Supplementary information: methods
Non-small-cell lung cancer (NSCLC) tumor tissue and immunohistochemistry
Hematoxylin and eosin (H & E) staining and immunohistochemical staining (IHC) of
CK7, TTF-1, and EGFR, were performed on the archival R2 lymph-node biopsy
specimen with standard techniques. The cytospin slide of the cerebrospinal fluid (CSF)
was stained with Papanicolou stain (Reagena, Finland).
Laser microdissection (LMD) and genomic DNA extraction
Tumor tissue and CSF were collected with the patient’s informed consent and in
accordance with the Institutional Review Board (IRB) approved protocol at the
University of Chicago. Genomic DNA was extracted from the tumor cells in the involved
R2 lymph node and CSF cytospin specimen using LMD. The paraffin-embedded tumor
block from the patient’s metastatic lymph node was cut onto Leica slides as 5µm thick
sections, deparaffinized in xylenes and stained for H&E. Approximately 300
histopathologically identified tumor cells were then microdissected using a Leica AS
LMD automated microscope with UV laser (Leica, Germany) directly into the cap of a
0.5ml PCR (polymerase chain reaction) tube (Figure 1). Genomic DNA from the tumor
cells was extracted using the PicoPure DNA Extraction Kit (Arcturus, Mountain View,
CA) for sequencing of the complete EGFR gene. The four tumor cells isolated from the
CSF (Figure 1) were placed directly into a 15µl of a digestion buffer solution (Arcturus)
and incubated at 65 °C overnight. 7.5µl of this solution was used to run a 10µl PCR
reaction of exon 21 and exon 22. The entire reaction volume of the first PCR was used to
run a second nested PCR reaction. The cycling conditions for the second PCR reaction
were similar to the first (see supplementary information for details of PCR conditions).
Leukocytes from the CSF were also microdissected to determine somatic nature of the
EGFR gene mutations identified.
PCR and DNA sequencing analysis
EGFR PCR conditions
The EGFR gene was amplified with nested PCR reactions from the DNA of laser
microdissected tumor cells. The first amplification was performed using external primer
pairs in a 10 µl reaction volume containing genomic DNA of 300 cells. The PCR cycling
program was: 95°C for 5 min; then 94°C for 30 s, 58°C for 45 s, 72°C for 45 s for 41
cycles, with a final extension at 72°C for 10 min. 1µl of the first reaction and specified
nested primers were used for the nested reaction. To avoid nonspecific PCR, a stepdown
cycling program was used for the second PCR reaction: 95°C for 5 min, 94°C for 30 s,
65–58°C for 30 s, 72°C 30s for 14 cycles (annealing temperature was decreased every
cycle by 0.5°C), 94°C for 30 s, 58°C for 30 s, 72°C 30 s, 72°C 10 min for 24 cycles.
Aliquots of all PCR products were examined by 1.5% agarose gel electrophoresis. PCR
products were purified using QIAquick PCR Purification Kit (Qiagen, Germany).
Direct DNA sequencing was performed in the Core DNA Sequencing Facility at
the University of Chicago (Applied Biosystems 3730XL 96-capillary automated DNA
sequencer) using standard techniques. Both forward- and reverse-strand sequencing
reactions were performed. EGFR genomic DNA PCR and sequencing primers are
available upon request. Mutation Surveyor software program (SoftGenetics, State
College, PA) was used to analyze the DNA sequencing results with subsequent manual
confirmation.
EGFR Expression constructs and site-directed mutagenesis
Expression plasmid constructs were prepared using the full-length wild-type EGFR
cDNA in pcDNA3, cloned in Xho I site. The EGFR mutations, L858R alone, E884K
alone, and the double mutant L858R+E884K (identified in our patient) were introduced
using the QuickChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA)
according to the manufacturer’s instructions. The plasmid vectors that resulted were
sequenced to confirm the presence of the corresponding mutations introduced.
In Vitro transfection and immunoblotting
In vitro transfection was performed using Cos-7 cells. Transient transfection was
performed as previously described using the Lipofectamine Plus reagent (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instructions. Cells were plated in 10cm
culture plates for transfection using 10g of plasmid vector for each of the constructs.
Transfected cells were split into six-well plates, 24 hours after transfection for epidermal
growth factor (EGF) stimulation studies. Cells were then starved in serum-free media for
16 hours prior to addition of the indicated concentrations of either gefitinib (gift from
AstraZeneca) or erlotinib (kindly provided by Dr. Balasz Halmos). Five hours after
incubation with the inhibitors, cells were stimulated with EGF as indicated (100ng/ml, 15
min) at 37oC. Whole cell lysates were then collected and separated by 7.5% SDS-PAGE
for immunoblotting. The following antibodies were used for the immunoblotting: anti-
phospho-EGFR [pY1068] (Cell Signaling Technology) for autophosphorylation of
EGFR, anti-EGFR (Santa Cruz) for equivalent receptor expression (data not shown), and
anti--actin (Sigma) as an internal loading control. Similar results were obtained from
two separate immunoblotting experiments. Quantitative analysis of the phospho-EGFR
signal was performed using the ImageJ software program.
EGFR external primers
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9
Exon 10
Exon 11
Exon 12
Exon 13
Exon 14
Exon 15
Exon 16
Exon 17
GACCGGACGACAGGCCACCTCGTC (sense)
GAAGAACGAAACGTCCCGTTCCTCC (antisense)
GTATTATCAGTCACTAAAGCTCAC (sense)
CACACTTCAAGTGGAATTCTGC (antisense)
GTTGAGCACTCGTGTGCATTAGG (sense)
CTCAGTGCACGTGTACTGGGTA (antisense)
GTTCACTGGGCTAATTGCGGGACTCTTGTTCGCAC (sense)
GGTAAATACATGCTTTTCTAGTGGTCAG (antisense)
GGTCTCAAGTGATTCTACAAACCAG (sense)
CCTTCACCTACTGGTTCACATCTG (antisense)
CATGGTTTGACTTAGTTTGAATGTGG (sense)
GGATACTAAAGATACTTTGTCACCAGG (antisense)
GAACACTAGGCTGCAAAGACAGTAAC (sense)
CCAAGCAAGGCAAACACATCCACC (antisense)
GGAGGATGGAGCCTTTCCATCAC (sense)
GAAGAGGAAGATGTGTTCCTTTGG (antisense)
GAATGAAGGATGATGTGGCAGTGG (sense)
GTATGTGTGAAGGAGTCACTGAAAC (antisense)
GGTGAGTCACAGGTTCAGTTGC (sense)
CAAAACATCAGCCATTAACGG (antisense)
CCACTTACTGTTCATATAATACAGAG (sense)
CATGTGAGATAGCATTTGGGAATGC (antisense)
CATGACCTACCATCATTGGAAAGCAG (sense)
GTAATTTCACAGTTAGGAATC (antisense)
GTAGCCAGCATGTCTGTGTCAC (sense)
CAGAATGCCTGTAAAGCTATAAC (antisense)
GTCCTGGAGTCCCAACTCCTTGAC (sense)
GGAAGTGGCTCTGATGGCCGTCCTG (antisense)
CATTTGGCTTTCCCCACTCACAC (sense)
GACCAAAACACCTTAAGTAACTGACTC (antisense)
CCAATCCAACATCCAGACACATAG (sense)
CCAGAGCCATAGAAACTTGATCAG (antisense)
GAAGCTACATAGTGTCTCACTTTCC (sense)
Exon 18
Exon 19
Exon 20
Exon 21
Exon 22
Exon 23
Exon 24
Exon 25
Exon 26
Exon 27
Exon 28a
Exon 28b
ACAACTGCTAATGGCCCGTTCTCG (antisense)
CAAATGAGCTGGCAAGTGCCGTGTC(sense)
GAGTTTCCCAAACACTCAGTGAAAC (antisense)
GCAATATCAGCCTTAGGTGCGGCTC (sense)
CATAGAAAGTGAACATTTAGGATGTG (antisense)
CCATGAGTACGTATTTTGAAACTC (sense)
CATATCCCCATGGCAAACTCTTGC (antisense)
CTAACGTTCGCCAGCCATAAGTCC (sense)
GCTGCGAGCTCACCCAGAATGTCTGG (antisense)
GAGCAGCCCTGAACTCCGTCAGACTG (sense)
CTCAGTACAATAGATAGACAGCAATG (antisense)
CAGGACTACAGAAATGTAGGTTTC (sense)
GTGCCTGCCTTAAGTAATGTGATGAC (antisense)
GACTGGAAGTGTCGCATCACCAATG (sense)
GGTTTAATAATGCGATCTGGGACAC (antisense)
GCAGCTATAATTTAGAGAACCAAGG (sense)
GGTTAAAATTGACTTCATTTCCATG (antisense)
CCTAGTTGCTCTAAAACTAACG (sense)
CTGTGAGGCGTGACAGCCGTGCAG (antisense)
CAACCTACTAATCAGAACCAGCATC (sense)
CCTTCACTGTGTCTGCAAATCTGC (antisense)
GCTCCTGCTCCCTGTCATAAGTC (sense)
GAAGTCCTGCTGGTAGTCAGGGTTG (antisense)
CTGCAGTGGGCAACCCCGAGTATC (sense)
CAGTCTGTGGGTCTAAGAGCTAATG (antisense)
EGFR PCR: nested primers
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9
Exon 10
Exon 11
Exon 12
Exon 13
Exon 14
Exon 15
Exon 16
Exon 17
Exon 18
Exon 19
Exon 20
Exon 21
GACAGGCCACCTCGTCGGCGTC (sense)
CAGCTGATCTCAAGGAAACAGG (antisense)
CAGGAATGGGTGAGTCTCTGTGTG (sense)
GTGGAATTCTGCCCAGGCCTTTC (antisense)
CTCGTGTGCATTAGGGTTCAACTGG (sense)
CCTTCTCCGAGGTGGAATTGAGTGAC (antisense)
GCTAATTGCGGGACTCTTGTTCGCAC (sense)
TACATGCTTTTCTAGTGGTCAG (antisense)
GATTCTACAAACCAGCCAGCCAAAC (sense)
CCTACTGGTTCACATCTGACCCTG (antisense)
GTTTGAATGTGGTTTCGTTGGAAG (sense)
CTTTGTCACCAGGCAGAGGGCAATATC (antisense)
GACAGTAACTTGGGCTTTCTGAC (sense)
CATCCACCCAAAGACTCTCCAAG (antisense)
CCTTTCCATCACCCCTCAAGAGG (sense)
GATGTGTTCCTTTGGAGGTGGCATG (antisense)
GATGTGGCAGTGGCGGTTCCGGTG (sense)
GGAGTCACTGAAACAAACAACAGG (antisense)
GGTTCAGTTGCTTGTATAAAG (sense)
ATTAACGGTAAAATTTCAGAAG (antisense)
CTGTTCATATAATACAGAGTCCCTG (sense)
AGAGATGCAGGAGCTCTGTGC (antisense)
GCAGTTTGTAGTCAATCAAAGGTGG (sense)
GTAATTTAAATGGGAATAGCCC (antisense)
CCAAGGTCATGGAGCACAGG (sense)
GTAAAGCTATAACAACAACCTGG (antisense)
CAACTCCTTGACCATTACCTCAAG (sense)
GATGGCCGTCCTGCCCACACAGG (antisense)
CCACTCACACACACTAAATATTTTAAG (sense)
GTAACTGACTCAAATACAAACCAC (antisense)
GAGTAGTTTAGCATATATTGC (sense)
GACAGTCAGAAATGCAGGAAAGC (antisense)
GAAGCTACATAGTGTCTCACTTTCC (sense)
CACAACTGCTAATGGCCCGTTCTCG (antisense)
CAAGTGCCGTGTCCTGGCACCCAAGC (sense)
CCAAACACTCAGTGAAACAAAGAG (antisense)
CCTTAGGTGCGGCTCCACAGC (sense)
CATTTAGGATGTGGAGATGAGC (antisense)
GAAACTCAAGATCGCATTCATGC (sense)
GCAAACTCTTGCTATCCCAGGAG (antisense)
CAGCCATAAGTCCTCGACGTGG (sense)
CATCCTCCCCTGCATGTGTTAAAC (antisense)
Exon 22
Exon 23
Exon 24
Exon 25
Exon 26
Exon 27
Exon 28a
Exon 28b
GACGGGTCCTGGGGTGATCTGGCTC (sense)
GATTACATTATCATTAGTCATTATC (antisense)
GTAGGTTTCTAAACATCAAGAAAC (sense)
GTGATGACATTTCTCCAGGGATGC (antisense)
CATCACCAATGCCTTCTTTAAGC (sense)
GCTGGAGGGTTTAATAATGCGATC (antisense)
GCAAACACACAGGCACCTGCTGGC (sense)
CATTTCCATGTGAGTTTCACTAGATGG (antisense)
CACCTTCACAATATACCCTCCATG (sense)
GACAGCCGTGCAGGGAAAAACC (antisense)
GAACCAGCATCTCAAGGAGATCTC (sense)
GAGCACCTGGCTTGGACACTGGAG (antisense)
CCTGTCATAAGTCTCCTTGTTGAG (sense)
GGTAGTCAGGGTTGTCCAGG (antisense)
CGAGTATCTCAACACTGTCCAGC (sense)
CTAAGAGCTAATGCGGGCATGGCTG (antisense)