Download Valor de la determinación de ADN tumoral circulante en plasma

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Static, potentially outdated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