Beatriz Bellosillo

Pathology Department & Cancer Research Program

Hospital del Mar & Institut Hospital del Mar d’ Investigacions Mèdiques (IMIM)

Estudio de biomarcadores en biopsialíquida en cáncer de colon

Molecular markers in solid tumors

BRAF NRAS

KIT

Papillary: BRAF

Metastatic melanomaMetastatic thyroid

Lung cancer

KRASNRASBRAF

BRAFBRCA1/BRCA2

Metastatic serous low-grade ovarian

Metastatic colon

H. Davies et al. Nature 2002

EGFRKRAS ALKROSRET

Predictive biomarkers are dynamic and evolve upon time

Response to treatment

Primary resistance to anti-EGFR therapy

Primary (innate) Resistance

no response to treatment

Biomarkers: KRAS & NRAS

40% KRASmut (12% also PI3Kmut)

6% NRASmut

Predictive biomarkers are dynamic and evolve upon time

Primary resistance to anti-EGFR therapy

6% NRASmut

Benefit from anti-EGFR therapy

Amado, JCO 2008Karapetis, NEJM 2008VanCutsem, NEJM 2009Peeters, JCO 2010Douillard, NEJM 2013

40% KRASmut (12% also PI3Kmut)

6% NRASmut 7% BRAFmut

Qua

drup

le n

egat

ive

Predictive biomarkers are dynamic and evolve upon time

Primary resistance to anti-EGFR therapy

6% NRASmut 7% BRAFmut

DeRoock, Lancet Oncol 2010

10% PI3KmutQua

drup

le n

egat

ive

Benefit from anti-EGFR therapy

Mechanisms of primary resistance in 116 KRAS wildtypecolorectal cancer tumors treated with cetuximab

Bertotti. Nature 2015

40% KRASmut (12% also PI3Kmut)

6% NRASmut 7% BRAFmut

Predictive biomarkers are dynamic and evolve upon timePrimary resistance to anti-EGFR therapy

10% PI3Kmut 3% HER2 mut 2% FGFR1 ampl

2% PDGFR

ampl

Benefit from anti-EGFR therapy

1% METampl

2% MEK1mut

4% HER2 ampl 1% EGFR mut

RAS testing has become integral to 1st line

treatment decision making

“The appropriate molecular analyses

National Comprehensive Cancer Network

(NCCN) 20161

Proposed ESMO consensus

2015/162

Current guidelines

1. NCCN Guidelines Version 2.2016 Colon Cancer Available at www.nccn.org/professionals/physician_gls/pdf/colon.pdf. Last accessed Feb 2016; 2. Van Cutsem E, et al. ‘The ESMO consensus on metastatic CRC – 2015’ (presented at WCGC 2015).

“The appropriate molecular analyses

are to be performed at the time of

diagnosis of mCRC and should comprise

a full RAS analysis, with simultaneous

BRAF analysis in a validated

laboratory/testing center to provide the

best diagnosis and prognostic decision

making”

“The panel strongly recommends

genotyping of tumor tissue (either primary

tumor or metastasis) in all patients with

metastatic colorectal cancer for RAS (KRAS

exon 2 and non-exon 2; NRAS) and BRAF at

diagnosis of stage IV disease”

Global implementation of RAS testing:

patients tested before 1st line treatment

85%

93%

86%

78% 79%

98%

91%

100%

95%

93%89%

81%

87%†

*Market research data from Q3 2015†KRAS 1. Merck, Data on file

EXON 1 EXON 2 EXON 3 EXON 4NRAS

EXON 1 EXON 2 EXON 3 EXON 4

12

KRAS

13 59 61 117 14

6

12 13 59 61 117 14

6

MUTATED

47%

NOT

MUTATED

53%

Samples recieved 1200

Samples analysed 1185

RAS mutational analysis in colorectal cancer (n=1200)

NO MUTATED

KRAS 12/13

KRAS 146

NRAS 61

KRAS 61

NRAS 12/13

KRAS 59

KRAS 117

nº of cases %

NOT MUTATED 621 52,41

KRAS 12/13 422 35,61

KRAS 146 44 3,71

NRAS 61 34 2,87

KRAS 61 24 2,03

NRAS 12/13 25 2,11

KRAS 59 5 0,42

KRAS 117 5 0,42

Samples analysed 1185

MUTATED 559

NOT MUTATED 621

396,87

13,7

NO MUTADO

KRAS

RAS + BRAF mutational analysis in colorectal cancer (n=197)

42,14

NRAS

BRAF

RAS + BRAF mutation testing with a threshold of 1% improved prediction of response to anti-EGFR therapy

Azuara et al. Mol Cancer Ther 2016

Cell-Free

Tumor DNA

Cell-Free

Normal DNA

Circulating

Tumor Cell

• Ausencia de tejido disponible

• Escasa,

• difícil acceso,

• rebiopsias

• Urgencia respuesta

• Heterogenicidad tumoral

• Phase I; 40%

Tumoral DNA (mutated)

Normal DNA(Wild-Type)

• Phase I; 40%• Phase II; 70%• Phase III; 60%• Registry study; 30%

Cell-Free DNA is Detectable Most Late Stage Cancers (100% in

mCRC)

(n = 177)

Bettegowda et al, Sci Tran Med Feb 2014

15

OncoBEAM® RAS CRC IVD KIT

EXON 1 EXON 2 EXON 3 EXON 4NRAS

EXON 1 EXON 2 EXON 3 EXON 4

12

KRAS

13 59 61 117 14

6

12 13 59 61 117 14

6

BEAMing (Beads, Emulsion, Amplification and Amplification)

Pre-Amplification

Emulsion PCRHybridization

Flow Cytometry

BEAMing (Beads, Emulsion, Amplification and Amplification)

Dressman et al. PNAS, 2003Diehl et al. PNAS, 2005

17Mutant signal

MutantDNA

Mutant &Wild-type DNA

Wild-type DNA

Wild

-typ

e si

gnal

Extracción de sangre

Emulsión PCR

Ruptura &

Hibridización

Preparación del

Plasma

Flujo de trabajo de BEAMing

ABI Veriti PCR

1 hr

3 hr

9 hr

Basado en el rendimiento de 6 pruebas / semana

Extracción del ADN

Amplificación por

PCR

Hibridización

Citometria de Flujo

Análisis de los datos

& Informe

QC: Cuantificación

del ADN por qPCR

“Realtime“

QC: Gel de Agarosa

Electroforesis

18

Rainin Pipettors

Qiagen DNA Extraction Manifold

Partec Cube 6

2 hr

9 hr

Total de respuesta en tiempo: 48 horas

Placa Magnética

Overview of concordance data presented at ECC and WCGC 2015:

RAS testing (BEAMing, Sysmex Inostics)

Abstract No. 402, P052Hahn S, et al.

92% overall concordance between

liquid and tissue-

Abstract No. 2012, P002Jones FS, et al.

93% overall concordance between

liquid and tissue-liquid and tissue-based RAS testing1

liquid and tissue-based RAS testing2

Fully in line with data from WCGC

20153

1. Hahn S, et al. ECC 2015 (Abstract No. 402);2. Jones FS, et al. ECC 2015 (Abstract No. 2012);3. Scott R, et al. WCGC 2015 (Abstract No. P-273).

Tissue RAS Result

Plasma Ras

Result

Positive Negative Total

Positive 25* 2 27

Negative 1 22 23

Total 26 24 50

Plasma/Tissue Correlation - H. del Mar Experience

Total 26 24 50

* 1 positive in tissue and plasma but in different codon

Positive Agreement: 25/26: 96.15%

Negative Agreement: 22/24: 91.66%

Overall Agreement: 47/50: 94%

J Vidal1,2*, L Muinelo Romay4,5*, A Dalmases2,3, A Abalo4,5, MC Vela3, M Abreu4,5, M Muset3, J Ruiz4, M Iglesias2,3, C Blanco4, E Lopez1, C Rodríguez4, F S. Jones6, D L Edelstein6, A Lukas7, J Albanell1,2, B

Bellosillo2,3, S Candamio4, C Montagut#1,2, R López4,5#

Tissue RAS Result

Plasma Ras

Result

Positive Negative Total

Positive 43* 5 48

Accuracy of Plasma RAS mutation testing for therapy selection and monitoring of colorectal cancer patients

Result

Negative 2 52 54

Total 45 57 102

* 1 positive in tissue and plasma but in different codon

Positive Agreement: 43/45: 95.6%

Negative Agreement: 52/57: 91.2%

Overall Agreement: 95/102: 93.1%

Proyecto BEAMing para RAS testing en España

Evaluación clínica de OncoBEAM (34 mutaciones de RAS)

Comparación de la determinación de la mutación RAS en ADN tumoral circulante y en muestra de tejido en pacientes con cáncer colorrectal metastásico

Participan 9 hospitales españoles

Tissue /plasma KRAS mutation concordance 76%

Tabernero et al, Lancet Oncology 2015

Tissue /plasma KRAS mutation concordance 76%

Tissue /plasma BRAF mutation concordance 97%

Tissue /plasma PIK3CA mutation concordance 88%

Determinación de BRAF -Idylla

1h 30 min

Tissue BRAF Result

Plasma

BRAF Result

Positive Negative Total

Positive 6 2 8

Negative 2 6 8

Total 8 8 16

Plasma/Tissue Correlation - H. del Mar Experience

Total 8 8 16

* 1 positive in tissue and plasma but in different codon

Positive Agreement: 6/8: 75%

Negative Agreement: 6/8: 75%

Overall Agreement: 12/16: 75%

primary resistance

to treatment

secondary resistance

to treatment

Challenge 3Acquired resistance to anti-EGFR therapy in CRC

response to

treatment

Jonker. N Engl J Med 2007; Amado. J Clin Oncol 2008

All KRAS wt patients treated with cetuximab / panitumumab presenttumor progression after 1 year

C1476T, S492R mutation

cetuximab

Ser492

EGFR

Montagut et al. Nature Medicine 2011

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✗✔ ✔

✔

✔✔

Serumsamples

✔ ✔

✗

✗

✗

Conclusiones

• El análisis de mutaciones en KRAS y NRAS es imprescindible

previo al inicio con terapia anti-EGFR

• Existe una buena correlación entre la determinación de

mutaciones en plasma y en tejido

• El análisis de ADN circulante es útil para determinar el perfil

mutacional en el momento de la aparición de resistencia al

tratamiento

Oncology Dpt , Hospital del MarOncology Dpt , Hospital del Mar

Clara MontagutJoan AlbanellJoana VidalAna RoviraJoaquim BellmuntLaura VisaEva López

Pathology Dpt, Hospital del Mar

Beatriz BellosilloSergi SerranoAlba DalmasesMar IglesiasMari Carmen Vela Gabriel PiquerMercè Muset