Supplemental Appendix
Methods: Trial duration and cut-off dates
ATLAS was conducted at 187 sites between January 10, 2006 and June 19, 2009. Two interim
efficacy analyses (at around 33% and 67% of progression-free survival [PFS] events) were
prospectively planned. At the second interim analysis (July 18, 2008; 69% of PFS events) the study
had met its primary endpoint, so this was considered the primary analysis. An additional exploratory
analysis of efficacy and safety was conducted on data collected up to and including January 28, 2009,
although additional updates for all efficacy analyses except overall survival (OS) were not conducted
on data collected after July 18, 2008. An additional OS data sweep was performed (data cut-off date:
June 19, 2009).
Methods: Patient Eligibility
Eligible patients were aged ≥ 18 years with histologically or cytologically confirmed advanced nonsmall-cell lung cancer (NSCLC) (stage IIIB with malignant pleural effusion or stage IV) or recurrent
metastatic disease, and an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of
0 or 1. Patients with a history of brain metastases were eligible for participation if their brain
metastases had been treated and they did not have an ongoing requirement for dexamethasone.
Methods: Randomization and Masking
During the initial chemotherapy phase, patients received bevacizumab and one of the protocolspecified chemotherapy doublets (investigator’s choice) for four cycles in an unblinded fashion. In the
post-chemotherapy phase, patients were randomized (1:1) centrally via an Interactive Voice Response
System, and stratified by initial chemotherapy choice, smoking status, and ECOG PS. The study
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sponsor, study investigators, and patients were blinded to treatment. Unblinding was allowed at any
time for safety reasons, or at disease progression.
Methods: A note on the CONSORT diagram
A total of 1145 patients were enrolled in the chemotherapy phase (Figure 1) from January 2006 to
June 2009. At the July 18, 2008 data cut-off, 743 patients (64.9%) had been randomized into the postchemotherapy phase (intent-to-treat [ITT] population). The collection of samples for biomarker
evaluation was optional. At the time of the first data cut-off (July 18, 2008), 177 patients in the
bevacizumab plus placebo arm and 190 patients in the bevacizumab plus erlotinib arm were evaluable
for biomarker status. As recruitment was not halted until after the Data and Safety Monitoring Board
review took place, by the time of the June 19, 2009 data cut-off an additional 25 patients had been
randomized; the resulting ITT population comprised 768 patients (67.1%). At the June 19, 2009 data
cut-off, 181 patients in the bevacizumab plus placebo arm and 191 patients in the bevacizumab plus
erlotinib arm were evaluable for biomarker status.
Methods: Biomarker Collection and Analysis
Where tumor content was > 40% of the sample, manual macrodissection was used. Where tumor
content was ≤ 40% of the sample, laser capture microdissection was employed (Arcturus PixCell II).
Analyses of epidermal growth factor receptor (EGFR) mutations in exons 18–21 and KRAS mutations
in exons 2 or 3 were performed using Somatic Mutation Scanning by Surveyor® Endonuclease
Digestion and Analysis on WAVE HS (Transgenomics Inc, Omaha, NB).1 Amounts of amplifiable
DNA were determined using quantitative polymerase chain reaction. Amplicons that were shown to
contain variants by Surveyor® analysis were examined by bi-directional sequencing to confirm and
characterize the genetic variation. EGFR gene copy number was measured by fluorescence in-situ
hybridization (FISH) using the PathVysion® kit from Abbott-Vysis (Des Plaines, IL). High gene copy
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number by FISH was defined as high polysomy (≥ 4 gene copies in ≥ 40% of cells) or amplification
(gene/chromosome ratio ≥ 2 or ≥ 15 gene copies in ≥ 10% of cells); clusters were not counted.2 EGFR
protein expression was measured by immunohistochemistry (IHC) using the PharmDxTM kit from
DAKO (Glostrup, Denmark); positive EGFR expression was defined as ≥ 10% of tumor cells staining
for EGFR.3
Results: A Note on Baseline Characteristics
Baseline characteristics and efficacy results for the biomarker evaluable population (BEP) were
similar to those in the overall ITT population.
Results: A Note on the Sample Size
Almost 50% (367/743) of patients were evaluable for biomarker status, in line with previous
biomarker analyses from major clinical trials in NSCLC.4,5
Discussion: NSCLC Guidelines
The guidelines recommend that all patients should be tested for EGFR mutation status and the ALK
fusion gene as a priority at diagnosis, regardless of clinical characteristics. The authors report limited
value in testing for KRAS mutations.6
3
BioLOGUE Group
Twelve clinicians and advisors with expertise in biomarkers and statistical analysis pooled the results
of the BR.21 and SATURN trials. EGFR mutation status was shown to be predictive for erlotinibtreated PFS, but only prognostic for OS, in this pooled analysis.
KRAS mutations in clinical studies
In the open-label, phase IV TRUST study (in which patients who had previously failed on
chemotherapy/radiotherapy or were unsuitable for these therapies were treated with erlotinib until
disease progression or unacceptable toxicity), none of the 17 patients with a KRAS mutation had a
tumor response.7 In a retrospective study of patients with EGFR wild-type NSCLC treated with an
EGFR tyrosine-kinase inhibitor (TKI) (any line), a PFS of 1.6 months was reported in patients with
KRAS mutation-positive NSCLC (n = 18), compared with 3.0 months in KRAS wild-type disease
(n = 49; p = 0.04).8 The study authors also noted that the group of patients with KRAS mutationpositive NSCLC had a highly variable response to EGFR TKI treatment (PFS range: 0.7–38.7
months). In a second retrospective study, investigators sequenced tumor specimens from 522 patients
with advanced NSCLC treated with EGFR TKIs (in the first-, second-, and third-line settings). This
study suggested that KRAS mutations were associated with shorter OS (HR: 1.7; 95% CI: 1.1–2·4;
p = 0.04].9
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