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Supplementary appendixThis appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors.
Supplement to: Heinemann V, von Weikersthal LF, Decker T et al. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab as first-line treatment for patients with metastatic colorectal cancer (FIRE-3): a randomised, open-label, phase 3 trial. Lancet Oncol 2014; published online Aug 1. http://dx.doi.org/10.1016/S1470-2045(14)70330-4.
Webappendix
1
Table S1: Study centres in order of the number patients included into the FIRE-3 study (highest number
to lowest number)
Principal investigator Name of site City
Prof. Dr. med. Volker Heinemann Ludwig-Maximilians-Universität München München
Dr. med. Ludwig Fischer von Weikersthal MVZ Gesundheitszentrum St. Marien GmbH Amberg
Prof. Dr. med. Thomas Decker Studienzentrum Onkologie Ravensburg Ravensburg
PD Dr. med. Alexander Kiani Klinikum Bayreuth GmbH Bayreuth
Dr. med. Ursula Vehling-Kaiser Praxis Hämatologie • Onkologie • Palliativmedizin Landshut
Prof. Dr. med. Elke Jäger Krankenhaus Nordwest Frankfurt
PD Dr. med. Tobias Heintges Lukaskrankenhaus Neuss Neuss
Dr. med. Christian Lerchenmüller Gemeinschaftspraxis f. Hämatologie u. Onkologie Münster
PD Dr. med. Christoph Kahl Städtisches Klinikum Magdeburg Magdeburg
PD Dr. med. Gernot Seipelt Onkologische Schwerpunktpraxis
und Tagesklinik
Bad Soden
Prof. Dr. med. Frank Kullmann Klinikum Weiden Weiden
Dr. med. Martina Stauch Praxis für Hämatologie und internistische Onkologie Kronach
Ao. Univ. Prof. Dr. med. Werner
Scheithauer
Univ.-Klinik für Innere Medizin I Wien
Dr. med. Jörg Hielscher Klinik für Chirurgie, Chemnitz gGmbH Chemnitz
Dr. med. Michael Scholz Klinikum Stuttgart Krankenhaus Bad Cannstatt Stuttgart (Bad
Cannstatt)
Dr. med. Sebastian Müller Gemeinschaftspraxis Ansbach
Dr. med. Birgitta Killing Klinikum Wetzlar-Braunfels Wetzlar
Prof. Dr. Norbert Niederle Klinikum Leverkusen gGmbH Leverkusen
Dr. med. Andreas Rost Klinikum Darmstadt Darmstadt
Prof. Dr. med. Heinz-Gert Höffkes Städtisches Klinikum Fulda Fulda
PD Dr. med. Markus Moehler Johannes-Gutenberg Universität Mainz Mainz
Dr. med. Reinhard Udo Lindig Klinikum der Universität Jena Jena
Dr. med. Thomas W. Kubin Klinikum Traunstein Traunstein
Prof. Dr. med. Claudio Denzlinger Marienhospital Stuttgart
Prof. Dr. med. Michael Geißler Städtische Kliniken Esslingen Esslingen
Uni. Prof. Dr. med. Hellmut Samonigg LKH-Uni.Klinikum Graz
Univ.Klinik für Innere Medizin
Graz
Dr. med. Manfred Reeb Gemeinschaftspraxis Kaiserslautern
PD. Dr. Rudolf Schmits Praxis Drs. Jacobs, Prof. Daus, PD Schmits Saarbrücken
Dr. med. Sandra Ketzler Klinikum Westfalen Knappschaftskrankenhaus Dortmund Dortmund
Dr. med. Jorge Riera Knorrenschild Universitätsklinikum Gießen und Marburg GmbH, Standort
Marburg
Marburg
Dr. med. Oswald Burkhard Gemeinschaftspraxis Worms
CA Prof. Dr. med. Georg Maschmeyer Klinikum Ernst von Bergmann gGmbH Potsdam
Dr. med. Gerhard Puchtler Klinikum Rosenheim Rosenheim
Dr. med. Egbert Eggers Kreiskrankenhaus Torgau "Johann Kentmann" gGmbH Torgau
Dr. med. Jan Schleicher Katharinenhospital Stuttgart Stuttgart
Dr. med. Wolfram Bohle Katharinenhospital Stuttgart Stuttgart
Prof. Dr. med. Hartmut Link Westpfalz-Klinikum GmbH Kaiserslautern
Dr. med. Hanns-Detlev Harich Gemeinschaftspraxis Ambulante Chemotherapie Hof
Dr. med. Elisabeth Maria Apollonia Schnoy Klinikum der Universität Regensburg Regensburg
Prof. Dr. med. Wolfgang Schepp Klinikum Bogenhausen München
Prof. Dr. med. Jürgen Siebler Universitätsklinikum Erlangen Erlangen
Prof. Dr. med. Markus Lerch Universitätsklinikum Greifswald
Ernst-Moritz-Arndt-Universität
Greifswald
Dr. med. Gerald Gehbauer Praxis für Hämatologie und Onkologie Ingolstadt
Dr. med. Joachim Zimber Internistische Gemeischaftspraxis Nürnberg
Dr. med. Erik Engel Hämatologisch-onkologische Praxis Altona (HOPA) Hamburg
Dr. med. Ingrid Raßmann Praxis Privatklinik Dr. Schindlbeck Herrsching
Dr. med. Burkhard Schmidt Gemeinschaftspraxis München
Dr. med. Ulrich Römmele Kreiskliniken Esslingen
Klinikum Kirchheim-Nürtingen
Nürtingen
Prim. Uni. Prof. Dr. med. Christian Dittrich Kaiser-Franz-Josefspital Wien
Dr. med. Jens Uhlig Praxis Naunhof
Dr. med. Johannes Meiler Universitätsklinikum Essen WTZ-Ambulanz
Essen
CA Dr. med. Ulrich Fleck DRK Krankenhaus Luckenwalde Luckenwalde
Dr. med. Thomas Wolff Hanseklinik Stralsund Stralsund
Barbara Kempf Klinikum Landshut Landshut
Prof. Dr. med. Michael Kiehl Klinikum Frankfurt (Oder) GmbH Frankfurt (Oder)
Prof. Dr. med. Michael Koenigsmann MediProjekt GbR (Praxis) Hannover
Prof. Dr. med. Holger Hebart Klinikum Schwäbisch Gmünd
Stauferklinik
Mutlangen
Prof. Angsar Weltermann A.ö. Krankenhaus der Elisabethinen Linz Linz
2
CA Dr. med. Helmut Lambertz Klinikum Garmisch-Partenkirchen Garmisch-
Partenkirchen
Dr. med. Oliver Stötzer Praxis für Hämatologie und internistische Onkologie München
PD Dr. med. Rudolf Pihusch MVZ Pihusch Rosenheim
Dr. med. Wolfgang Bair Schlossbergklinik Oberstaufen
Gesellschaft der Schwesternschaft München vom BRK e.V.
Oberstaufen
Dr. med. Johannes Spes Kreiskliniken Altötting-Burghausen Altötting
Prof. Dr. med. Günther Schlimok Klinikum Augsburg Augsburg
Prof. Dr. med. Thomas Herrmann Klinikum Idar-Oberstein GmbH Idar-Oberstein
Dr. med. Rudolf Schlag Gemeinschaftspraxis Würzburg
Dr. med. Michael Schwittay Praxis Rötha
OA Dr. med. Johannes Andel LKH Steyr Steyr
Dr. med. Mathias Schulze Praxis für Innere Medizin Zittau
Dr. med. Christopher Haberl Klinikum St. Elisabeth Straubing
Dr. med. Ludwig Prügl Praxis Dr. Ludwig Prügl Zwiesel
Dr. med. Ulrike Söling Gemeinschaftspraxis Kassel
Dr. med. Matthias Respondek Gastroenterologische und Hämatologisch-Onkologische Praxis Stuttgart
Dr. med. Jens T. Siveke Klinikum Rechts der Isar München
Dr. med. Johann Weiß Schwerpunktpraxis Hämatologie/Internistische Onkologie Weiden
PD Dr. med. habil. Detlef Quietzsch Klinikum Chemnitz Chemnitz
Dr. med. Wolfgang Schneider-Kappus Praxis für Onkologie Ulm
Prof. Dr. med. Winfried Gassmann St. Marien-Krankenhaus Siegen Siegen
Dr. med. Matthias Demandt Gemeinschaftspraxis Straubing
Prof. Dr. med. Dirk Behringer Augusta-Krankenanstalt GmbH Bochum
Dr. med. Edgar Breunig Stiftung Juliusspital Würzburg Krankenhaus Würzburg
Dr. med. Gerlinde Maria Michl Praxis München
PD Dr.med. Nimrod Schwella Klinikum Ludwigsburg
Akademisches Lehrkrankenhaus der Universität Heidelberg
Ludwigsburg
Prof. Dr. med. Michael Müller Niels-Stensen-Kliniken Marienhospital Osnabrück
Osnabrück
Dr. med. Helmut Hitz Onkologische Schwerpunktpraxis München
Dr. med. Hans Rainer Slawik Gemeinschaftspraxis Augsburg
Dr. med. Christian Constantin Klinikum Lippe-Lemgo Lemgo
PD Dr. med. habil. Jürgen Truckenbrodt Georgius Agricola Klinikum Zeitz Zeitz
Dr. med. Volker Hagen St- Johannes-Hospital Dortmund Dortmund
Dr. med. Heinz-Albert Dürk St-Marienhospital Hamm
Dr. med. Florian Fauth Gemeinschaftspraxis Hanau am Main
Dr. med. Marc Walther SRH-Zentralklinikum Suhl gGmbH Suhl
Prim. Uni. Prof. Dr. med. Heinz Ludwig Wilhelminenspital Wien
Dr. med. Armin Schulz-Abelius Klinikum Altenburger Land GmbH Altenburg
Prim. Uni. Prof. Dr. med. Josef Thaler Klinikum Wels-Grieskirchen Wels
Dr. med. Bettina Peuser Onkologische Praxis am Diakonissenhaus Leipzig
Prof. Dr. med. Wolfgang E. Schmidt Universitätsklinik St. Josef Hospital
Klinikum der Ruhr-Universität Bochum
Bochum
Dr.med. Gerhard Feder Kreisklinik Ebersberg gGmbH Ebersberg
Dr. med. Herbert Kappauf Internistische Schwerpunktpraxis Hämatologie-Onkologie Starnberg
Prof. Dr. med. habil. Axel Matzdorff Caritasklinik St. Theresia Saarbrücken
PD Dr. med. Johann Mittermüller Internisten - Hämatologie - Onkologie Germering
Dr. med. Michael Perker Gemeinschaftspraxis Weilheim
PD Dr. med. Mathias Rummel Zentrum Innere Medizin der Justus-Liebig-Universität Gießen
Prof. Dr. med. Rainer Porschen Klinikum Bremen-Ost gGmbH Bremen
Prof. Dr. med. Albrecht Johannes Georg Pfeiffer
Klinikum Memmingen Memmingen
Dr. med. Birgit Luhn OncoResearch Lerchenfeld UG Hamburg
PD Dr. med. Dirk Graf Heinrich-Heine-Universität Düsseldorf Düsseldorf
Prof. Dr. med. Roland Repp Klinikum am Bruderwald,Sozialstiftung Bamberg Bamberg
OA Dr. med. Thomas Hoffmann Sophien- und Hufeland-Klinikum Weimar gGmbH
Klinik für Innere Medizin II
Weimar
Dr. med. Bernhard Koch St. Vincenz-Krankenhaus Datteln
Prof. Dr. med. Jörg Hoffmann St. Marienkrankenhaus Akadem. Lehrkrankenhaus der Johannes-Gutenberg-Universität
Mainz
Ludwigshafen
Dr.med. Stefan Schanz Kreisklinikum Siegen
Akadem. Lehrkrankenhaus der Uni Marburg
Siegen
Prof. Dr. med. Josef Menzel Klinikum Ingolstadt Ingolstadt
Dr. med. Henry Simon Kreiskliniken Esslingen gGmbH, Paracelsus-Krankenhaus Ruit Ostfildern
Prof. Dr. med. Olaf Koch Klinkum Osnabrück Osnabrück
Dr. med. Thomas Ettrich Universitätsklinik Ulm Ulm
3
Methods
KRAS and NRAS mutation screen
RAS mutation analyses were undertaken at the Molecular Pathology department of the Institute for Pathology of
the University of Munich under the supervision of Andreas Jung and Thomas Kirchner. The procedures for
KRAS and NRAS mutation testing were quality certified by the Quality Initiative in Pathology of the German
Society for Pathology and the Association of German Pathologists.
For the determination of the mutation status at hotspots within exons 2, 3 and 4 of both the KRAS and NRAS
genes, tumour-containing areas were marked on a haematoxylin and eosin (H&E) stained tissue section and the
marks transferred onto an unstained dewaxed serial section. The area was microscopically macrodissected and
the remaining tissue was stained by H&E and compared with the original H&E section. Microscopic
macrodissection was taken as valid when more than 200 cells of which at least 30% were tumour cells had been
selected. DNA was isolated from the tissue, in a final volume of 20 µl, using Qiagen FFPE micro kits together
with a QiaCube robot. 1 µl of the DNA solution was subjected to polymerase chain reaction (PCR)
amplification using KRAS or NRAS exon-specific primer pairs (table S1) and HotStar Taq Polymerase (Qiagen).
Each reaction included: 1 x PCR buffer, 1·5 mM MgCl2, 200 µM dNTP mix, 400 nM of each primer and 1 unit
of Taq polymerase; applying the amplification protocol: 1 x 15 minutes at 95° C, and 50 cycles (30 seconds at
95° C, 30 seconds at 60° C, 30 seconds at 72° C) followed by 1 x 2 minutes at 72° C. PCR products were
sequenced by pyrosequencing using amplicon specific sequencing primers (table S1) and Pyromark Gold kits
(Qiagen) following for each step the manufacturer’s recommendations. Analysis of the final data was carried out
independently by Andreas Jung and Sebastian Stintzing. Discordant cases were resolved jointly.
4
Table S2: PCR and sequencing primers for KRAS and NRAS mutation analyses
Gene Exon Primers
KR
AS
2
Forward1
Reverse1 Sequencing
Codons 12, 13
5ʹ NNNGGCCTGCTGAAAATGACTGAA 3ʹ
5ʹ Biotin~TTAGCTGTATCGTCAAGGCACTCT 3ʹ 5ʹ TGTGGTAGTTGGAGCT 3ʹ
3
Forward Reverse
Sequencing
Codon 61
5ʹ CAATTGATGGAGAAACCTGTCTCTT 3ʹ 5ʹ Biotin~TCCTCATGTACTGGTCCCTCATT 3ʹ
5ʹ TCTCTTGGATATTCTCGAC 3ʹ
4
Forward Reverse
Sequencing
Codon 146
5ʹ GGCTCAGGACTTAGCAAGAAGTTA 3ʹ Biotin~AGTTATGATTTTGCAGAAAACAGA 3ʹ
GAATTCCTTTTATTGAAAC 3ʹ
NR
AS
2 Forward
Reverse
Sequencing
Codons 12, 13
5ʹ CTTGCTGGTGTGAAATGACTGAG 3ʹ
5ʹ Biotin ~GGATTGTCAGTGCGCTTTT 3ʹ
5ʹ TGGTGGTGGTTGGAG 3ʹ
3 Forward
Reverse
Sequencing
Codons 59, 61
5ʹ AAACCTGTTTGTTGGACATACTG 3ʹ
5ʹ Biotin~TATTGGTCTCTCATGGCACTGT 3ʹ
5ʹ TTGTTGGACATACTGGAT 3ʹ
4
Forward
Reverse Sequencing
Forward Reverse
Sequencing
Codon 117
5ʹ Biotin~ATGATGTACCTATGGTGCTAGTGG 3ʹ
5ʹ CGTAACTCTTGGCCAGTTCG 3ʹ 5ʹ TCCTTGTTGGCAAATC 3ʹ
Codon 146
5ʹ CGAACTGGCCAAGAGTTACG 3ʹ 5ʹ Biotin~TGAAAGCTGTACCATACCTGTCTG 3ʹ
5ʹ TCCATTCATTGAAACCT 3ʹ
5
Table S3: Third-line treatment regimens
FOLFIRI plus cetuximab
n=107
FOLFIRI plus bevacizumab
n=118
Bevacizumab 37 (34·6) 23 (19·5)
Cetuximab or panitumumab 36 (33·6) 62 (52·5)
Irinotecan 30 (28·0) 42 (35·6)
Oxaliplatin 27 (25·2) 33 (28·0)
5-fluorouracil or capecitabine 83 (77·6) 76 (64·4)
Other 15 (14·0) 7 (5·9)
Data are number (%).
6
Table S4: Efficacy in patients with RAS mutant tumours according to treatment group*
Variable RAS mutant population (n=178)†
FOLFIRI plus
cetuximab n=92
FOLFIRI plus
bevacizumab n=86
p value‡
Objective response, n (%)
Complete response 0 3 (3·5) 0·11
Partial response 35 (38·0) 41 (47·7) 0·23
Stable disease 30 (32·6) 31 (36·0) 0·64
Progressive disease 13 (14·1) 6 (7·0) 0·15
Not evaluable 14 (15·2) 5 (5·8) 0·05
Response rate
n (%) 35 (38·0) 44 (51·2) 0·10
95% CI 28·1–48·8 40·1–62·1
Odds ratio (95% CI) 0·59 (0·32–1·06)
Progression-free survival
Progression events, No. (%) 86 (93·5) 77 (89·5)
Median, months (95% CI) 7·5 (6·1–9·0) 10·1 (8·9–12·2)
Hazard ratio (95% CI) 1·31 (0·96–1·78)
p value§ 0·085
Overall survival
Deaths, n (%) 74 (80·4) 67 (78·8)
Median, months (95% CI) 20·3 (16·4–23·4) 20·6 (17·0–26·7)
Hazard ratio (95% CI) 1·09 (0·78–1·52)
p value§ 0·60
*FOLFIRI denotes 5-fluorouracil, folinic acid and irinotecan. †Comprising patients excluded from the intention-
to-treat population because of a tumour KRAS exon 2 mutation (or with no initial result, but where a KRAS exon
2 mutation was subsequently identified) combined with those patients from the current analysis with other RAS
mutations (n=113 plus n=65, respectively). ‡The p value was calculated with the use of a two-sided Fisher’s
exact test. §p values were calculated with the use of log-rank tests.
7
Figure S1: Exploratory subgroup analysis of overall survival in patients without tumour KRAS exon 2
mutation
8
Figure S2: Kaplan-Meier estimates of progression-free (A) and overall survival (B) in the overall RAS
mutant population, according to treatment group
Pro
ba
bil
ity o
f P
rog
res
sio
n-f
ree
Su
rviv
al
0·0
0·25
0·50
0·75
1·0
12 24 4836 60 72
Progression-free Survival (months)
92
86
No. at risk
23
30
3
2
2 2 2FOLFIRI + cetuximab
FOLFIRI + bevacizumab
Hazard ratio (95% CI): 1·31 (0·96–1·78)
p=0·085
A RAS Mutant Population
Median
(months)
95% CI
FOLFIRI plus cetuximab 7·5 6·1─9·0
FOLFIRI plus bevacizumab 10·1 8·9─12·2
9
Pro
ba
bil
ity o
f O
ve
rall
Su
rviv
al
0·0
0·25
0·50
0·75
1·0
12 24 4836 60 72
Overall Survival (months)
92
86
No. at risk
73
66
27
31
16
166
6
2
3
FOLFIRI + cetuximab
FOLFIRI + bevacizumab
Hazard ratio (95% CI): 1·09 (0·78–1·52)
p=0·60
B RAS Mutant Population
Median
(months)
95% CI
FOLFIRI plus cetuximab 20·3 16·4─23·4
FOLFIRI plus bevacizumab 20·6 17·0─26·7
10
Reference
1 Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutation
detection by pyrosequencing. J Mol Diagn 2005; 7: 413-21.
Recommended