Quantitative in vivo immunohistochemistry of epidermal growth factor receptor using a receptor concentration imaging approach

Abstract

As receptor-targeted therapeutics become increasingly used in clinical oncology, the ability to quantify protein expression and pharmacokinetics in vivo is imperative to ensure successful individualized treatment plans. Current standards for receptor analysis are performed on extracted tissues. These measurements are static and often physiologically irrelevant; therefore, only a partial picture of available receptors for drug targeting in vivo is provided. Until recently, in vivo measurements were limited by the inability to separate delivery, binding, and retention effects, but this can be circumvented by a dual-tracer approach for referencing the detected signal. We hypothesized that in vivo receptor concentration imaging (RCI) would be superior to ex vivo immunohistochemistry (IHC). Using multiple xenograft tumor models with varying EGFR expression, we determined the EGFR concentration in each model using a novel targeted agent (anti-EGFR affibody-IRDye800CW conjugate) along with a simultaneously delivered reference agent (control affibody-IRDye680RD conjugate). The RCI-calculated in vivo receptor concentration was strongly correlated with ex vivo pathologist-scored IHC and computer-quantified ex vivo immunofluorescence. In contrast, no correlation was observed with ex vivo Western blot analysis or in vitro flow-cytometry assays. Overall, our results argue that in vivo RCI provides a robust measure of receptor expression equivalent to ex vivo immunostaining, with implications for use in noninvasive monitoring of therapy or therapeutic guidance during surgery.

Figure 1

The standard methods of quantifying protein expression in tissue and cell samples are compared in terms of type of sample preparation, detection technique and sensitivity range. In each row, the relevance to in vivo tissues and tissue integrity decreases from left to right. IHC = immunohistochemistry, IF = immunofluoresence, EGFR = epidermal growth factor receptor, AR = amplification ratio, and OD = optical density. The sensitivity ranges for IHC and IF are reported as pathologist score (top) and computer analyzed (bottom).

Figure 2

The concentration of in vivo EGFR is determined by RCI in five different tumor models (rows). The tumor lines are presented in the order of predicted EGFR concentration (fading purple triangle on far left). The first column shows the fluorescence uptake of the EGFR targeted tracer in the tumor (boundary designated by white dashed line) and surrounding tissues at 40 minutes post-injection. The second column is the corresponding untargeted tracer uptake in the same tissue at the same time point. The third column presents maps of the EGFR concentration (nM), determined using the RCI method. The forth column show the time trajectories of the average fluorescence within the tumor region of the targeted (red) and untargeted (green) tracers. The curves are normalized to the first time point.

Figure 3

A box plot of the RCI determined in vivo receptor concentration for each tumor model is shown. Each box represents the 25–75% percentile range, the filled square is the mean, the solid line dividing the box is the median, and the open circles are the individual tumors within each group. The A431, AsPC-1(Or), AsPC-1(SQ) and U251 tumor models were found to be statistically different than muscle (p < 0.05, denoted by *) and the EGFR-negative 9L tumor (p < 0.05, denoted by #). The 9L tumor model was not significantly different than the muscle.

Figure 4

The receptor concentration determined using anti-EGFR affibody RCI is compared to four standard methods used to determine protein concentration: A) pathologist scored ex vivo immunohistochemistry (IHC, r = 0.69, p = 2×10⁻⁵, m = 0.29 ± 0.06, b = 0.8 ± 0.2), B) ex vivo immunofluorescence (r = 0.62, p = 5×10⁻⁴, m = 1.0 ± 0.2, b = 2.1 ± 0.9), C) ex vivo Western blot (r = 0.35, p = 0.08, m = 0.04 ± 0.03, b = 0.06 ± 0.08), and D) in vitro flow cytometry (r = 0.43, p = 0.017, m = 9×10⁴ ± 3×10⁴, intercept = 4×10⁴ ± 1.3×10⁵). The linear regression (solid line) is displayed as well as the 95% confidence bands (dashed lines) and the 95% prediction bands (dotted lines).

Figure 5

Ex vivo tissue sections of the five tumor models with varying expression of EGFR studied. Column 1: hemotoxylin and eosin (H&E, column 1), Column 2: EGFR immunohistochemistry (IHC, column 2), and Column 3: EGFR immunofluorescence (IF, column 1). The images are ordered from highest expressing (top row) to lowest expressing (bottom row). Note in the AsPC-1(Or) model, the tumor cells are interspersed and surrounded by normal pancreas acinar cells that are EGFR negative (arrow).