A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification.

BACKGROUND: Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. Quantification of recovery efficiency after these steps can facilitate quality assurance and improve reliability when comparing serial samples. METHODS: We developed an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulfite modification (CEREBIS) after cfDNA extraction and/or subsequent bisulfite modification from plasma. The strategic placement of cytosine bases in the 180 bp CEREBIS enabled PCR amplification of the construct by a single primer set both after plasma DNA extraction and following subsequent bisulfite modification. RESULTS: Plasma samples derived from 8 organ transplant donors and 6 serial plasma samples derived from a liver transplant recipient were spiked with a known number of copies of CEREBIS. Recovery of CEREBIS after cfDNA extraction and bisulfite modification was quantified with high analytical accuracy by droplet digital PCR. The use of CEREBIS and quantification of its recovery was useful in identifying problematic extractions. Furthermore, its use was shown to be invaluable towards improving the reliability of the analysis of serial samples. CONCLUSIONS: CEREBIS can be used as a spike-in control to address the preanalytical variable of recovery efficiency both after cfDNA extraction from plasma and following bisulfite modification. Our approach can be readily implemented and its application may have significant benefits, especially in settings where longitudinal quantification of cfDNA for disease monitoring is necessary.

Cerebrospinal fluid liquid biopsy for detecting somatic mosaicism in brain.

Brain somatic mutations are an increasingly recognized cause of epilepsy, brain malformations and autism spectrum disorders and may be a hidden cause of other neurodevelopmental and neurodegenerative disorders. At present, brain mosaicism can be detected only in the rare situations of autopsy or brain biopsy. Liquid biopsy using cell-free DNA derived from cerebrospinal fluid has detected somatic mutations in malignant brain tumours. Here, we asked if cerebrospinal fluid liquid biopsy can be used to detect somatic mosaicism in non-malignant brain diseases. First, we reliably quantified cerebrospinal fluid cell-free DNA in 28 patients with focal epilepsy and 28 controls using droplet digital PCR. Then, in three patients we identified somatic mutations in cerebrospinal fluid: in one patient with subcortical band heterotopia the LIS1 p. Lys64* variant at 9.4% frequency; in a second patient with focal cortical dysplasia the TSC1 p. Phe581His*6 variant at 7.8% frequency; and in a third patient with ganglioglioma the BRAF p. Val600Glu variant at 3.2% frequency. To determine if cerebrospinal fluid cell-free DNA was brain-derived, whole-genome bisulphite sequencing was performed and brain-specific DNA methylation patterns were found to be significantly enriched (P = 0.03). Our proof of principle study shows that cerebrospinal fluid liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable.

NTRK and ALK rearrangements in malignant pleural mesothelioma, pulmonary neuroendocrine tumours and non-small cell lung cancer.

OBJECTIVES: Gene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK have been identified in many types of cancer, including non-small cell lung cancer (NSCLC). Data in malignant pleural mesothelioma (MPM), lung neuroendocrine tumors (NETs) and small-cell lung cancer (SCLC) are lacking. Given the activity of NTRK, ROS-1 and ALK inhibitors in tumors harboring gene fusions, we sought to explore such rearrangements in these less common tumors in addition to NSCLC. METHODS: Archival tumor tissue from patients with MPM, lung NETs, SCLC and NSCLC were used to create tissue microarrays. Immunohistochemistry (IHC) was performed using a cocktail of antibodies against TRK, ROS1 and ALK. IHC positive samples underwent RNA sequencing using the ArcherDX FusionPlex CTL diagnostic assay. Clinical data were obtained through retrospective chart review. RESULTS: We performed IHC on 1116 samples: 335 MPMs, 522 NSCLCs, 105 SCLCs and 154 lung NETs. There were 23 IHC positive cases (2.1%) including eight MPMs (2.4%), eight NETs (5.2%), five SCLC (4.8%) and two NSCLC (0.4%). The following fusions were detected: one MPM with an NTRK ex10-TPM3 ex8, another MPM with an ALK ex20-EML4ex13, one lung intermediate-grade NET (atypical carcinoid) with an ALK ex20-EML4 ex6/intron6, and two NSCLCs with an ALK ex20-EML4 ex6/intron6 rearrangement. None of the patients received targeted treatment. CONCLUSIONS: To our knowledge, we report for the first time NTRK and ALK rearrangements in a small subset of MPM. An ALK rearrangement was also detected in lung intermediate-grade NET (or atypical carcinoid). Our data suggest that IHC could be a useful screening test in such patients to ensure that all therapeutic strategies including targeted therapy are utilized.

Publisher Correction: A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standard dose osimertinib for erlotinib refractory T790M-negative EGFR-mutant non-small cell lung cancer with leptomeningeal disease.

BACKGROUND: Leptomeningeal spread in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations who experience disease progression on TKIs portends a poor prognosis. Mutation profiling of tumour DNA in cerebrospinal fluid (CSF) samples can be used to determine the presence of the EGFR T790M resistance mutation, indicating that osimertinib, a CNS-penetrating 3rd generation TKI may be efficacious. METHODS: Eight patients on EGFR TKIs who progressed with cytology-proven leptomeningeal disease at our institution were studied. EGFR mutations were profiled in CSF using droplet digital PCR (ddPCR) and compared to matched plasma samples. Clinical characteristics and survival outcomes on subsequent therapies tailored to ddPCR analysis were reported. RESULTS: None of the four patients who developed leptomeningeal disease while receiving 1st generation EGFR TKIs developed the EGFR T790M mutation in CSF. One patient who did not have extra-cranial disease and was EGFR T790M-negative in both plasma and CSF was nevertheless treated with standard-dose osimertinib, and achieved a rapid and durable response lasting 9 months to date. Three patients developed leptomeningeal disease on osimertinib, with one patient developing the EGFR C797S mutation in a cis-allelic conformation with the EGFR T790M mutation in plasma. CONCLUSIONS: Standard-dose osimertinib resulted in a clinically meaningful response in a patient with EGFR T790M-negative 1st generation EGFR TKI refractory leptomeningeal disease. Next generation sequencing and ddPCR has a role at identifying the C797S mutation and its allelic conformation with the T790M mutation with clinical implications.

The Measurement of Donor-Specific Cell-Free DNA Identifies Recipients With Biopsy-Proven Acute Rejection Requiring Treatment After Liver Transplantation.

BACKGROUND: Assessment of donor-specific cell-free DNA (dscfDNA) in the recipient is emerging as a noninvasive biomarker of organ rejection after transplantation. We previously developed a digital polymerase chain reaction (PCR)-based approach that readily measures dscfDNA within clinically relevant turnaround times. Using this approach, we characterized the dynamics and evaluated the clinical utility of dscfDNA after liver transplantation (LT). METHODS: Deletion/insertion polymorphisms were used to distinguish donor-specific DNA from recipient-specific DNA. Posttransplant dscfDNA was measured in the plasma of the recipients. In the longitudinal cohort, dscfDNA was serially measured at days 3, 7, 14, 28, and 42 in 20 recipients. In the cross-sectional cohort, dscfDNA was measured in 4 clinically stable recipients (>1-y posttransplant) and 16 recipients (>1-mo posttransplant) who were undergoing liver biopsies. RESULTS: Recipients who underwent LT without complications demonstrated an exponential decline in dscfDNA. Median levels at days 3, 7, 14, 28, and 42 were 1936, 1015, 247, 90, and 66 copies/mL, respectively. dscfDNA was higher in recipients with treated biopsy-proven acute rejection (tBPAR) when compared to those without. The area under the receiver operator characteristic curve of dscfDNA was higher than that of routine liver function tests for tBPAR (dscfDNA: 98.8% with 95% confidence interval, 95.8%-100%; alanine aminotransferase: 85.7%; alkaline phosphatase: 66.4%; gamma-glutamyl transferase: 80.1%; and bilirubin: 35.4%). CONCLUSIONS: dscfDNA as measured by probe-free droplet digital PCR methodology was reflective of organ health after LT. Our findings demonstrate the potential utility of dscfDNA as a diagnostic tool of tBPAR.

A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma.

Low-passage, serum-free cell lines cultured from patient tumour tissue are the gold-standard for preclinical studies and cellular investigations of glioblastoma (GBM) biology, yet entrenched, poorly-representative cell line models are still widely used, compromising the significance of much GBM research. We submit that greater adoption of these critical resources will be promoted by the provision of a suitably-sized, meaningfully-described reference collection along with appropriate tools for working with them. Consequently, we present a curated panel of 12 readily-usable, genetically-diverse, tumourigenic, patient-derived, low-passage, serum-free cell lines representing the spectrum of molecular subtypes of IDH-wildtype GBM along with their detailed phenotypic characterisation plus a bespoke set of lentiviral plasmids for bioluminescent/fluorescent labelling, gene expression and CRISPR/Cas9-mediated gene inactivation. The cell lines and all accompanying data are readily-accessible via a single website, Q-Cell (qimrberghofer.edu.au/q-cell/) and all plasmids are available from Addgene. These resources should prove valuable to investigators seeking readily-usable, well-characterised, clinically-relevant, gold-standard models of GBM.

Somatic GNAQ mutation in the forme fruste of Sturge-Weber syndrome.

OBJECTIVE: To determine whether the GNAQ R183Q mutation is present in the forme fruste cases of Sturge-Weber syndrome (SWS) to establish a definitive molecular diagnosis. METHODS: We used sensitive droplet digital PCR (ddPCR) to detect and quantify the GNAQ mutation in tissues from epilepsy surgery in 4 patients with leptomeningeal angiomatosis; none had ocular or cutaneous manifestations. RESULTS: Low levels of the GNAQ mutation were detected in the brain tissue of all 4 cases-ranging from 0.42% to 7.1% frequency-but not in blood-derived DNA. Molecular evaluation confirmed the diagnosis in 1 case in which the radiologic and pathologic data were equivocal. CONCLUSIONS: We detected the mutation at low levels, consistent with mosaicism in the brain or skin (1.0%-18.1%) of classic cases. Our data confirm that the forme fruste is part of the spectrum of SWS, with the same molecular mechanism as the classic disease and that ddPCR is helpful where conventional diagnosis is uncertain.

GRIDSS: sensitive and specific genomic rearrangement detection using positional de Bruijn graph assembly.

The identification of genomic rearrangements with high sensitivity and specificity using massively parallel sequencing remains a major challenge, particularly in precision medicine and cancer research. Here, we describe a new method for detecting rearrangements, GRIDSS (Genome Rearrangement IDentification Software Suite). GRIDSS is a multithreaded structural variant (SV) caller that performs efficient genome-wide break-end assembly prior to variant calling using a novel positional de Bruijn graph-based assembler. By combining assembly, split read, and read pair evidence using a probabilistic scoring, GRIDSS achieves high sensitivity and specificity on simulated, cell line, and patient tumor data, recently winning SV subchallenge #5 of the ICGC-TCGA DREAM8.5 Somatic Mutation Calling Challenge. On human cell line data, GRIDSS halves the false discovery rate compared to other recent methods while matching or exceeding their sensitivity. GRIDSS identifies nontemplate sequence insertions, microhomologies, and large imperfect homologies, estimates a quality score for each breakpoint, stratifies calls into high or low confidence, and supports multisample analysis.

EGFR and KRAS mutations do not enrich for the activation of IL-6, JAK1 or phosphorylated STAT3 in resected lung adenocarcinoma.

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) against EGFR mutant lung adenocarcinoma develops after a median of nine to thirteen months. Upregulation of the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway may be a potential source of resistance to EGFR TKIs. We undertook a detailed assessment of the IL-6/JAK1/phosphorylated STAT3 (pSTAT3) pathway in resected lung adenocarcinoma specimens, with special interest in whether the presence of an EGFR mutation enriched for pSTAT3 positivity. Tumours from 143 patients with resected lung adenocarcinoma were assessed. EGFR and KRAS mutation status were scanned for with high-resolution melting and confirmed by polymerase chain reaction. Immunohistochemisty (IHC) was performed for IL-6, gp130, JAK1 and pSTAT3. Two methods for assigning IHC positivity were assessed (the presence of any positivity, and the presence of positivity at an H score >40). We found statistically significant associations between IL-6, JAK1 and pSTAT3 measured by IHC, consistent with the activation of the pathway in clinical specimens. No relationship was demonstrated between members of this pathway and oncogenic mutations in EGFR or KRAS. However, a proportion of tumours with EGFR mutations showed staining for IL-6, JAK1 and pSTAT3. No correlations with clinicopathologic features or survival outcomes were found for IL-6, JAK1 or pSTAT3 staining. The presence of EGFR or KRAS mutations did not enrich for the activation of IL-6, JAK1 or pSTAT3. pSTAT3 may still play a role in resistance to EGFR TKIs in clinical practice.

De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features.

Combination Osimertinib and Gefitinib in C797S and T790M EGFR-Mutated Non-Small Cell Lung Cancer.

INTRODUCTION: Osimertinib, a third-generation EGFR tyrosine kinase inhibitor has demonstrated efficacy in tumors harboring the EGFR T790M resistance mutation. Inevitably, resistance to third-generation inhibitors results in disease progression, with the EGFR C797S mutation being one of several resistance pathways identified to date. On the basis of preclinical data, we report what is the first known case of a patient harboring the T790M and C797S mutations in trans treated with combination gefitinib and osimertinib. METHODS: On development of progressive disease after multiple therapies, the patient's plasma was sequenced using the Oncomine Lung cfDNA Assay (Thermo Fisher Scientific, Waltham, MA). Subsequent monitoring of circulating tumor DNA in plasma was performed by droplet digital polymerase chain reaction. RESULTS: Sequencing showed that the T790M and C797S mutations were in trans. Within 2 weeks of commencement of combination therapy, rapid clinical improvement occurred. Accompanying this, a rapid decline in the C797S mutation subclone in plasma was detected. However, the levels of the EGFR exon 19 deletion driver mutation and the T790M resistance mutation in the circulating tumor DNA continued to rise and the patient died from progressive disease 6 weeks after commencement of combination therapy. There were no adverse events seen with the combination therapy. CONCLUSION: This is, to the best of our knowledge, the first reported case of combination EGFR tyrosine kinase inhibitor therapy tailored to the allelic conformation of T790M and C797S mutation that resulted in brief clinical improvement without toxicity.

Reducing Artifactual EGFR T790M Mutations in DNA from Formalin-Fixed Paraffin-Embedded Tissue by Use of Thymine-DNA Glycosylase.

BACKGROUND: False-positive EGFR T790M mutations have been reported in formalin-fixed lung tumors, but the cause of the false positives has not been identified. The T790M mutation results from a C>T change at the cytosine of a CpG dinucleotide. The presence or absence of methylation at this cytosine has different consequences following deamination, resulting in a thymine or uracil, respectively, both of which however result in an artifactual change. Uracil-DNA glycosylase (UDG) can be used to eliminate DNA templates with uracil residues but is not active against artifactual thymines. We therefore investigated the use of thymine-DNA glycosylase (TDG) to reduce artifactual T790M mutations. METHODS: Formalin-fixed normal lung tissues and lung squamous cell carcinomas were tested to measure the frequency of false-positive EGFR mutations by use of droplet digital PCR before and after treatment with either UDG or TDG. Methylation at the cytosine at EGFR T790 was assessed by pyrosequencing and by analysis of public databases. RESULTS: Artifactual EGFR T790M mutations were detected in all of the archival formalin-fixed normal lung and lung squamous cell carcinomas at mutant allele frequencies of 1% or lower. The cytosine at EGFR T790 showed high levels of methylation in all lung cancer samples and normal tissues. Pretreatment of the formalin-fixed DNA with either UDG or TDG reduced the false EGFR T790M mutations, but a greater reduction was seen with the TDG treatment. CONCLUSIONS: Both U:G and T:G lesions in formalin-fixed tissue are sources of false-positive EGFR T790M mutations. This is the first report of the use of TDG to reduce sequence artifacts in formalin-fixed DNA and is applicable to the accurate detection of mutations arising at methylated cytosines.

Reduced abundance of the E3 ubiquitin ligase E6AP contributes to decreased expression of the INK4/ARF locus in non-small cell lung cancer.

The tumor suppressor p16INK4a, one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15INK4b, p16INK4a, and p19ARF) was decreased in E6AP-/- mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16INK4a-low protein abundance profile correlated with low methylation of the gene encoding p16INK4a (CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC.

Digital PCR of Genomic Rearrangements for Monitoring Circulating Tumour DNA.

Identifying circulating tumour DNA (ctDNA) for monitoring of cancer therapy is dependent on the development of readily designed, sensitive cancer-specific DNA markers. Genomic rearrangements that are present in the vast majority of cancers provide such markers.Tumour DNA isolated from two fresh-frozen lung tumours underwent whole genome sequencing. Genomic rearrangements were detected using a new computational algorithm, GRIDSS. Four genomic rearrangements from each tumour were chosen for further study using rearrangement-specific primers. Six of the eight rearrangements tested were identified as tumour-specific, the remaining two were present in the germline. ctDNA was quantified using digital PCR for the tumour genomic rearrangements in patient blood. Interestingly, one of the patients had no detectable ctDNA either prior to or post surgery although the rearrangements were readily detectable in the tumour DNA.This study demonstrates the feasibility of using digital PCR based on genomic rearrangements for the monitoring of minimal residual disease. In addition, whole genome sequencing provided further information enabling therapeutic choices including the identification of a cryptic EGFR exon 19 deletion in one patient and the identification of a high somatic mutation load in the other patient. This approach can be used as a model for all cancers with rearranged genomes.

Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment.

AZD9291, a T790M specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has demonstrated impressive response rates in tumours harbouring the EGFR T790M resistance mutation. Emergence of resistance to AZD9291 has been shown to occur through several different mechanisms including the development of new mutations (e.g. C797S) in the EGFR tyrosine kinase domain. We studied two patients with paired tumour biopsies and blood samples pre- and post-progression on AZD9291 to explore possible resistance mechanisms. Pre- and Post-AZD9291 tumour biopsies as well as serial plasma samples were collected from two patients on the AURA clinical study (AZD9291 First Time in Patients Ascending Dose study). Droplet digital PCR (ddPCR) assays were used to quantify T790M, the driver EGFR mutation, and the C797S mutation in genomic DNA from paired tumour biopsies and plasma cell-free DNA. In the first patient, both EGFR T790M and L858R became undetectable in the plasma within 1 month after treatment with AZD9291. However, the T790M and the original L858R mutation re-emerged with radiologically confirmed resistance to AZD9291. In patient two, the levels of T790M were undetectable at the time of radiological resistance to AZD9291 but increasing levels of the original EGFR exon 19 deletion was detected. MET amplification was detected in a biopsy performed on progression. The EGFR C797S mutation was not detected in either patient at the time of relapse. ddPCR of cell free DNA enables real time monitoring of patients on 3rd generation TKIs. As resistance mechanisms are variable, monitoring levels of the initial activating EGFR mutation may facilitate more reliable detection of progression.

Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization.

BACKGROUND: Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalin-fixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented and also contains DNA lesions that are the sources of sequence artifacts. Sequence artifacts can be difficult to distinguish from true mutations, especially in the context of tumor heterogeneity, and are an increasing interpretive problem in this era of massively parallel sequencing. Understanding of the sources of sequence artifacts in FFPE tissues and implementation of preventative strategies are critical to improve the accurate detection of actionable mutations. CONTENT: This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis. In particular, fragmentation, base modification (including uracil and thymine deriving from cytosine deamination), and abasic sites are discussed as indirect or direct sources of sequence artifacts. We discuss strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts. These strategies are applicable for the detection of actionable mutations in both single amplicon and massively parallel amplicon sequencing approaches. SUMMARY: Because FFPE tissues are usually the only available material for DNA analysis, it is important to maximize the accurate informational content from FFPE DNA. Careful consideration of each step in the work flow is needed to minimize sequence artifacts. In addition, validation of actionable mutations either by appropriate experimental design or by orthogonal methods should be considered.

Multicenter randomized, open-label phase II trial of sequential erlotinib and gemcitabine compared with gemcitabine monotherapy as first-line therapy in elderly or ECOG PS two patients with advanced NSCLC.

AIM: The potential beneficial interaction between erlotinib and chemotherapy may require sequencing or pharmacodynamic separation. The aim of this study was to evaluate the efficacy and tolerance of sequential erlotinib and gemcitabine versus gemcitabine monotherapy as first-line therapy in elderly or ECOG PS-2 patients with advanced non-small cell lung carcinoma. METHODS: The primary objective of this multicenter randomized Phase II study was progression-free survival (PFS). Secondary objectives were overall response rate (ORR), disease control rate, response duration, overall survival and safety. Patients were randomized to either gemcitabine (1250 mg/m2 Day 1, 8 q28 days) followed by erlotinib (150 mg/day on day 15 through day 28), (EG-arm), or gemcitabine monotherapy (1000 mg/m2 Days 1, 8, 15 q28 days), (G-arm) for up to six cycles. RESULTS: Fifty-four patients were recruited, 28 G-arm and 26 EG-arm. Overall, efficacy results were not significantly different between study arms. Median PFS and ORR for the G- versus EG-arms were 8.0 versus 10.3 weeks (hazard ratio 1.3; 95% confidence interval [0.63;2.68]; P=0.48) and 7.1 versus 3.8 percent respectively (difference -3.30; 95% confidence interval [-17.5;10.9]). The majority of adverse events (AEs) in both arms were Grade 1-2. The commonest AEs recorded in the EG- and G-arms were rash-like events (65 percent) and nausea (42 percent) respectively. Four patients (17 percent) in EG-arm and five (16 percent) in G-arm experienced at least one treatment-related serious AE. CONCLUSIONS: In this study, patients with non-small cell lung carcinoma at ECOG PS-2 or aged ≥70 years derived no efficacy advantage from sequential erlotinib in combination with gemcitabine relative to gemcitabine alone. No unexpected safety findings were noted.