Valor de la determinación de ADN tumoral circulante en plasma mediante la tecnología Beaming. Aplicaciones clínicas presentes y futuras Clara Montagut Hospital del Mar, Barcelona

Putting RASpatient testing into context: Improved selection has enhanced the benefit of anti-EGFR therapies

Extended benefit of anti-EGFR therapy

Overall patient population

2

KRAS (exon 2) wt population

RAS wt population

OS benefit* CRYSTAL1,2 PRIME3 FIRE-3†4,5

PFS benefit* OPUS6,7 PEAK8

*Greater benefit in the RAS wt population than the KRAS (exon 2) wt population or ITT population

1. Van Cutsem E, et al. J Clin Oncol 2011;29:2011–2019; 2. Van Cutsem E, et al. J Clin Oncol 2015;33:692–700; 3. Douillard J-Y, et al. N Engl J Med 2013;369:1023–1034; 4. Heinemann V, et al. Lancet Oncol 2014;15:1065–1075; 5. Stintzing S, et al. ESMO 2014 (Abstract No. LBA11), updated information presented at the meeting: https://content.webges.com/library/esmo/browse/search/3PY#9faw02SG (accessed Feb 25 2016); 6. Bokemeyer C, et al. Ann Oncol 2011;22:1535– 1546; 7. Bokemeyer C, et al. Eur J Cancer 2015;51:1243–1252; 8. Schwartzberg, et al. J Clin Oncol 2014;32:2240–2247; 9. Erbitux® SmPC June 2014; 10. Vectibix® SmPC February 2015.

RAS detection Platform – Hospital del Mar KRAS exon 2: Therascreen KRAS exon 3,4 + NRAS exon 2,3,4: Pyrosequencing KRAS 12-13 KRAS 59 114; 32%

Mutation frequencies

KRAS 61 KRAS EXON 4

201; 56%

NRAS 12-13 1; 0% 7; 2%

10; 3%

10; 3% 15; 4%

NRAS 61

NO MUTADAS

Turnaround time

CALENDAR DAYS

40

MEAN MINIMUM

30

MAXIMUM 20 10 0 NOVEMBER DECEMBER JANUARY FEBRUARY

MARCH

APRIL

Global implementation of RAS testing: Average waiting times for results

11 days

16 days

11 days

14 days

14 days

6 days

12 days

9 days†

12 days

14 days

16 days

Patients typically wait 1–2 weeks or more before RAS test results are available, potentially leading to unnecessary delays in treatment initiation

10 days

The traditional tissue-based mutation testing process is familiar and established but contains challenges Invasive procedure

Obtain tumor tissue block

Selection bias Tumor heterogeneity

Manual microdissection

DNA isolation & purification

Assessment of RAS gene status

Assessment of DNA quantity

Tissue not always accessible Barriers to monitoring therapeutic effect

Treatment decision Archival tissue Static, potentially outdated mutation profile, often degraded or unavailable

Biopsia líquida: DNA tumoral circulante

ADN Tumoral (Mutado) Capacidad de Detección (ADN mutado/ ADN total) 100%

10%

1%

0.1%

Secuenciación Sanger

Pyrosecuenciación Real-Time PCR

0.01%

BEAMing

ADN Normal (Wild-Type)

Inostics Laboratory Validations (internal). Diehl F, et. al Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients. Gastroenterology. 2008 Aug;135(2):489-98.

BEAMing (Beads, Emulsion, Amplification and Amplification) PreAmplification

Emulsion PCR Hybridization

Flow Cytometry

Wild-type signal

Wild-type DNA Mutant & Wild-type DNA

Mutant DNA

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

Mutant signal

9

Aplicaciones Clínicas de la biopsia líquida en cáncer colorrectal

Aplicaciones Clínicas de los Test de ADN tumoral 1. Determinación RAS al diagnóstico circulante (Cáncer de Colorrectal) 2. Monitorizar respuesta / resistencia al tratamiento

Strengthening the evidence for use of liquid biopsy Overview of concordance data presented at ECC and WCGC (Sysmex Inostics)* 2015:testing RAS Abstract No. 402, P052 Hahn S, et al.

Abstract No. 2012, P002 Jones FS, et al.

92% overall concordance between liquid and tissuebased RAS testing1

93% overall concordance between liquid and tissuebased 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).

Concordance of RAS mutation detection in plasma and tumor tissue samples from metastatic colorectal cancer patients for selection fo anti-EGFR therapy Joana Vidal, Alba Dalmases, Gabriel Piquer, Roser Correa, Mar Iglesias, Gemma López, Frederick S. Jones, Joan Albanell, Beatriz Bellosillo, Clara Montagut Hospital del Mar, Barcelona

Objective To assess the value of plasma RAS mutation test for the selection of anti-EGFR therapy in mCRC patients by analysing its concordance with tissue RAS mutation testing

Design Retrospective analysis of determination of RAS mutations in paired plasma and tumor tissue samples obtained from primary tumor from mCRC patients anti-EGFR therapy - naïve

Material and methods Determination of KRAS and NRAS mutations (exon 2,3,4) in FFPE tissue samples: standard-of-care (SOC) by pyrosequencing tissue BEAMing in selected cases in Plasma: BEAMing OncoBEAM CRC KIT 34

Cut-off of 0.02% mutant fraction threshold for BEAMing, and 2% for SOC

Tissue RAS Result Plasma Ras Result

Positive

Negative

Total

Positive

17*

2

19

Negative

2

17

19

Total

19

19

38

Positive Agreement: 17/19: 89,47% Negative Agreement: 17/19: 89,47% Overall Agreement: 34/38: 89,47%

Proyecto BEAMing para RAS testing en España Evaluación clínica de OncoBEAM mediante el panel extendido de 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

Monitorización de la Resistencia a tratamiento anti-EGFR

Clonal dynamics and anti-EGFR treatment

Mutations of resistance emerge during anti-EGFR treatment KRAS KRAS

wt Basal tumor

wt

KRAS

wt EGFR

Response to treatment

Progression to treatment

S492R

I491M

K467T

G465R

S464L

R451C

PIK3CA exon 20

PIK3CA exon 9

BRAF exon 15

Pre-Treatment NRAS exon 4

NRAS exon 3

NRAS exon 2

KRAS exon 4

KRAS exon 3

KRAS exon 2

EGFR exon 12

PIK3CA exon 20

PIK3CA exon 9

BRAF exon 15

NRAS exon 4

NRAS exon 3

NRAS exon 2

KRAS exon 4

KRAS exon 3

KRAS exon 2

Patient #

Emergence of mutations of resistance during cetuximab treatment in colorectal cancer Post-Treatment EGFR exon 12

4

5

11 8

13

15

16

17

18

20

21

23

26

27

31

33

34

35

36

37

Montagut et al. Nat Med 2012 Arena&Bellosillo et al. Clin Cancer Res 2015

Clonal dynamics and anti-EGFR treatment

Mutations in KRAS emerge during anti-EGFR treatment and decline when treatment is suspendend KRAS KRAS

wt Basal tumor

wt

KRAS

wt EGFR

Response to treatment

Off treatment

Progression to treatment

Liquid biopsy for longitudinal monitoring of RAS mutations in blood of patients Rechallenge with cetuximab

Siravegna et al. Nat Med 2015

Aim: To assess the clinical relevance of monitoring mutations in serial plasma samples from RAS wt mCRC patients treated with cetuximab-based therapy in 1st line Cetuximab-based treatment in 1st line Diagnosis

During treatment

Progression

Post-progression

Tumor sample

Plasma sample

Baseline

Monthly

End of treatment

+3 / +6 months

Caso clínico Varón de 54 años

Antecedentes Familiares - sin antecedentes familiares de cáncer colorectal

Antecedentes Personales - Sin alergias medicamentosas conocidas - Hábitos tóxicos: ninguno - Fiebre reumática a los 18 años - Tratamiento habitual: ninguno

.

Enfermedad Oncológica Primer síntoma: • rectorragias + Sd tóxico

Exploración física • • • •

PS 0 AR: MVC sin ruidos sobreañadidos ACV: rítmico sin soplos ABD: no se palpan masas ni megalias, no semiología ascitis, peristaltismo presente

Analítica general: • Hb 11.6; perfil hepático normal, LDH 413 • CEA 2.3

.

Exploraciones complementarias Fibrocolonoscopia - tumoración no estenosante en recto (8cm margen anal)

Anatomía Patológica ADENOCARCINOMA intestinal moderadamente diferenciado RAS nativo

.

Exploraciones complementarias TAC tóraco-abdominal

• hígado:

segm VIII: 2.17cm segm VII: 1.53cm

segm VI: 2.4cm

segm V: 0.87cm

• 2 adenopatías en tronco celíaco (1 y 1,2cm)

Diagnóstico: adenocarcinoma de colon RAS nativo, estadio IV por metástasis hepáticas y adenopatías pericelíacas PLAN: Inicia FOLFOX + cetuximab Diagnóstico

Respuesta Parcial

FOLFOX + cetuximab

Progresión

¿Cuál de estas afirmaciones es verdadera?

A. La determinación de las mutaciones de RAS en sangre periférica es un proceso poco agresivo y rápido. B. La determinación longitudinal de RAS en sangre periférica nos puede dar información sobre la eficacia del tratamiento en este paciente. C. Es preciso usar técnicas de alta sensibilidad como BEAMing para poder determinar las mutaciones de RAS en sangre periférica. D. Todas son verdaderas.

Detección de adquisición de mutaciones de RAS en sangre periférica mediante BEAMing en paciente tratado con anti-EGFR tumor load (%) NRAS p.Q61L

150

% mutant alleles

100

5 50

0

0

Baseline

1st CT scan

PD

FOLFOX+CETUX

27-FEB-2012

01-MAY-2012

FOLFIRI+Aflibercept

01-AGO-2013

CT scan: PR

Last administration of anti-EGFR

22-DEC-2014

Tumor load (% of baseline)

10

Take-home message • La determinación de RAS al diagnóstico es obligatorio para identificar a los pacientes candidatos a tratamiento anti-EGFR.

• La detección de RAS en biopsia líquida ha demostrado alata correlación con la determinación en tejido en pacientes con CCRM. • La rapidez en los resultados de RAS en biopsia líquida permitiran una toma de decisiones óptima y sin retrasos. • La determinación de RAS en biopsias líquidas seriadas permite monitorizar la dinámica clonal de los tumores, detectar precozmente la aparición de resistencias y seleccionar el tratamiento adecuado en cada momento.

Gracias [email protected]

Clonal dynamics in a CRC patient treated with anti-EGFR therapy

tumor load (%) EGFR p.S492R

% mutant alleles

Tumor load (% of baseline)

KRAS p.Q61H

Baseline

1st CT scan 1st line

1st line PD

FOLFOX+CETUX

25-JUN-2009

1st CT scan 2nd line

2nd line PD

PANIT

21-AGO-2009 17-MAY-2010 1-NOV-2010 9-DEC-2010

CT scan: PR

13-APR-2011

CT scan: PR First administration of anti-EGFR (2nd line)

Last administration of anti-EGFR (1st line)

Last administration of anti-EGFR (2nd line)

Joana Vidal.Unpublished data

Experience of liquid biopsy RAS testing (BEAMing) Nurse Blood Extraction

Liquid biopsy RAS testing, together with tissue RAS determination, is routinely performed in all mCRC patients eligible for cetuximab/panitumumab treatment Liquid biopsy included in Electronic Medical Record

Oncologist

Biopsy

Test in tissue Test in plasma Pathologist

Molecular Testing

32