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.