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 Additional blood samples were taken at timed intervals until erlotinib treatment was withdrawn.ResultsThe amount of plasma DNA with sensitizing EGFR mutations was found to be reduced after the first cycle of erlotinib treatment in 22 of 23 patients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

Objective-To investigate the role of glucosamine-mediated endoplasmic reticulum (ER) stress and Grp78 (BiP) in the intracellular degradation of apolipoprotein B100 (apoB100) in cultured hepatocytes. Methods and Results-Glucosamine treatment (2.5 to 10 mmol/L) of HepG2 cells increased levels of the ER chaperones, 78-kDa glucose-regulated protein (Grp78) and Grp94, in a dose-dependent manner and led to significant decreases in both cellular and secreted apoB100 by up to 97% (PϽ0.01). In contrast, no changes were observed in ER resident (ER60, PTP-1B) or secretory (albumin, apoE) control proteins. Glucosamine-induced apoB degradation was similarly observed in primary hamster hepatocytes and McA-RH7777 cells. Glucosamine treatment led to reduced tranlocational efficiency of apoB100 in the ER and enhanced its ubiquitination and proteasomal degradation. Adenoviral overexpression of Grp78 also led to significantly decreased levels of newly synthesized apoB100 in a dose-dependent manner (PϽ0.01). Grp78-induced downregulation of apoB100 was sensitive to inhibition by the proteasome inhibitor, lactacystin, but not lysosomal protease inhibitors, E64 and leupeptin, suggesting that overexpression of Grp78 selectively induced proteasomal degradation of apoB100. Conclusion-These findings suggest that binding and retention by Grp78 may play a critical role in proteasomal targetingand the ER quality-control of misfolded apoB. Key Words: apolipoprotein B Ⅲ degradation Ⅲ glucosamine Ⅲ Grp78 Ⅲ proteasome H epatic biogenesis of apoB is a complex process involving regulation by multiple post-transcriptional control mechanisms. 1-3 Intracellular availability of core lipoprotein lipid substrates, particularly triglyceride, appears to dictate the intracellular fate of newly synthesized apoB protein. 4 In the absence of lipid, a significant proportion of newly synthesized apoB100 is degraded in cultured hepatoma cells, 5 as well as in primary hepatocytes from hamsters, 6 rats, 7 and rabbits. 8 The bulk of apoB degradation appears to be mediated by the ubiquitin-proteasome degradative system. 9 High exogenous free fatty acid flux (particularly in cultured hepatocytes) or SREBP1-mediated de novo lipogenesis appear to protect apoB from proteasomal degradation. 10 Ubiquitination and proteasomal degradation of apoB begins cotranslationally and involves the interaction of misfolded apoB with cytosolic chaperones, Hsp70 and Hsp90. 11 Association with these cytosolic chaperones may be important in unfolding and subsequent targeting of the apoB polypeptide to the ubiquitin-proteasome pathway. 11 Mechanisms that target misfolded apoB or luminal lipoprotein-associated apoB to either proteasomal or nonproteasomal degradative pathways are currently unknown. Ample evidence is available showing the association of newly synthesized apoB polypeptide with endoplasmic reticulum (ER) chaperones. Several laboratories have observed that apoB100 is tightly associated with the 78-kDa glucoseregulated protein/immunoglobulin heavy chain-binding protein (Grp78/BiP...

Objective-Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway. Methods and Results-HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins. Conclusion-Inhibition

HepaticapolipoproteinB(apoB)lipoproteinproductionismetabolicallyregulatedviathephosphoinositide 3-kinase cascade; however, the role of the key negative regulator of this pathway, the tumor suppressor phosphatase with tensin homology (PTEN), is unknown. Here, we demonstrate that hepatic protein levels of apoB100 and microsomal triglyceride transfer protein (MTP) are significantly down-regulated (73% and 36%, respectively) in the liver of PTEN liver-specific knockout (KO) mice, and this is accompanied by increased triglyceride (TG) accumulation and lipogenic gene expression, and reduced hepatic apoB secretion in freshly isolated hepatocytes. MTP protein mass and lipid transfer activity were also significantly reduced in liver of PTEN KO mice. Overexpression of the dominant negative mutant PTEN C/S124 (adenovirus expressing PTEN C/S mutant [AdPTENC/S]) possessing constitutive phospoinositide 3-kinase activity in HepG2 cells led to significant reductions in both secreted apoB100 and cellular MTP mass (76% and 34%, respectively), and increased messenger RNA (mRNA) levels of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Reduced apoB100 secretion induced by AdPTENC/S was associated with increased degradation of newly-synthesized cellular apoB100, in a lactacystin-sensitive manner, suggesting enhanced proteasomal degradation. AdPTENC/S also reduced apoB-lipoprotein production in McA-RH7777 and primary hamster hepatocytes. Our findings suggest a link between PTEN expression and hepatic production of apoB-containing lipoproteins. We postulate that perturbations in PTEN not only may influence hepatic insulin signaling and hepatic lipogenesis, but also may alter hepatic apoB-lipoprotein production and the MTP stability. On loss of PTEN activity, increased lipid substrate availability in the face of reduced hepatic lipoprotein production capacity can rapidly lead to hepatosteatosis and fatty liver.

AimTo conduct a methods correlation study of three different assays for the detection of mutations at EGFR gene in human formalin-fixed paraffin-embedded tumour (FFPET) specimens of non-small cell lung carcinomas (NSCLC).MethodsWe conducted a 2-site method comparison study of two european conformity (CE) in vitro diagnostic (IVD)-marked assays, the cobas EGFR Mutation Test and the Therascreen EGFR29 Mutation Kit, and 2× bidirectional Sanger sequencing. We blind-tested 124 NSCLC FFPET specimens with all three methods; the cobas test was performed at both sites. Positive (PPA) and negative percent agreements (NPA) were determined for the cobas test versus each of the other two methods. Specimens yielding discordant test results between methods were further tested using quantitative massively parallel pyrosequencing (MPP).ResultsPPA between cobas and Sanger was 98.8%; NPA was 79.3%. Overall there were seven discordant results. MPP confirmed an exon 19 deletion in two cases and L858R mutation in four cases. PPA between cobas and Therascreen was 98.9% and NPA was 100%. There was one discordant result. Reproducibility of the cobas test between the two sites was 99.2%.ConclusionsThe invalid rates for the cobas test and Therascreen were lower than Sanger sequencing. The cobas and Therascreen assays showed a high degree of concordance, and both were more sensitive for the detection of exon 19 deletion and L858R mutations than Sanger. The cobas test was highly reproducible between the two testing sites, used the least amount of DNA input and was the only test with automated results reporting.