Biomarcadores

en cáncer colorectal

Perspectiva Clínica

Hacia una medicina molecular

Ramon Salazar MD, PhD Catalan Institute of Oncology

Terapia anti-EGFR

1. Van Cutsem E, et al. J Clin Oncol 2011; 29:2011-9;

2. Bokemeyer C, et al. Ann Oncol 2011; 22:1535-46;

3. Douillard JY, et al. J Clin Oncol 2011; 29(Suppl):3510 (oral presentation).

*Significant P-value; **Non-significant P-value;

pmab, panitumumab; cmab, cetuximab

• KRAS exon 2 mutational status is a key predictor of anti-EGFR mAbs efficacy and a detrimental effect has been observed for anti-EGFRs + FOLFOX in patients with KRAS MT tumours

• mCRC patients with WT KRAS exon 2 may benefit from anti-EGFR mAbs in combination with chemotherapy in all lines of treatment and as single agent in later lines

WT KRAS exon 2

CRYSTAL1 OPUS2 PRIME3

FOLFIRI FOLFIRI + cmab

FOLFOX FOLFOX + cmab

FOLFOX FOLFOX + pmab

RR, % 43 57 p=0.0001 34 57 p=0.002 48 57*

PFS, months 8.4 9.9 HR 0.70* 7.2 8.3 HR 0.56* 8.6 10 HR 0.80*

OS, months 20 23.5 HR 0.80* 18.5 22.8 HR 0.85** 19.7 23.9 HR 0.88**

MT KRAS

RR, % 36 31 52 34 0.02 41 40

PFS, months 7.7 7.4 HR 1.17** 8.6 5.5 HR 1.72* 9.2 7.4 HR 1.27*

OS, months 16.7 16.2 HR 1.03** 17.5 13.4 HR 1.29** 19.2 15.5 HR 1.17**

KRAS testing predicts efficacy to cetuximab and panitumumab in major

registration trials

DNA mutation in XXXX gene

DNA: normal

Ala Ala Ser Asn Leu Gln Trp Arg Protein: normal

DNA: mutated

Ala Ala Ser Asn Leu STOP Protein. truncated

MUTATION 9604C>T = Gln3126Stop in XXXX

N

Control

Patient

Sanger

Sensitivity of mutation detection 10-20%

ARMS Sensitivity of detection~1%

Mutations in codons 12, 13 and 61

(20 mutations panel)

Mutations in codons 12 and 13

(7 mutations panel)

TaqMelt

Sensitivity of mutation detection ~1-5%

Mutation detection kits in KRAS

Douillard J-Y, et al. N Engl J Med 2013;369:102334.

Percentages have been rounded. 7 patients harboured either

KRAS or NRAS codon 59 mutations. RAS and BRAF ascertainment rate: 89%.

KRAS, NRAS extended panel and BRAF mutation hotspots in

the PRIME trial (& others)

NRAS

KRAS

EXON 2 EXON 3 EXON 4 EXON 1

12 13 61 117 146

EXON 2 EXON 3 EXON 4 EXON 1

12 13 61 117 146

BRAF

EXON 15 EXON 16… EXON 1…

600

40% 4% 6%

3% 4% 0%

59

59

8%

Among WT KRASexon 2 patients, an additional

17% of tumours with RAS mutations were found

12 13 61 117 146 59

12 13 61 117 146 59

600

Overall RAS

ascertainment

rate: 90%

Other RAS mutations: CRYSTAL & OPUS

Ciardiello F et al. Ann Oncol 2014; Tejpar S et al. Ann Oncol 2014

Study N Method Sensitivity Other RAS

mt, %

CRYSTAL 430 BEAMing† 0.01% 14.7

OPUS 118 BEAMing† 0.01% 26.3

FIRE-3 407 Pyrosequencing 1-5% 16.0

PRIME 620 Dideoxy sequencing/WAVE 5% 17.4

PEAK 221 Dideoxy sequencing/WAVE 5% 23.1

WT KRAS ex 2 WT All RAS

Study PRIME (+/- Panitumumab)

N 656 512

PFS HR / p 0.8 / 0.02 0.72 / 0.04

OS HR / p 0.83 / 0.072 0.78 / 0.043

Study CRYSTAL (+/- Cetuximab)

N 666 367

PFS HR / p 0.7 / 0.001 0.56 / 0.0002

OS HR / p 0.8 / 0.009 0.69 / 0.002

Study FIRE-3 (Cetuximab vs Bevacizumab)

N 592 342

PFS HR / p 1.06 / 0.54 0.93 / 0.54

OS HR / p 0.77 / 0.017 0.7 / 0.011

Douillard JY, NEJM 2013; Ciardiello F, Ann Oncol 2014; Schwartzberg L, JCO 2013 (A 3631); Stintzing S, EJC 2013 (Proc ECCO)

All RAS mutations: additional efficacy effect

RAS mutations: Which is the right

sensitivity cut-off?

Ciardiello F et al. Ann Oncol 2014

RAS mutations: Which is the right

sensitivity cut-off?

Ciardiello F et al. Ann Oncol 2014

?

RAS mutations: Which is the right

sensitivity cut-off?

Ciardiello F et al. Ann Oncol 2014

?

?

RAS mt clone RAS wt clone

Sensitive tumor

RAS mutations: Which is the right

sensitivity cut-off?

Resistant tumor

Sensitivity cut-off?

Cobas 4800 (ROCHE) – Cobas Kits Sensitivity 1-5%

PyroMark (QIAGEN) – TherascreenPyro Kits Sensitivity 1%

BioMark Digital PCR Arrays (Fluidigm) Sensitivity 0.1%

LightCycler 480 Sensitivity 1-5 %

Sensitivity 5-10%

The puzzle of RAS testing

Slide provided by R. Salazar

BEAMing Sysmex Inostics Sensitivity 0.01%

ICORAS Panel – LightCycler and Fluidigm –

38 mutations (RAS+BRAF+PI3KCA)

Common mutations detected by the KRAS, NRAS, RAS Extension and BRAF Pyro Kit

81 mutations panel (RAS+BRAF) Sensitivity 5%

ICO-HUB National Referral Diagnostic Lab

Clinical Trial “ULTRA” (TTD)

N=80+

N=28+

N=82+

LightCycler Roche (ICO) Wave Surveyor (Others)

PyroMark Qiagen

Clinical Practice (routine)

Analysis Platforms Comparative Study (TTD-RTICC)

Cobas 4800 (ROCHE) – Cobas Kits Sensitivity 1-5%, extended panel

PyroMark (QIAGEN) – TherascreenPyro Kits Sensitivity 1%, 81 mut panel

BioMark Digital PCR Arrays (Fluidigm) Sensitivity 0.1%

LightCycler 480 Sensitivity 1-5 %

38 mut panel (sizeable up to 48)

Plataformas Next Generation Sequencing

Illumina MiSeq

Illumina HiSeq

Life Technologies Solid & Ion Torrent

Junior 454-Roche

Construcción librería para capturar genes a analizar

Access Array Target-specific Primers

Multiplicom Somatic 1MASTR

Illumina Trusight Tumor

Diseño personalizado, se ha de validar. Más flexibilidad, más económico

26 genes Está validado y en uso en varios laboratorios Menos flexibilidad, más caro

3 genes NRAS, KRAS, BRAF Está validado y en uso en varios laboratorios Menos flexibildad, máscaro

Agilent Sure Select

CONCORDANCE: High sensitivity methodologies for mutational analysis in

circulating tumor DNA

BEAMing technology Sensitivity 0.01

Fludigm technology Sensitivity 0.1

PlasmaSeq technology Sensitivity 0.1?

Vilar E & Tabernero J, Nature 2012

“Under pressure” clonal selection vs evolution

28

Treatment of Resistance in present

or future clinical trials

RAS mutants:

Cetuximab + Irinotecan in KRAS G13D mutant CRC….

MEK inhibitors + pan HER inhibitors

BRAF mutants:

BRAF + EGFR + PIK3Ca

S492R mutants:

Panitumumab

C-MET amplifications

C-MET inhibitors ?

Tumor debulking in unresectable metastasic disease

proof of principle cfDNA load as surrogate endpoint of

effective debulking of clones with resistance potential

Acknowledgements & Collaborations Agendia

Iris Simon

Paul Roepman

Annuska Glas

Sun Tian

Laura V´Veer

IDIBELL CRC Group, HUB & ICO CRTeam

Víctor Moreno

Adriana López-Doriga

Rebeca Sanz-Pamplona

UMC Leiden

Rob Tollenaar

Wilma Mesker

H. Del Mar BCN

Clara Montagut

NKI R. Bernards Lab

Anirudh Prahallad

Ian Majewski

Magali Michaut Slotervaart Hospital

Johan Westerga

Sjoerd Bruin

Rechts der Isar Munich Robert Rosenberg Ulricht Nitsche

Hospital Gasthuisberg Leuven Sabine Tejpar

Swiss Inst Bioinformat

Vlad Popovici Mauro Delorenzi

VHIO BCN

Josep Tabernero

J. Capdevila

Rodrigo Dienstmann

Gabriel Capellà

Alberto Villanueva

David García-Molleví

Dani Azuara

Marga Nadal

E. Kreisler, S.Biondo

Xavier Sanjuan

Antonio Soriano

Cristina Santos

Gemma Soler

Merche Martínez V.

Sonia Abenia

Alex Teulé

Julieta Grasselli

Maria Cambray

Xavi Pérez

JR Germà

UBB

LRT UGR

HUB UFCCR

ICO CRT

SOM ORT UIC

IRB

E Batlle E.Sancho

PEBC

M Esteller Ana Martínez

Idibell G. Thomas MDACC S. Kopetz

www.iconcologia.net

Institut Català d’Oncologia

ICO l’Hospitalet Hospital Duran i Reynals Gran Via de l’Hospitalet, 199-203 08907 l’Hospitalet de Llobregat

ICO Badalona Hospital Germans Trias i Pujol Ctra. del Canyet s/n 08916 Badalona

ICO Girona Hospital Doctor Trueta Av. França s/n 17007 Girona

ramonsalazar@iconcologia.net

PCR Digital

770 nanopocillos: se realiza una PCR en tiempo real en cada uno de ellos

PCR Digital

770 nanopocillos: se realiza una PCR en tiempo real en cada uno de ellos