Purpose: Blood-based circulating-free (cf) tumor DNA may be an alternative to tissue-based EGFR mutation testing in NSCLC. This exploratory analysis compares matched tumor and blood samples from the FASTACT-2 study.Experimental Design: Patients were randomized to receive six cycles of gemcitabine/platinum plus sequential erlotinib or placebo. EGFR mutation testing was performed using the cobas tissue test and the cobas blood test (in development). Blood samples at baseline, cycle 3, and progression were assessed for blood test detection rate, sensitivity, and specificity; concordance with matched tumor analysis (n ¼ 238), and correlation with progression-free survival (PFS) and overall survival (OS).Results: Concordance between tissue and blood tests was 88%, with blood test sensitivity of 75% and a specificity of 96%. Median PFS was 13.1 versus 6.0 months for erlotinib and placebo, respectively, for those with baseline EGFR mut þ cfDNA [HR, 0.22; 95% confidence intervals (CI), 0.14-0.33, P < Conclusions: Blood-based EGFR mutation analysis is relatively sensitive and highly specific. Dynamic changes in cfDNA EGFR mutation status relative to baseline may predict clinical outcomes.

BackgroundThe feasibility of monitoring epidermal growth factor receptor (EGFR) mutations in plasma DNA from patients with advanced non–small cell lung cancer (NSCLC) during treatment with erlotinib and its relation to disease progression was investigated.MethodsThe amount of EGFR-mutant DNA was tested in plasma DNA from patients with advanced NSCLC with allele-specific polymerase chain reaction assays. Blood samples from 23 patients with adenocarcinoma of NSCLC that carried tyrosine kinase inhibitor-sensitizing EGFR mutations were taken immediately before treatment with erlotinib. Additional blood samples were taken at timed intervals until erlotinib treatment was withdrawn.ResultsThe amount of plasma DNA with sensitizing EGFR mutations was found to be reduced after the first cycle of erlotinib treatment in 22 of 23 patients (96%). No patients presented with the resistant T790M mutation in the pretreatment sample, but at the time of disease progression the mutation was detected in plasma from 9 patients (39%). The quantitative data from the current study demonstrated that when a T790M mutation emerged in the blood it was accompanied by an increase in the original sensitizing EGFR mutation. When T790M was detected, it was found to be present in all subsequent blood samples from that patient. Most interestingly, the results of the current study demonstrated that monitoring the EGFR mutations in the blood allows for the detection of the T790M mutation up to 344 days before disease progression is clinically evident (range, 15-344 days).ConclusionsThe results of the current study demonstrated that serial monitoring of EGFR mutations in plasma DNA is feasible and may allow for the early detection of resistance mutations. These results warrant further studies to explore the clinical usefulness of such analysis.

BackgroundLung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma samples with allele-specific PCR assays.MethodsPairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence of mutations in exons 18–21 of the EGFR gene, employing the cobas® EGFR Tissue Test and cobas® EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA).ResultsTest results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue was 179/196 (91%) (kappa value: 0.621).ConclusionMutational analysis of the EGFR gene in plasma samples is feasible with allele-specific PCR assays and represents a non-invasive supplement to biopsy analysis.Trial registrationM-20080012 from March 10, 2008 and reported to ClinicalTrials.gov: NCT00815971.

Accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) results in ER stress and lipid overload-induced ER stress has been implicated in the development of insulin resistance. Here, evidence is provided for a molecular link between hepatic apolipoprotein B100 (apoB100), induction of ER stress, and attenuated insulin signaling. First, in vivo upregulation of hepatic apoB100 by a lipogenic diet was found to be closely associated with ER stress and attenuated insulin signaling in the liver. Direct in vivo overexpression of human apoB100 in a mouse transgenic model further supported the link between excessive apoB100 expression and hepatic ER stress. Human apoB100 transgenic mice exhibited hypertriglyceridemia and hyperglycemia. In vitro, accumulation of cellular apoB100 by free fatty acid ( P erturbations in lipid metabolism and lipid signaling underlie the pathogenesis of a cluster of chronic metabolic diseases, including insulin resistance, type 2 diabetes, fatty liver disease, and atherosclerosis.The atherogenic dyslipidemia commonly associated with insulin-resistant states consists of hypertriglyceridemia, a high level of very low-density lipoprotein (VLDL), a low level of high-density lipoprotein (HDL) cholesterol, 1 and elevated small, low-density lipoprotein (LDL). High dietary fat intake has been shown to induce insulin resistance (IR) and the lipid synthetic rate by way of increased free fatty acid (FFA) flux, as well as assembly and secretion of both VLDL-apolipoprotein B (apoB) and triglyceride (TG) in animal and human models. 2,3 The association of IR and increased VLDL secretion is thought to be derived from increased FFA delivery to the liver resulting from increased lipolysis in adipose tissue, a phenomenon accompanied by increased hepatic lipogenesis, increased hepatic microsomal triglyceride transfer protein (MTP) level and activity, and loss of apoB regulation by insulin.Recent studies implicate hepatic ER stress as a central abnormality linking obesity, hepatic IR, and hepatic steatosis. 4,5 Ozcan et al. 4 have demonstrated that obesityinduced ER stress leads to hepatic IR by activating c-Jun N-terminal kinase (JNK) through inositol-requiring enzyme-1 (IRE-1), with subsequent inhibition of insulin receptor signaling. ER stress has also been linked to increased hepatic lipogenesis. 6,7 However, loss of MTP activity in mouse hepatocytes by either gene disruption or