BackgroundResponse 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).MethodsThis 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.ResultsThis 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.ConclusionsThis 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.

Background: Although the ingestion of chilli has been associated with gastroesophageal reflux (GER) symptoms, there are no studies that have explored the effect of a chronic ingestion of different kinds of chilli with a variable content of capsaicin as a cause of GER. Methods: The effect of chilli on esophageal 24-hour pH monitoring was studied in 12 healthy subjects without GER symptoms before and after of ingestion one of two kinds of chilli. Patients were randomized to ingest 3 g daily of cascabel chilli (Capsicum annum coraciforme containing 880 ppmof capsaicin) or ancho chilli (Capsicum annum grossum containing 488 ppm of capsaicin). Results: After chilli ingestion, the Johnson De Meester Index (JDI) increased significantly [basal: 7 (1–14), after chilli: 13 (2–69), p = 0.0047]. When considering both kinds of chilli separately, the JDI varied, although nonsignificantly, with the ancho chilli [basal: 3 (1–8), after chilli: 10 (2–69), p = 0.11], and significantly with the cascabel chilli [basal: 10 (5–14), after chilli: 18 (2–44), p = 0.028]. Conclusion: Our results suggest that the chronic ingestion of chilli induces GER, and that the magnitude of the induced reflux seems to be related to the kind of chilli.

2587 Background: EGFR is an oncogene product whose extracellular domain (ECD) can be either up-regulated or down-regulated in serum of patients with various cancers. In colon cancer patients with positive tissue EGFR expression, 2 EGFR ECD-targeting monoclonal antibody drugs, cetuximab (Erbitux) and panitumumab (Vectibix), are widely used chemotherapeutic agents. Patient responses to these drugs vary, and the mechanism of this variability remains unelucidated. Methods: A Slow Off-Rate Modified Aptamer (SOMAmer) targeting the EGFR ECD was selected via Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Using this SOMAmer as a capturing reagent and based on published studies ( Gold et al. PLoS ONE; Dec 7, 2010:10.1371/journal.pone.0015004), we developed a quantitative serum EGFR assay to reliably quantify EGFR in serum. Results: Recovery tests using various amounts of purified EGFR spiked into serum demonstrated a full level of EGFR. Intra and inter assay variability were tested and showed minimum variability. The detection range is 0.95 ng/ml to 600 ng/ml. Interestingly, when serum samples from patients taking cetuximab or panitumumab at the time of blood collection were tested, we observed markedly lower levels of EGFR-captured SOMAmer. ELISA assays from 2 different vendors showed normal to high levels of EGFR in these samples. We further showed that pretreatment of normal serum with either cetuximab or panitumumab can dose-dependently reduce the EGFR SOMAmer signal. The data suggest that our EGFR SOMAmer assay detects serum EGFR molecules that are unbound by cetuximab or panitumumab. Some treated patient samples had more drastic reductions in circulating serum EGFR than others. Conclusions: Various assay development parameters such as accuracy, detection range, and intra and inter assay variability showed that a SOMAmer-based assay detecting serum EGFR can be used in a clinical setting. Our data suggest that this assay can accurately measure drug-unbound EGFR in patients, which may serve as a surrogate drug efficacy indicator, and this may help physicians to adjust the drug dosage. Further studies will be necessary to investigate this possibility.

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7601 Background: Support for low-dose helical computed tomography (CT) screening for lung cancer in a high risk population has emerged from the National Lung Screening Trial (NLST). However, the low specificity of CT raises concerns regarding the cost and potential morbidity associated with resection of benign nodules. Non-invasive lung cancer biomarkers may serve as a useful complement to imaging, providing a simple means to further clarify the diagnosis of suspicious pulmonary nodules. SOMAmer-based chip technology was previously used to identify a panel of serological biomarkers capable of accurately classifying NSCLC. Here we assess the analytical and clinical performance of an automated assay that uses SOMAmer reagents and qPCR to simultaneously quantify a subset of these markers (n=12). Methods: Biomarker levels were measured in sera from 43 subjects with stage I-III NSCLC and 63 long-term smoker controls. qPCR results were analyzed by relative (ΔΔCT) quantitation, which uses calibrator serum and a set of normalizers. Linear mixed-effects models were fit to identify key sources of analytical variability (variance components), including plate-to-plate, within-sample, and between sample effects. Clinical performance for distinguishing NSCLC from control sera was established by a random forest predictive model. Results: Median within-sample %CV for the 12 markers was 6.7%. Variance components analyses suggest that, on average, 5.7% of the variance for a given biomarker was due to within-sample effects, 5.2% to plate-to-plate effects, and 86.2% to between-sample effects. A 10-marker random forest model exhibited an AUC [95% CI] of 0.915 [0.905, 0.924], classifying NSCLC from control sera with 79% sensitivity and 90% specificity. Conclusions: We have developed a highly reproducible automated assay designed for the clinical laboratory. Combining SOMAmer-based protein capture and qPCR-based quantification, the assay monitors the levels of 12 potential lung cancer markers. An algorithm-based model incorporating 10 of these markers showed good accuracy for distinguishing NSCLC from control sera. Further studies are warranted to evaluate the performance of the test in classifying pulmonary nodules.