Supplementary material for “EGFR and KRAS mutations detected in plasma are
associated with patient outcomes in erlotinib-treated non-small cell lung cancer”; Mack et
al.
PCR and direct sequencing for EGFR
To detect mutations in EGFR exons 19, 20 and 21, a nested PCR assay was used.
External primer sequences for each exon have been previously published (16):
exon 19:
e19F1 sense 5’-GTGCATCGCTGGTAACATCC,
e19R1 antisense 5’-TGTGGAGATGAGCAGGGTCT,
exon 20:
e20F1 sense 5’-ATCGCATTCATGCGTCTTCA-3’,
e20R1 antisense 5’-ATCCCCATGGCAAACTCTTG)
exon 21:
e21F1 sense 5’- GCTCAGAGCCTGGCATGAA-3’
e21R1 antisense 5’-CATCCTCCCCTGCATGTGT-3’)
The following internal primers were used for nested PCR:
exon 19:
e19F2 sense 5’-CATGTGGCACCATCTCACA–3’,
e19R2 antisense 5’-CCACACAGCAAAGCAGAAAC-3’),
exon 20:
e20F2 sense 5’-ACCATGCGAAGCCACACTGA-3’
e20R2 antisense 5’-CGCAGACCGCATGTGAGGAT-3’),
exon 21:
e21F2 sense 5’-CCTCACAGCAGGGTCTTCTC-3’,
e21R2 antisense 5’-AATGCTGGCTGACCTAAAGC-3’).
Each PCR reaction comprised 20 uL total volume and included 1x cloned Pfu buffer, 200
uM dNTP, 0.5 uM forward and reverse primers, 1 unit of Pfu DNA polymerase and 1 ul of
DNA template. After an initial DNA denaturation of 5 min at 95 oC, 35 cycles of PCR were
performed under the following conditions: 95oC for 30 sec, 55oC for 30 sec, and 72oC for
30 sec. The nested PCR with internal primers utilized the same PCR conditions. PCR
products were extracted and purified from agarose gels using a QIAquick gel extraction kit
(Qiagen) and sequenced in both forward and reverse directions (ABI 3730 Capillary
Electrophoresis Genetic Analyzer). For mutant-positive samples, confirmatory sequencing
was conducted on DNA extracted from a second paraffin-embedded tissue section, when
available.
Scorpion-ARMS for EGFR and KRAS mutations
The EGFR test kit was supplied with probes specific for exon 19 deletions, T790M and
L858R mutations. The KRAS test kit was supplied with 8 probes, 7 targeting mutations in
codons 12 and 13. Each kit also contains a control probe specific to a non-polymorphic
site used to assess total DNA content. Five ul of template DNA was used in each reaction.
In addition, every probe set contains an exogenous assay, labeled with HEX, to control for
the presence of inhibitors that may lead to false negative results.
All reactions were
performed on an iQ5 Real-Time PCR detection system (BioRad, Hercules, CA) using
these conditions: 95oC for 10 min, 45 cycles of 95oC for 30s, 61oC for 60s with fluorescent
readings (set for FAM and HEX) collected after each cycle. Data was analyzed using iQ5
Optical System software (version 2.0) (BioRad). The cycle threshold (CT) was defined as
the fractional cycle number at which the fluorescence passes the fixed threshold, set
above baseline, as determined by the software. For each probe set, a cut-off deltaCT,
defined as the CT of the mutant minus the CT of the control, has been established by the
manufacturer as the point at which a positive signal could be due to background
fluorescence of the primer on wild-type DNA. Any specimen where the control probe had
a CT value of ≥40 was repeated and/or disqualified as insufficient. For a patient specimen
to be considered positive for mutation it must have: 1) a mutation CT <45, 2) a deltaCT
lower than the cut-off for that probe and 3) a smooth amplification curve in the log phase
parallel to the control probe. For all patients testing positive, an additional genomic DNA
specimen was independently extracted to confirm presence of mutation. For sensitivity
experiments, DNA extracted from NSCLC cell lines H1650 (E746-A750del) and H1975
(L858R and T790M) were used as positive controls for EGFR, while DNA from A549
(G12S) was used as a positive control for KRAS. NSCLC cell lines H460 and A549 were
used as wild-type controls for KRAS and EGFR, respectively.