressed as mean ± SEM. The 25th and 75th percentiles were also listed for each biomarker, respectively. The results were reported as the average of 3 independent measurements of these 4 biomarkers, with each measurement in triplicate. e-h: The correlations between QDB and IHC results were analyzed using Spearman's rank correlation analysis, with P < .0001 for all analysis. For ER, PR, and Ki67, these specimens were further subgrouped based on their IHC scores, and correlation of their subgroup averages with the matching IHC scores were analyzed again with Pearson's correlation analysis. P < .0001 for all analysis.

Figure 2.

Three-dimensional distribution of 852 FFPE specimens based on the absolute and quantitative levels of ER, PR, and Her2. (a)The 3-dimensional scatterplot using absolutely quantitative ER, PR, and Her2 protein levels as X, Y, and Z axes was created with Origin 9.1 software. Specimens were segregated into 3 groups as described in the text. (b)The 3D distribution of the specimens was narrowed down constantly into a small block with ER<0.2 nmol/g, PR <0.8 nmol/g, and Her2 <0.3 nmol/g where the specimens were found distributed randomly inside.

Figure 3.

Evaluation of Ki67 levels of specimens grouped by their spatial distribution. (a) Comparison of Ki67 levels among 3 spatial groups of HR, Her2, and corner groups. The absolute and quantitative Ki67 levels of all 852 specimens were measured with QDB method using the same lysates for ER, PR, and Her2 measurements. The specimens were grouped into HR, Her2, and corner groups based on the observed cutoffs at Figure 2b (Her2 group: Her2 ≥0.3 nmol/g; HR group: ER≥0.2 nmol/g, and/or PR≥0.8 nmol/g; corner group: ER<0.2 nmol/g, PR<0.8 nmol/g, and Her2 < 0.3 nmol/g). The results were analyzed using unpaired two-tailed Student's t-test. (b) The spatial distribution of specimens using ER, Her2, and Ki67 levels as X, Y, and Z axes. Those specimens with PR level <0.8 nmol/g are arbitrarily colored red, while those with PR level ≥0.8 nmol/g are blue. We observed that specimens with the highest Ki67 levels are dominantly red around the intersection of ER and Her2, suggesting a lack of co-expression of ER, PR, and Her2.

Figure 4.

Evaluating the sensitivity and specificity of QDB method using receiver operative characteristics (ROC) analysis based on provided Her2 IHC scores from local hospitals. (a) Specimens with provided IHC scores were grouped as negative (IHC scores of 0 and 1 + ) or positive (IHC score of 3 + ), and were used for receiver operative characteristics (ROC) analysis using Graphpad Software. We were able to achieve area under the curve (AUC) at 0.932 ± 0.014, 95%CI at 0.904 to 0.960 (P < .0001). Using 0.3 nmol/g as the cutoff, we achieved sensitivity at 84.8% and specificity at 97.2%. The concordant rate was at 93.8% (560 out of 597, specimens with Her2 score of 2 + were excluded from analysis) with IHC results. (b) To better illustrate the effectiveness of this cutoff (indicated by the dashed line) at separating negative specimens from positive ones, specimens were plotted in log scale and grouped based on their respective IHC scores. All those specimens with their Her2 levels measured as 0 were arbitrarily set as 0.001 nmol/g to avoid being omitted in the log scale plot.

Figure 5.

The “seesaw” hypothesis. The unique distribution patterns of the breast cancer specimens in the ER–PR–Her2 space suggest a “seesaw” relationship, with predominant activation of ER/PR pathway in the HR group (a), predominant activation of Her2 pathway in the Her2 group (b), and lack of activation of both pathways in the corner group (c). Possibly, a shared factor, or factors, might be needed for the activation of both pathways. Consequently, inhibition of one pathway may liberate this common factor to allow activation of the other pathway in vivo. Thus, this factor may serve as novel target to develop a new class of drugs without incurring acquired resistance among breast cancer patients.