Supplemental Files

SUPPLEMENTAL FILES

Supplemental Figure S1. Sample inclusion flow chart.

Supplemental Figure S2. Boxplot of circulating free DNA concentration in plasma (in ng/µl) regarding M stage (up), and number of metastatic sites (down) in all samples (n=106). Y axis is in logarithmic scale.

Supplemental Table S1. Processes used in the different participating labs for tumor DNA extraction, storage, shipment, and biomarker analysis.

Supplemental Table S2. Primers used for amplicon sequencing by IonTorrent next-generation sequencing

Supplemental Table S3. Multiplex reaction conditions.

Supplemental Table S4. NGS mutation screening results in the 68 paired samples

Supplemental Table S5. IonTorrent NGS test results in cfDNA, for each individual amplicon and overall, taking tDNA as a reference and restricted to stage IV samples (n=50)

Supplemental Table S6.NGS test results in tDNA and cfDNA for all amplicons concomitantly using the ITVC strategy (5A), or the in-house strategy (5B); and corresponding calculated sensitivity for all matched samples analyzed by NGS (n=68)

Supplemental Figure S1. Sample inclusion flow chart

cfDNA: Circulating Free DNA; tDNA: tumoral DNA; NGS: Next-Generation Sequencing

Supplemental Figure S2. Boxplot of circulating free DNA concentration in plasma (in ng/µl) regarding M stage (up), and number of metastatic sites (down) in all samples (n=106). Y axis is in logarithmic scale.

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Supplemental Files VR1CCR_V3

Supplemental Table S1. Processes used in each participating labsfor tumor DNA extraction, storage, shipping, and biomarker analysis.

Lab / n / % / micro-
dissection / DNA extraction / Storage / Shipping / EGFR / KRAS / HER2 / BRAF / PI3KCA
L1 / 1 / 1% / No / FFPE DNA tissu kit (Qiagen) / -20°C / RT / RT-PCR / Pyroseq / RFLP / Pyroseq
L2 / 1 / 1% / Yes / NucleoSpin FFPE DNA(Macherey-Nagel) / -20°C / RT / PCR Taqman (exons18,20,21) + RFLP (exon19) / PCR snaphot / RFLP / PCR snaphot / PCR snaphot
L3 / 1 / 1% / Yes / EZ1 DNA kit (Quiagen) / -20°C / RT / RFLP (exon 19+20) + PCR Taqman (L858R) + HRM / Sequencing (exon 18) / HRM / Taqman / HRM / Sequencing / HRM / Taqman (V600x)
L4 / 1 / 1% / -20°C / F / Sequencing / HRM / HRM
L5 / 1 / 1% / No / QIAampl DNA micro kit (Qiagen) / -20°C / RT / Sequencing (19 à 21) + PCR Taqman (exon 18, 20, 21) + RFLP (exon 19) / PCR Taqman / Sequencing / PCR Taqman
L6 / 1 / 1% / No / QIAampl DNA micro kit (Qiagen) / -20°C / RT / Pyroseq / PCR Taqman / Pyroseq / Pyroseq / Pyroseq
L7 / 2 / 3% / No / iPrep instrument (Life technologies) / -20°C / RT / Allele specific PCR / Sanger Sequencing / Allele specific PCR
L8 / 2 / 3% / Yes / Maxwell 16 FFPE Plus LEV DNA purification kit (Promega, Ref AS1135) / -20°C / RT / PCR Taqman (exons 18,20,21) + RFLP (exons 19,20) / PCR Taqman / RFLP / PCR Taqman / PCR Taqman
L9 / 2 / 3% / -20°C / F
L10 / 3 / 4% / Magtration PLC (Bionobis) / -20°C / RT / Pyroseq (exon 18, 20, 21) + RFLP (19, 20) / Pyroseq / Pyroseq
L11 / 3 / 4% / No / Maxwell® 16 FFPE Plus LEV DNA Purification Kit (Promega) / -20°C / RT / HRM + Pyroseq / HRM + pyroseq / HRM+ Sanger Pyroseq / HRM +pyroseq / HRM + Pyroseq
L12 / 4 / 6% / -20°C / F / Pyroseq / Pyroseq / Pyroseq / Pyroseq / Pyroseq
L13 / 4 / 6% / No / Eeasymag (Biomerieux) / +4°C / RT / HRM +séquençage / PCR Taqman / HRM +séquençage
L14 / 4 / 6% / No / QIAampl DNA micro kit (Qiagen) / -80°C / F / Sequencing / PCR snapshot / Fragment analysis / PCR snapshot / PCR Snapshot
L15 / 4 / 6% / -20°C / RT / Sequencing + RFLP (exon 19) + PCR Taqman (exon 20) / PCR Taqman / RFLP
L16 / 6 / 9% / No / QIAampl DNA micro kit (Qiagen) / -20°C / F / Fragment analysis (exons 19 and 20); Fret+PNA (exon 21 - L858R); Sequencing (others) / Fret+PNA / Fragment analysis / Fret / Sequencing
L17 / 7 / 10% / -20°C / RT / PCR Taqman / PCR Scorpio / PCR Taqman
L18 / 21 / 31% / Yes / QIAampl DNA micro kit (Qiagen) / -20°C / RT / Sequencing / PCR snapshot / Sequencing / Sequencing / Sequencing

Pyroseq – Pyrosequencing; HRM - High Resolution Melting; RFLP - Restriction Fragment Length Polymorphism; (RT)PCR - (Real Time) Polymerase Chain Reaction; RT - Room temperature; F – Frozen; Fret + PNA - Förster resonance energy transfer donor for the detection of DNA hybridization in conjunction with a fluorescein-labeled pyrrolidinyl peptide nucleic acid (PNA) probe.

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Supplemental Files VR1CCR_V3

Supplemental Table S2. Primers used for amplicon sequencing by IonTorrent next-generation sequencing

Gene / Exon / Genomic region / Codons / Primer / Sequence / Amplicon size(bp)
EGFR / 18 / chr7: 55241636-55241755 / 696-728 / EGFR18v2F / GTGGAGAAGCTCCCAACCAA / 120
EGFR18v2R / CCTGTGCCAGGGACCTTAC
19 / chr7: 55242381-55242537 / 729-761 / EGFR19F / GCCAGTTAACGTCTTCCTTCT / 157
EGFR19R / CCACACAGCAAAGCAGAAAC
20 / chr7: 55248968-55249092 / 762-796 / EGFR20F / GCCTCTCCCTCCCTCCAG / 125
EGFR20v2R / CAGCCGAAGGGCATGAGC
21 / chr7: 55259459-55259565 / 840-874 / EGFR21v2F / AGCCAGGAACGTACTGGTGA / 107
EGFR21v2R / TGCCTCCTTCTGCATGGTAT
KRAS / 2 / chr12: 25398216-25398329 / 1-38 / KRAS2v2F / GCCTGCTGAAAATGACTGAA / 114
KRAS2v2R / TTGGATCATATTCGTCCACAA
3 / chr12: 25380220-25380317 / 48-79 / KRAS3v2F / TGGAGAAACCTGTCTCTTGGA / 98
KRAS3v2R / AAAGAAAGCCCTCCCCAGT
BRAF / 11 / chr7: 140481373-140481480 / 444-478 / BRAF11v2F / GACGGGACTCGAGTGATGAT / 108
BRAF11v2R / TACCATGCCACTTTCCCTTG
15 / chr7: 140453078-140453180 / 586-619 / BRAF15v2F / TGAAGACCTCACAGTAAAAATAGGTG / 103
BRAF15v2R / CCACAAAATGGATCCAGACA
HER2/ERBB2 / 19 / chr17: 37880177-37880282 / 741-769 / HER2-19v2F / CCTGATGGGGAGAATGTGAA / 106
HER2-19v2R / AGAGGGTGGAGGGGCTTAC
20 / chr17: 37880963-37881061 / 769-796 / HER2-20v2F / GTACCCTTGTCCCCAGGAAG / 99
HER2-20v2R / ACCAGCTGCACCGTGGATGT
PIK3CA / 9 / chr3: 178936029-178936130 / 525-555 / PIK3AC9v2F / GGGAAAATGACAAAGAACAGC / 102
PIK3CA9v2R / GCACTTACCTGTGACTCCATAGAA
20 / chr3: 178952038-178952142 / 1039-1058 / PIK3AC20v2F / TGAGCAAGAGGCTTTGGAGT / 105
PIK3CA20v2R / GCATGCTGTTTAATTGTGTGG

Supplemental Table S3. Multiplex reaction conditions.

Product / Volume (µL)
Multiplex#1
5X PCR buffer without MgCl2 / 4
25mM MgCl2 / 1.2
dNTP mix (final 0.4mM) / 1.6
PIK3AC20v2F (0.4 µM final) / 0.8
PIK3CA20v2R (0.4 µM final) / 0.8
EGFR21v2F (0.4 µM final) / 0.8
EGFR21v2R (0.4 µM final) / 0.8
ERBB2-19v2F (0.8 µM final)* / 1.6
ERBB2-19v2R (0.8 µM final)* / 1.6
GoTaq HotStart DNA polymerase (5U/µl) / 0.1
Template DNA / 4
Water, molecular biology grade / 2.7
Total / 20
Multiplex#2
5X PCR buffer without MgCl2 / 4
25mM MgCl2 / 1.2
dNTP mix (final 0.4mM) / 1.6
HER2-20v2F (0.4 µM final) / 0.8
HER2-20v2R (0.4 µM final) / 0.8
EGFR18v2F (0.8 µM final) / 1.2
EGFR18v2R (0.8 µM final) / 1.2
EGFR19F (0.8 µM final) / 0.8
EGFR19R (0.8 µM final) / 0.8
GoTaq HotStart DNA polymerase (5U/µl) / 0.1
Template DNA / 4
Water, molecular biology grade / 1.1
Total / 20
Multiplex#3
5X PCR buffer without MgCl2 / 4
25mM MgCl2 / 1.2
dNTP mix (final 0.4mM) / 1.6
PIK3AC9v2F (0.4 µM final) / 0.8
PIK3CA9v2R (0.4 µM final) / 0.8
BRAF11v2F (0.4 µM final) / 0.8
BRAF11v2R (0.4 µM final) / 0.8
EGFR20F (0.8 µM final) / 1.6
EGFR20v2R (0.8 µM final) / 1.6
GoTaq HotStart DNA polymerase (5U/µl) / 0.1
Template DNA / 4
Water, molecular biology grade / 2.7
Total / 20
Multiplex#4
5X PCR buffer without MgCl2 / 4
25mM MgCl2 / 1.2
dNTP mix (final 0.4mM) / 1.6
BRAF15v2F (0.4 µM final) / 0.8
BRAF15v2R (0.4 µM final) / 0.8
KRAS2v2F (0.4 µM final) / 1.2
KRAS2v2R (0.4 µM final) / 1.2
KRAS3v2F (0.4 µM final) / 1.2
KRAS3v2R (0.4 µM final) / 1.2
GoTaq HotStart DNA polymerase (5U/µl) / 0.1
Template DNA / 4
Water, molecular biology grade / 4.3
Total / 20

* ERBB2 was amplified in an independent PCR reaction in half of the samples.

Supplemental Table S4. Mutation screeningresults in the 68 paired samples

Stage IV / Gene / Mutation description / Tum Freq (%) / Cf DNA Freq (%) / Cf DNA cov / ITVC / N ampl tested / Median cov on all tested ampl
#1 / Y / WT / 12 / 3079
#2 / Y / EGFR / chr7:55241708G>C; ENST00000344576:c.2156G>C; p.G719A / 62 / 44 / 1007 / Y / 12 / 1103
#3 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 21 / 31 / 1431 / Y / 12 / 1273
#4 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 19 / 10 / 1854 / 12 / 809
#5 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 21 / 7 / 833 / 12 / 478
#6 / Y / WT / 12 / 352
#7 / Y / WT / 11 / 3380
#8 / Y / EGFR / chr7:55242465del15;ENST00000344576:c.2235_2249del;p.K746_A750del / 20 / 17 / 761 / 12 / 475
#9 / Y / KRAS / chr12:25398284C>A; ENST00000256078:c.35G>T; p.G12V / 11 / 0.6 / 80113 / 12 / 541
#10 / Y / EGFR / chr7:55259515T>G; ENST00000344576:c.2573T>G; p.L858R / 12 / 1.5 / 611 / 12 / 405
#11 / Y / EGFR / chr7:55242465del15;ENST00000344576:c.2235_2249del;p.K746_A750del / ND / 12 / 20178 / 12 / 20283
#12 / WT / 12 / 617
#13 / EGFR / chr7:55242465del15;ENST00000344576:c.2235_2249del;p.K746_A750del / 15 / ND / 35583 / 12 / 577
#14 / Y / EGFR / chr7:55242465del15;ENST00000344576:c.2235_2249del;p.K746_A750del / 40 / ND / 37816 / 12 / 4233
#15 / Y / EGFR / chr7:55242466del15; ENST00000344576:c.2236_2250del; p.E746_A750del / 37 / ND / 25737 / 12 / 6803
#16 / WT / 12 / 8319
#17 / Y / EGFR / chr7:55259515T>G; ENST00000344576:c.2573T>G; p.L858R / 9 / 3 / 68047 / 12 / 401
#18 / WT / 1 / 59327
#19 / Y / ERBB2 / chr17:37880981ins12; ENST00000269571:c.2310_2311ins12; p.E770_A771ins12 / 40 / 0.5 / 51046 / 1 / 51046
#20 / Y / EGFR / chr7:55259515T>G; ENST00000344576:c.2573T>G; p.L858R / 83 / 1.3 / 79294 / 1 / 79294
#21 / Y / EGFR / * / chr7:55241708G>C; ENST00000344576:c.2156G>C; p.G719A / 19 / 1 / 62516 / 12 / 1310
#22 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 14 / 37 / 25981 / Y / 12 / 5715
#23 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 41 / 0.2 / 34769 / 12 / 478
#24 / Y / WT / 12 / 1989
#25 / WT / 12 / 16611
#26 / Y / WT / 12 / 6635
#27 / Y / WT / 12 / 3095
#28 / Y / EGFR / chr7:55242466del18; ENST00000344576:c.2236_2253del; p.E746_T751del / 74 / ND / 24690 / 12 / 6619
#29 / WT / 1 / 68896
#30 / Y / BRAF / chr7:140453136A>T; NM_004333:c.1799T>A; p.V600E / ND / ND / 1 / 81003
#31 / Y / EGFR / chr7:55259524T>A; ENST00000344576:c.2582T>A; p.L861Q / 59 / 0.4 / 10510 / 12 / 5800
#32 / Y / EGFR / chr7:55242466del18; ENST00000344576:c.2236_2253del; p.E746_T751del / 6 / ND / 18416 / 12 / 5280
#33 / WT / 12 / 8071
#34 / KRAS / chr12:25398284C>T; ENST00000256078:c.35G>A; p.G12D / 27 / 1.5 / 79583 / 11 / 1012
#35 / ERBB2 / chr17:37880981ins12; ENST00000269571:c.2310_2311ins12; p.E770_A771ins12 / 18 / ND / 20410 / 12 / 7766
#36 / Y / WT / 12 / 3772
#37 / PIK3CA / chr3:178936092A>C; NM_006218.1:c.1634A>C; p.E545A / ND / 13 / 82722 / 12 / 606
#38 / EGFR / chr7:55248998ins9; ENST00000344576:c.2296_2297ins9; p.V769_D770ins9 / 49 / ND / 33210 / 12 / 8331
#39 / WT / 12 / 4609
#40 / Y / WT / 12 / 5610
#41 / Y / KRAS / chr12:25398284C>G; ENST00000256078:c.35G>C; p.G12A / 15 / ND / 43596 / 12 / 1255
#42 / Y / EGFR / chr7:55259515T>G; ENST00000344576:c.2573T>G; p.L858R / ND / ND / 12 / 3169
#43 / EGFR / chr7:55242469del18; ENST00000344576:c.2239_2256del; p.L747_S752del / 37 / ND / 65096 / 12 / 7422
#44 / EGFR / * / chr7:55248998ins9; ENST00000344576:c.2296_2297ins9; p.V769_D770ins9 / 19 / ND / 57714 / 12 / 2369
#45 / Y / EGFR / chr7:55241707; ENST00000344576:c.2155G>A; p.G719S / 12 / 0.5 / 19361 / 12 / 20911
#46 / Y / BRAF / chr7:140453136A>T; NM_004333:c.1799T>A; p.V600E / 15 / ND / 88080 / 12 / 588
#47 / Y / EGFR / chr7:55259515T>G; ENST00000344576:c.2573T>G; p.L858R / 15 / 4 / 1175 / Y / 12 / 721
#48 / Y / BRAF / chr7:140453136A>T; NM_004333:c.1799T>A; p.V600E / 47 / ND / 14982 / 12 / 2341
#49 / Y / ERBB2 / * / chr17:37880981ins12; ENST00000269571:c.2310_2311ins12; p.E770_A771ins12 / 25 / 11 / 52841 / 1 / 52841
#50 / Y / EGFR / chr7:55259515T>G; ENST00000344576:c.2573T>G; p.L858R / 8 / 2 / 72624 / 12 / 226
#51 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 59 / 11 / 28760 / Y / 12 / 7631
#52 / EGFR / chr7:55242469del18; ENST00000344576:c.2239_2256del; p.L747_S752del / ND / 0.4 / 76313 / 12 / 5454
#53 / Y / KRAS / chr12:25398284C>T; ENST00000256078:c.35G>A; p.G12D / 11 / 18 / 389 / Y / 12 / 492
#54 / Y / EGFR / chr7:55242469del18; ENST00000344576:c.2239_2256del; p.L747_S752del / 31 / ND / 32981 / 12 / 548
#55 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 86 / 19.5 / 13446 / Y / 12 / 3829
#56 / Y / ERBB2 / chr17:37880981ins12; ENST00000269571:c.2310_2311ins12; p.E770_A771ins12 / 43 / 0.3 / 9882 / 12 / 4219
#57 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 18 / ND / 36092 / 12 / 817
#58 / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 56 / 0.6 / 33098 / 12 / 561
#59 / Y / WT / 12 / 223
#60 / Y / EGFR / chr7:55259515T>G; ENST00000344576:c.2573T>G; p.L858R / 14 / ND / 55798 / 9 / 11932
#61 / Y / WT / 12 / 2036
#62 / Y / EGFR / chr7:55242464del15; ENST00000344576:c.2234_2248del; p.K745_A750del / 55 / 0.4 / 76559 / 9 / 7991
#63 / EGFR / chr7:55242466del18; ENST00000344576:c.2236_2253del; p.E746_T751del / 12 / ND / 44725 / 12 / 1284
#64 / Y / PIK3CA / * / chr3:178936091G>A; NM_006218.1:c.1633G>A; p.E545K / 5 / ND / 84646 / 1 / 84646
#65 / WT / 12 / 15391
#66 / Y / ERBB2 / * / chr17:37880981ins12; ENST00000269571:c.2310_2311ins12; p.E770_A771ins12 / 28 / ND / 19620 / 1 / 19620
#67 / Y / EGFR / chr7:55242465del15;ENST00000344576:c.2235_2249del;p.K746_A750del / 95 / 0.9 / 28373 / 12 / 18456
#68 / Y / EGFR / chr7:55259515T>G; ENST00000344576:c.2573T>G; p.L858R / 27 / ND / 86220 / 12 / 20732

* Indicates a mutation found in tDNA but that was not detected in routine analysis.

Tum - Tumor; Freq – frequency of the considered mutation; ITVC- Ion Torrent Variant Caller; Ampl - Amplicon; Cov – Coverage; WT- Wild Type; ND- Not Detected (indicates a false positive if figured in the tDNA column and a false-negative if figured in the cfDNA column); Y- Yes.
Supplemental Table S5. IonTorrent NGS test results in cfDNA, for each individual amplicon and overall, taking tDNA as a reference and restricted to stage IV samples (n=50)

n / cfDNA+ / cfDNA- / Sensitivity / Concordance rate
tDNA+ / tDNA- / tDNA- / tDNA+
PI3KCA exon 9 / 45 / 44 / 1 / 98%
PI3KCA exon 20 / 44 / 44 / 100%
EGFR exon 18 / 43 / 3 / 40 / 100% / 100%
EGFR exon 19 / 43 / 10 / 1 / 26 / 6 / 63% / 84%
EGFR exon 20 / 44 / 44 / 100%
EGFR exon 21 / 45 / 6 / 37 / 2 / 75% / 96%
BRAF exon 11 / 44 / 44 / 100%
BRAF exon 15 / 44 / 42 / 2 / 93%
KRAS exon 2 / 39 / 2 / 40 / 1 / 67% / 108%
KRAS exon 3 / 42 / 42 / 100%
ERBB2 exon 19 / 44 / 44 / 100%
ERBB2 exon 20 / 46 / 3 / 42 / 1 / 75% / 98%
Overall [95%CI] / 50 / 24 / 1 / 12 / 13 / 65%
[49%;78%] / 72%
[58%;83%]
All 12 amplicons together [95%CI] / 41 / 20 / 1 / 10 / 10 / 67%
[49%;81%] / 73%
[58%;84%]

Supplemental table S6 – NGS test results in tDNA and cfDNA for all amplicons concomitantly using the ITVC strategy (5A), or the in-house strategy (5B); and corresponding calculated sensitivity for all matched samples analyzed by NGS (n=68)

5A
ITVC strategy / tDNA
MT / WT
cfDNA / MT / 7 / 3
WT / 38 / 20
Sensitivity = 16%
5B
In house strategy / tDNA
MT / WT
cfDNA / MT / 26 / 3
WT / 19 / 20
Sensitivity = 58%

MT - Mutated; WT – Wild Type

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Supplemental Files VR1CCR_V3