Measurement of cetuximab and panitumumab-unbound serum EGFR extracellular domain using an assay based on slow off-rate modified aptamer (SOMAmer) reagents.

Response to cetuximab (Erbitux®) and panitumumab (Vectibix®) varies among individuals, and even those who show response ultimately gain drug resistance. One possible etiologic factor is differential interaction between the drug and target. We describe the development of an assay based on Slow Off-rate Modified Aptamer (SOMAmer(™)) reagents that can distinguish drug-bound from unbound epidermal growth factor receptor (EGFR).This quantitative assay uses a SOMAmer reagent specific for EGFR extracellular domain (ECD) as a capturing reagent. Captured SOMAmer is quantitated using PCR. Linearity and accuracy (recovery) of the assay were assessed using normal sera and purified EGFR ECD.This EGFR ECD assay showed linearity between 2.5 and 600 ng/mL. Average recovery was 101%. The assay detected EGFR but showed little cross-reactivity to other ErbB proteins: 0.4% for ErbB2, 6.9% for ErbB3, and 1.3% for ErbB4. Preincubation of normal serum with either cetuximab or panitumumab resulted in a dose-dependent decrease in EGFR ECD levels measured using the SOMAmer assay; preincubation did not affect measurement with an ELISA.This SOMAmer-based serum EGFR ECD assay accurately and specifically measures EGFR in serum. Detection of significant amounts of drug-unbound EGFR in patients undergoing cetuximab or panitumumab treatment could be an indicator of poor drug response. Further studies are needed to evaluate the utility of the assay as an indicator of drug efficacy or as a guide to dosing

EGFR SOMamer assay detects cetuximab and panitumumab-unbound fraction of EGFR.

<p>Normal serum samples were incubated with varying amounts of panitumumab [PANEL A] or cetuximab [PANEL B] for 30 min at room temperature. These samples were then diluted 30 fold, followed by EGFR SOMAmer capture and quantitative PCR (qPCR) (gray bars). EGFR ELISA was also performed on drug-treated samples on the same day (white bars). Triplicate samples were tested at each condition. The vertical lines at each bar represent standard deviations among replicates.</p

Spiked-in purified EGFR to serum is accurately detected by the EGFR SOMAmer assay.

<p>Thirty-fold diluted serum samples were appropriately spiked with three different amounts of purified EGFR ECD (final concentration of 30, 150, and 300 ng/mL plus the endogenous serum EGFR ECD). We then performed the EGFR SOMAmer assay followed by the qPCR. The percent recovery is calculated by dividing SOMAmer-measured EGFR by the expected EGFR level (spiked plus endogenous serum EGFR level), multiplied by one hundred. The codes on the X-axis represent sample number and concentration of spiked EGFR. For example, S_1_300 is serum sample #1 with 300 ng/mL of spiked purified EGFR protein. Each sample was measured three times. The error bars represent standard deviations.</p

EGFR SOMAmer specificity test.

<p>EGFR SOMAmer reagent was incubated with one of the ErbB proteins followed by labeling of bound protein with Alexa fluor 647. After extensive washing, photocleavage dissociates SOMAmer:protein complex from the bound beads, and the complex is separated by polyacrylamide gel electrophoresis under denaturing conditions. Lane 1, pulled down EGFR; Lane 2, EGFR standard; lane 3, pulled down ErbB2; lane 4, ErbB2 standard; lane 5, pulled down ErbB3; lane 6, ErbB3 standard; lane 7, pulled down ErbB4; lane 8, ErbB4 standard, and lane 9, MW size standards The EGFR SOMAmer showed limited cross-reactivity with ErbB family proteins. Relative to EGFR, the SOMAmer “pulled down” limited amounts of ErbB2 (0.4%), ErbB3 (6.9%), and ErbB4 (1.3%).</p

Intra and inter assay variability of EGFR ECD SOMAmer assay.

<p>Inter-assay variability was determined from 20 runs each of control samples with low (∼27 ng/mL), middle (∼53 ng/mL), or high (∼310 ng/mL) concentrations of EGFR. Intra-assay variability was determined by testing each control sample at least 20 times in a single run.</p

Comparison of ELISA and SOMAmer EGFR levels.

<p>Serum EGFR ECD levels were measured using EGFR SOMAmer assay (y-axis) and ELISA (x-axis). Black diamond data points showed correlation between the two methods and were used for R² calculation. Grey diamond data points showed much reduced EGFR SOMAmer levels and were not used for R² calculation.</p

Detection of drug-unbound EGFR in patents receiving treatment with an EGFR monoclonal antibody.

<p>Detection of drug-unbound EGFR in patents receiving treatment with an EGFR monoclonal antibody.</p

Linear range of the EGFR SOMAmer assay.

<p>The dilution series was prepared by diluting purified EGFR in 5-fold steps from 600 to 2.5 ng/mL. Each dilution was further diluted to 30 fold using SB17T buffer to mimic the serum dilution. The EGFR SOMAmer assay and quantitative PCR (qPCR) were done as described in Materials and Methods. Triplicate runs were performed per each dilution.</p

Determination of <i>cis</i> vs <i>trans</i> orientation using next-generation sequencing (NGS).

<p>The Integrative Genomics Viewer (IGV) graphic report is from a patient with 2 adjacent variants on a single DNA molecule visualized with NGS on the MiSeq/QSAP platform. The <i>cis</i> orientation is clearly visible, as each strand contains either both or neither of the mutations.</p

Alignment of a 64 bp-deletion (41246533-41246596del; c.952_1015del) in a validation sample.

<p>The Integrative Genomics Viewer (IGV) graphic reports show detection of the deletion using MiSeq platform with QSAP (panel A) but not the PGM platform with Torrent Suite variant calling software (panel B).</p