Gefitinib treatment in EGFR mutated Caucasian NSCLC: circulating-free tumor DNA
as a surrogate for determination of EGFR status
Online Supplemental Appendix
Methods
DNA extraction and EGFR mutation analysis
Tumor DNA was extracted using the Qiagen QIAamp DNA Mini Kit while circulating-free
tumor DNA (ctDNA) was extracted from plasma using the Qiagen QIAamp Circulating
Nucleic Acid Kit. Modifications to sample processing and kit instructions are described in
Online Supplemental Appendix Table 1.
Epidermal growth factor receptor (EGFR) mutation status of all samples was assessed using a
Scorpion® Amplification Refractory Mutation System (ARMS®)-based EGFR mutation
detection kit (Therascreen® EGFR RGQ PCR kit, Qiagen, Crawley, UK), which detects 29
mutations across the EGFR gene. For tumor samples, all mutations in the kit were analyzed.
Modifications to tumor/ctDNA analysis kit instructions are described in Online Supplemental
Appendix Table 1.
A comparison of the DxS kit used for ctDNA EGFR mutation analysis in the current study
and the previous version of the Dx kit used in the IPASS study 1,2 is shown in Online
Supplemental Appendix Table 2.
Results
Patients
Demographics and baseline characteristics by EGFR mutation status derived from baseline
tumor samples (full analysis set population), plasma 1 samples, and plasma 2 samples are
presented in Online Supplemental Appendix Table 3, and were similar for all populations.
Exon 19 deletions and L858R point mutations were the most common mutations, as
determined by baseline tumor, plasma 1, and plasma 2 samples (Online Supplemental
Appendix Table 3).
EGFR mutation status of ctDNA samples: false-positive result
1
Of the 547 patients considered to have an EGFR mutation-negative status by analysis of
tumor DNA, one patient was considered to have an EGFR mutation-positive status (L858R)
by analysis of plasma 1 ctDNA, giving a false-positive rate of 0.2% (1/547). However, this
was subsequently found to be due to sample drift during analysis, rather than true
amplification (see analysis plots in Supplemental Online Appendix Figures 1A and B).
Manual calling, recommended in the Therascreen® EGFR RGQ PCR kit insert, was not
performed prospectively, and therefore this issue was not identified in real-time.
Retrospective quality control of all EGFR mutation-positive plasma 1 samples, with unevaluable matched tumor results, confirmed no other samples were affected by this issue.
2
Online Supplemental Appendix Table 1 Methodological and technical details for DNA extraction and EGFR mutation testing of tumor and plasma ctDNA
samples
DNA extraction
Sample type
Original step in protocol
Change to kit instructions
Rationale
Tumor
Perform de-paraffinization using xylene
Scrape tissue into tube containing 200 µl 0.5% TWEEN 20 and centrifuge
briefly
Incubate sample at 90°C for 10 min with regular inversion mixing
Incubate sample at 55°C for 5 min
Add 4 µl of 10 mg/ml Proteinase K and incubate for 1 hour at 55°C with
occasional mixing. Repeat twice over the course of 12-24 hours
Heat sample to 99°C for 10 min
Transfer sample into a chilled tube at 4°C and centrifuge at 10,500g for 15 min
Place sample on ice for 5 min
Aspirate the liquid from below the wax layer into a clean tube
Lab experience of alternative
procedure
Incubate with Proteinase K at 56°C until
completely lyzed (1-3 hours or
overnight)
Centrifuge QIAamp Mini columns at
6,000g in order to reduce centrifuge
noise. Centrifuging at full speed will not
affect DNA yield
Elute in 200 µl of buffer AE
Incubate with Proteinase K at 56°C until completely lyzed (overnight)
Lab preference
Centrifuge spin columns at full speed (20,000g)
Lab preference
Elute in 150 µl of buffer AE
Experience that reduced yields
and DNA concentration are
likely from FFPE compared to
fresh tissue
NA
Centrifuge plasma samples at 3000 rpm for 2 min prior to extraction and transfer
supernatant to clean tube
To remove any contamination
(cellular debris/protein) which
may clog the column
Plasma
3
Add 1550 µl buffer AVE to 310 µg of
carrier RNA (0.2 µg/µl). 1 µg of carrier
RNA added to each reaction
Following addition of buffer ACL,
incubate samples at 60°C for 30 min
Add 310 µl buffer AVE to 310 µg of carrier RNA (1 µg/µl). 5.6 µg of carrier
RNA added to each reaction
As previous version of manual
(v1)
Following addition of buffer ACL, incubate samples at 60°C for 1 hour
Experience that longer
incubation can result in
increased yield
Elute cfDNA in 20-150 µl of buffer AVE
Elute cfDNA in 55 µl of buffer AVE
NA: volume within kit
recommended range
EGFR mutation analysis
Tumor
Plasma
Quantify DNA using Rotor-Gene® Q and
dilute DNA which has a ct <23
NA
Lab experience of alternative
procedure
Assess DNA before analysis and dilute
samples with a control ct <23
Quantify DNA using Q-PCR and dilute DNA which has a copy number of
>4 ng/µl
Repeat entire plate if any NTC HEX signals >37
Repeat entire assay if sample HEX >37 (if there is no sample FAM signal)
Repeat entire plate if mixed standard falls outside of 26.26-30.95
Repeat entire assay if mixed standard ∆ct fail observed
Dilute and repeat sample if control ct <23
Repeat samples with a diagnostic ct >38 in triplicate (all 3 results must be
identical to assign a positive call)
ctDNA not quantified prior to analysis. Add 5 μl of neat ctDNA, eluted from
the QIAamp preparation, to each reaction
Analyse all 7 assays, with a maximum of
7 samples per plate
Analyze 3 assays (T790M, deletions, and L858R), with a maximum of 16
samples per plate
To conserve ctDNA while
focusing on the common and
clinically relevant mutations
Run 40 cycles of PCR
Run 50 cycles of PCR, however only the first 40 cycles should be analyzed as
per protocol
To examine the shape of the
amplification curve and to
determine if any very late
amplification was observed in
ctDNA samples
NA
Experience that it is unlikely
that ctDNA samples have
control ct <23 and therefore a
step to conserve DNA
4
Repeat entire plate if any NTC HEX
signals >37a
Repeat entire assay if sample HEX >37
(if there is no sample FAM signal)a
Repeat entire plate if mixed standard
falls outside of 26.26-30.95a
Repeat entire assay if mixed standard ∆ct
fail observeda
Dilute and repeat sample if control ct
<23a
Repeat samples with a diagnostic ct >38
in triplicate (all 3 results must be
identical to assign a positive call)a
Repeat all samples for relevant assay
To conserve ctDNA
Repeat sample for relevant assay
Repeat plate if it contains any mutation-positive samples
Repeat sample for relevant assay if mutation-positive
Dilute and repeat sample if mutation-positive
Do not repeat samples if ct >38
ctDNA, circulating-free tumor DNA; CT, cycle threshold; EGFR, epidermal growth factor receptor; FFPE, formalin-fixed, paraffin-embedded; NA, not applicable; NTC, no
template control
a
Note that the amendments to the described methodology were to the AstraZeneca FFPE Therascreen protocol only.
5
Online Supplemental Appendix Table 2 A comparison of the mutation test kits used for cfDNA EGFR mutation analysis in the current study and the
IPASS study
IPASS
Current study
{Douillard, 2014 6235 /id}
1,2
Sample type
Serum
Plasma
Sample volume used for extraction
~2 ml
~2 ml
Extraction kit
QIAamp DNA Mini Kit (Qiagen, Crawley, UK)
Cat. # 51304
QIAamp Circulating Nucleic Acid Kit v2 (Qiagen, Crawley,
UK)
Cat. # 55114
Methodology
Scorpion® Amplification Refractory Mutation System
(ARMS®)-based EGFR mutation detection kit
Scorpion® Amplification Refractory Mutation System
(ARMS®)-based EGFR mutation detection kit
Kit name
DxS EGFR mutation test kit (DxS, Manchester, UK)
Note: no longer available
Therascreen® EGFR RGQ PCR kit v1 (Qiagen, Crawley, UK)
Cat. # 870111
Mutation cut-offs, ∆ct
Modified for ctDNA (tumor)
ctDNA as per standard protocol (tumor)
8 (8)
12 (9)
14 (11)
6.38 (6.38)
9.06 (9.06)
8.58 (8.58)
50 cycles run, called only within 40 cycles
50 cycles run, called only within 40 cycles
T790M
Exon 19 Deletions
L858R
Number of cycles run (called)
ctDNA, circulating-free tumor DNA; CT, cycle threshold; EGFR, epidermal growth factor receptor
6
Online Supplemental Appendix Table 3 Key demographic and baseline characteristics by EGFR mutation status derived from patients with baseline tumor
samples, and duplicate plasma ctDNA samples
Tumor samples
(FAS, N=106)
Characteristic
Plasma 1 samples (N=1060)
Plasma 2 samples (N=1060)
EGFR mutation status
EGFR mutation status
Positive
(n=82)
Negative
(n=702)
Unknown
(n=276)
Positive
(n=65)a
Negative
(n=160)a
Unknown
(n=835)
Age group (years), %
≥18–<65
49.1
45.1
49.4
44.9
43.1
47.5
48.4
≥65–<75
26.4
30.5
31.9
33.7
30.8
31.9
32.5
≥75
24.5
24.4
18.7
21.4
26.2
20.6
19.2
Male
29.2
29.3
66.0
61.6
33.8
55.0
65.5
Female
70.8
70.7
34.0
38.4
66.2
45.0
34.5
Caucasianb
100.0
100.0
100.0
99.6
100.0
100.0
99.9
0.0
0.0
0.0
0.4
0.0
0.0
0.1
Gender, %
Race, %
Black or African
American
7
Tumor samples
(FAS, N=106)
Characteristic
Plasma 1 samples (N=1060)
Plasma 2 samples (N=1060)
EGFR mutation status
EGFR mutation status
Positive
(n=82)
Negative
(n=702)
Unknown
(n=276)
Positive
(n=65)a
Negative
(n=160)a
Unknown
(n=835)
Histology, %
Adenocarcinoma
(NOS)
86.8
86.6
60.8
60.9
89.2
68.1
59.8
Adenocarcinoma
bronchioloalveolar
9.4
7.3
6.3
5.4
6.2
8.1
5.7
Adenosquamous
carcinoma
1.9
1.2
1.4
2.5
1.5
1.3
1.8
Large cell
carcinoma (NOS)
0.9
0.0
1.0
1.8
0.0
0.6
1.3
Squamous cell
carcinoma (NOS)
0.0
1.2
24.4
23.2
0.0
18.1
24.8
Undifferentiated
carcinoma
0.0
2.4
2.3
2.2
1.5
1.9
2.4
Adenocarcinoma
tubulopapillary
0.9
-
-
-
-
-
-
Missing
0.0
0.0
0.4
1.8
0.0
0.0
1.0
Other
0.9
1.2
3.4
2.2
1.5
1.9
3.2
8
Tumor samples
(FAS, N=106)
Characteristic
Plasma 1 samples (N=1060)
Plasma 2 samples (N=1060)
EGFR mutation status
EGFR mutation status
Positive
(n=82)
Negative
(n=702)
Unknown
(n=276)
Positive
(n=65)a
Negative
(n=160)a
Unknown
(n=835)
Disease stage at screening,
%
IIIA
1.9
0.0
0.7
0.7
0.0
0.6
0.7
IIIB
5.7
3.7
24.8
22.8
4.6
18.8
24.8
IV
92.5
96.3
74.2
72.8
95.4
80.6
73.1
Missing
0.0
0.0
0.3
2.5
0.0
0.0
1.1
Other
0.0
0.0
0.0
1.1
0.0
0.0
0.4
0
45.3
41.5
35.3
33.0
43.1
38.1
34.0
1
48.1
51.2
55.1
51.1
47.7
52.5
54.5
2
6.6
7.3
8.8
13.0
9.2
8.1
10.2
3
0.0
0.0
0.0
0.7
0.0
0.0
0.2
Missing
0.0
0.0
0.7
2.2
0.0
1.3
1.1
Performance status, %
Smoking status, %
9
Tumor samples
(FAS, N=106)
Characteristic
Plasma 1 samples (N=1060)
Plasma 2 samples (N=1060)
EGFR mutation status
EGFR mutation status
Positive
(n=82)
Negative
(n=702)
Unknown
(n=276)
Positive
(n=65)a
Negative
(n=160)a
Unknown
(n=835)
Current
5.7
3.7
30.8
24.3
6.2
26.9
28.6
Former
30.2
32.9
48.7
48.9
32.3
44.4
49.3
Never
64.2
63.4
19.9
24.6
61.5
28.8
20.8
Missing
0.0
0.0
0.6
2.2
0.0
0.0
1.2
Exon 19 deletions
65.1
68.3
-
-
67.7
-
-
L858R
31.1
31.7
-
-
32.3
-
-
L861Q
1.9
-
-
-
-
-
G719X (G719S/A/C)
1.9
-
-
-
-
-
EGFR mutation subtype, %
a
In order to achieve a similar measure of accuracy for analysis of plasma 2 samples and avoid sample waste, EGFR mutation status was determined for 224 plasma 2 samples
whose corresponding plasma 1 sample was EGFR mutation status-known (all EGFR mutation-positive plasma 1 for whom plasma 2 was available, and a matched number of
EGFR mutation-negative ). Of these 224 plasma 2 samples chosen for analysis, 65 were assigned an EGFR mutation-positive status.
10
b
Caucasians were considered to be patients of European, North African, or Middle Eastern descent only for the purpose of this study
ctDNA, circulating-free tumor DNA; EGFR, epidermal growth factor receptor; FAS, full analysis set; NOS, not otherwise specified
11
Supplemental Online Appendix Figure 1 Therascreen® EGFR RGQ PCR analysis results for (A) false-positive plasma 1 ctDNA sample and (B) a typical
example of a true L858R signal
(A)
12
(B)
Sample analysis performed using Qiagen® Rotor-Gene® Q Series Software version 2.0.2
ctDNA, circulating-free tumor DNA; EGFR, epidermal growth factor receptor
13
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14