Are Gene Signatures Ready for Use in the Selection of Patients for Adjuvant Treatment?

Cáncer colorrectal: Nueva clasificación molecular. ¿Qué hacemos ahora? Implicaciones en la práctica asistencial

Dr. Ramon Salazar Institut Català d’Oncologia L’Hospitalet de Llobregat, Barcelona Molecular criteria (tumor)

– CIN, MSI (dMMR) – Individual mutations/amplifications/fusions…. • (RAS/BRAF…) – Supervised signatures • (Oncotype/Coloprint…) – CRCSC Consensus Classification • (Intrinsec subtypes)

PROGNOSIS & PREDICTION Clinical setting 2 MSI (stage II/III) – ACCENT DBase

MMR data available in 7803 patients (IHC and/or MSI testing)

Stage II: MSI Good Prognosis 5FU no benefit in MSI & marginal benefit in MSS

Stage III: MSI Good Prognosis 5FU benefits both MSI and MSS Sargent et al., ASCO Annual Meeting 2014 C07 (FULV+/-Ox) and C08 (Ffox+/-Bev): Stage II and III Colon Cancer

• MSI & BRAF: poor prognostic after relapse • Oxaliplatin: benefits stage III both MSS & MSI, no benefit in stage II

Gavin P G et al. Clin Cancer Res 2012;18:6531-6541 *Pogue-Geile K et al, JNCI 2013: 105 , 989-992 5 Phase 1B study of vemurafenib in combination with irinotecan and cetuximab in patients with BRAFV600E-mutated advanced cancers and metastatic colorectal cancer David S. Hong1, Van Morris2, Badi El-Osta1, Siqing Fu1, Michael Overman2, Sarina Piha-Paul1, Vivek Subbiah1, Bryan Kee2, Apostolia Tsimberidou1, Ralph Zinner1, David Fogelman2, Jorge Bellido1, Imad Shureiqi2, Funda Meric-Bernstam1, Scott Kopetz2

ASCO 2015 HERACLES TRIAL A: Lapatinib + Herceptin in HER ++/+++ Fish >50%

Responses by HER2 IHC Score 35% RR 78% Dis. control

Presented By Salvatore Siena at 2015 ASCO Annual Meeting Molecular criteria (tumor)

– CIN, MSI (MMR)/CIMP/hypermutated – Individual mutations • (RAS/BRAF…) – Prognostic Signatures (stage II) • (Supervised trainning e.g. Oncotype/Coloprint…) – CRCSC Consensus Classification • (Intrinsec subtypes)

PROGNOSIS & PREDICTION

8 ONCOTYPE DX® IN CALGB 9581 CALGB 9581 trial: edrecolomab vs observation – stage II colon cancer

The 12-gene RS was calculated by using the prespecified genes and algorithm previously validated in QUASAR

Multivariable Analysis: Relationship of RS, T Stage, and MMR with Risk of Recurrence

Recurrence risk % patients 5-year RR Recurrence risk % patients 5-year RR groups groups Low RS NR 12% (10% - 15%) Low RS 44% 13% (10% - 16%)

Int RS NR 15% (12% - 17%) Int RS 34% 16% (13% - 19%)

High RS NR 18% (14% - 22%) High RS 22% 21% (16% - 26%)

Recurrence risk in the overall population. NR = not reported Subgroup analysis in T3-MSS patients

Venook et al., J Clin Oncol 2013 ONCOTYPE DX® IN NSABP C07 (FULV+/-Ox) st II & III Algorithm for chemotherapy decision Colon Cancer Oncotype Dx®/ColoPrint®/Veridex/GeneF® Colon Stage (others II run-ups: ColoStage, microRNA, CDX2)Stage III recommended if uncertain decision: MSI MSS NO MSI Determination • T3 MSS if chemo considered only if high riskneeded signature CT may be discussed Other Markers: T3/T4, Other Factors: age, patient wish... • T4 MSS if follow-Prognosticup w/o signatures?* chemo considered only if low risk signature NO CT Good prognosis, FOLFOX resistance to FU CRCSC – Original Individual Group Classifiers Dissecting Colorectal Cancer in multiple subtypes

Guinney, Dienstmann et al, Nat Med 2015 Proposed taxonomy of colorectal cancer

Prognosis

Liver Surgery outcome? ? Dissecting Colorectal Cancer in multiple subtypes

Relapse-free survival

Guinney, Dienstmann et al, Nat Med 2015 A B C

Salazar et al, J Clin Oncol 2011: 29:17-24 Dissecting Colorectal Cancer in multiple subtypes

Survival after relapse

Guinney, Dienstmann et al, Nat Med 2015 Dissecting Colorectal Cancer in multiple subtypes

Guinney, Dienstmann et al, Nat Med 2015 Proposed taxonomy of colorectal cancer

Prognosis

Drug ? ? ? ? response prediction Dissecting Colorectal Cancer in multiple subtypes

Supervised immune and stromal infiltration analysis CMS1 CMS2 CMS3 CMS4

Becht et al, Clin Car Res 2016 Dissecting Colorectal Cancer in multiple subtypes

Supervised immune infiltration analysis

CMS1 CMS2 CMS3 CMS4

Becht et al, Clin Car Res 2016 Dissecting Colorectal Cancer in multiple subtypes

6 cells Broad Institute Pharmacogenomic collection

Unpublished Proposed taxonomy of colorectal cancer

Prognosis

Drug Inmune Oncogene Metabolism TGF- response checkpoints drivers & inhibition prediction blockade Amplifications DNA damage + Inmune checkpoint blockade Summary

• Prognostic Clinically Relevant Molecular Variables – MSI in Stage II – BRAF and MSI in stage IV – Supervised & Intrinsec Signatures? • Predictive Clinically Relevant Molecular Variables – MSI in stage II (5FU) and IV (PD1) – RAS and BRAF in stage IV – CMS 1-4 (Clinical Trials) – Still unknown what combination of genomic features (mutation + MSI + gene expression + immune + stromal) will provide will provide the best prediction of drug response.

CRSC: IDIBELL CRC Group, Acknowledgements & Collaborations HUB & ICO CRTeam Víctor Moreno Agendia UMC Leiden UBB Adriana López-Doriga Iris Simon Rob Tollenaar Rebeca Sanz-Pamplona Paul Roepman Wilma Mesker Susanna Aussó, Annuska Glas NKI R. Bernards Lab LRT Gabriel Capellà Sun Tian Anirudh Prahallad UGR Alberto Villanueva Laura V´Veer Begoña Diosdado Dani Azuara Slotervaart Magali Michaut M. Nadal, M. Martínez Hospital VHIO BCN Johan Westerga HUB E. Kreisler, S.Biondo Josep Tabernero Xavier Sanjuan Sjoerd Bruin UFCCR Hospital Gasthuisberg Rodrigo Dienstmann Antonio Soriano Leuven E.Élez, Fiorella Ruiz, Cristina Santos Sabine Tejpar Ariadna Garcia, Marta Gemma Soler Vilaro, ICO Rechts der Isar H. Del Mar BCN CRT Merche Martínez V. Munich Clara Montagut SOM Alex Teulé Robert Rosenberg UIC Julieta Grasselli Ulricht Nitsche IRB Idibell E Batlle M Esteller Xavi Pérez Swiss Inst Bioinformat E.Sancho JL Manzano Vlad Popovici G.Thomas

Mauro Delorenzi MDACC S. Kopetz BACK up slides…. Other RAS mutations: CRYSTAL & OPUS

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

Ciardiello F et al. Ann Oncol 2014; Tejpar S et al. Ann Oncol 2014 RAS mutations: Which is the right sensitivity cut-off?

? ?

Ciardiello F et al. Ann Oncol 2014 The puzzle of RAS testing

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

PyroMark (QIAGEN) – TherascreenPyro Kits Sensitivity 1%

Sensitivity 5-10%

BioMark Digital PCR Arrays (Fluidigm) Sensitivity 0.1%

BEAMing Sysmex Inostics LightCycler 480 Sensitivity 0.01% Sensitivity 1-5 %

Slide provided by R. Salazar Response to anti-EGFR according to fraction of mutant alleles by quantitative digital PCR

p<0.005

alleles

mutated % of %

CR PR SD PD n=1 n=29 n=26 n=37

D.Azuara, C. Santos, et al. Nanofluidic digital PCR and extended genotyping of RAS and BRAF for improved selection of metastatic colorectal cancer patients to anti-EGFR therapies MOLECULAR CANCER THERAPEUTICS. In Press Improvement in response prediction by assessing mutation status of each gene

Vilar E & Tabernero J, Nature 2012 Treatment of Resistance (primary or secondary) 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 or WNT inhibitors  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 33

Adjuvant chemotherapy - benefit QUASAR

QUASAR col. group, Lancet 2007 Risk stratification in stage II colon cancer

• Clinicopathological features • Microsatellite instability, KRAS and BRAF • Supervised Prognostic signatures – Do gene signatures currently add information to the decision making progress? • Intrinsic molecular subtypes

ACCENT – MSI (stage II)

MMR data available in 7803 patients (IHC and/or MSI testing)

dMMR patients had better outcome without adjuvant treatment  5FU resistance should not be considered for chemo

Sargent et al., ASCO Annual Meeting 2014 ACCENT – MSI (stage III)

Treated dMMR stage III retained favourable prognosisMMR should not guide decission in stage III MOSAIC and NSABP-C07 Oxaliplatin seem to benefit equally both MSS and MSI patients*  both MSI and MSS should be considered for chemo (OXL based) Sargent et al., ASCO Annual Meeting 2014 *Gavin PG et al., Clin Cancer Res 2012; Fléjou JF et al., ASCO Annual Meeting 2013

C07 (FULV+/-Ox) and C08 (Ffox+/-Bev): Stage II and III Colon Cancer

• MSI: prognostic factor with HR 0.48 for RFS (and OS) • MSI: oxaliplatin similar benefit in pMMR and dMMR • BRAF (& MSI): poor prognostic after relapse • MSI: predictive of BEV benefit OS HR 0.52 p:0.02 favours BEV *

Gavin P G et al. Clin Cancer Res 2012;18:6531-6541 *Pogue-Geile K et al, JNCI 2013: 105 , 989-992 STADES II & III

RFS DFS OS

100 100 100

)

(

( %

80 80 80

(

i l

60 60 i 60

b b

LV5FU2 LV5FU2 b LV5FU2

b b

FOLFOX4 o FOLFOX4 FOLFOX4

r r

40 40 o 40

S F

F 20 20 20

R D 0 0 0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (years) Time (years) Time (years) Nb at risk Stage II and III dMMR LV5FU2 51 44 40 40 37 37 36 32 29 27 20 51 44 40 40 37 37 36 32 29 27 20 51 47 45 43 42 41 39 34 31 29 22 FOLFOX4 44 40 39 38 38 37 36 35 34 32 MOSAIC26 44 40 39 38 38 37study 36 35 34 32 26 44 41 40 39 39 39 38 37 36 24 28 STADE II STADE III DFS DFS 100 100

80 80

) )

% %

( (

60 y 60

t t

i i

l l

i i b

LV5FU2 b LV5FU2

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40 FOLFOX4 b 40 FOLFOX4

o o

r r

p p

S S

F F D 20 D 20

0 0 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Time (years) Time (years) Nb at risk Nb at risk LV5FU2 22 21 20 20 19 19 18 16 14 13 9 LV5FU2 29 23 20 20 18 18 18 16 15 14 11 FOLFOX4 26 25 24 24 24 23 23 23 23 21 17 FOLFOX4 18 15 15 14 14 14 13 12 11 11 9

Fléjou JF, ASCO 2013

Algorithm for chemotherapy decision Colon Cancer

*AreStage II gene signatures readyStage for III use in the selection of patients MSI MSS NO MSI Determination for adjuvant treatment? needed CT may be discussed Other Markers: T3/T4, BRAF, KRAS? Other Factors: age, patient wish... Prognostic signatures?*

NO CT Good prognosis, FOLFOX resistance to FU ONCOTYPE DX® IN QUASAR

Recurrence risk % patients 3-year RR groups

Low RS 43.7% 12% (0% - 16%)

Int RS 30.7% 18% (13% - 24%)

High RS 25.6% 22% (16% - 29%)

Kerr et al., ASCO Annual Meeting 2009 Gray et al., J Clin Oncol 2011 ONCOTYPE DX® IN CALGB 9581 CALGB 9581 trial: edrecolomab vs observation – stage II colon cancer

The 12-gene RS was calculated by using the prespecified genes and algorithm previously validated in QUASAR

Multivariable Analysis: Relationship of RS, T Stage, and MMR with Risk of Recurrence

Recurrence risk % patients 5-year RR Recurrence risk % patients 5-year RR groups groups Low RS NR 12% (10% - 15%) Low RS 44% 13% (10% - 16%)

Int RS NR 15% (12% - 17%) Int RS 34% 16% (13% - 19%)

High RS NR 18% (14% - 22%) High RS 22% 21% (16% - 26%)

Recurrence risk in the overall population. NR = not reported Subgroup analysis in T3-MSS patients

Venook et al., J Clin Oncol 2013 ONCOTYPE DX® IN NSABP C07 (FULV+/-Ox) st II & III ColoPrint® Whole Genome analysis on 44K Agilent microarrays 18 genes identified that correlate Distant Metastasis-free Survival, 18 most stable genes selected

18 gene signature 70% N = 188 (SI:24; SII:100; SIII:56; SIV:8) RC: 92; LC:74; Rectum: 17)

No Chemo: 145; Chemo: 31

30% DMFS HR = 3.41 (P> 0.0001) 5-year DMFS 82%(95CI, 76-89%) low-risk 50%(95CI, 38-66%) high-risk

Salazar et al., J Clin Oncol 2011 Algorithm for chemotherapy decision Colon Cancer Oncotype Dx®/ColoPrint®/Veridex/GeneF® Colon Stage II (others run-ups: microRNA) Stage III recommended if uncertain decision: MSI MSS NO MSI Determination • T3 MSS if chemo considered only if high riskneeded CT may be discussed signature Other Markers: T3/T4, BRAF, KRAS? • T4 MSS ifOther follow Factors: age,-up patient w/o wish... Prognosticchemo signatures?* considered only NOif CT low risk signature Good prognosis, FOLFOX resistance to FU