Transcript BRAF - Cipomo

ISTITUTO NAZIONALE PER LO
STUDIO E LA CURA DEI TUMORI
FONDAZIONE G. Pascale – NAPOLI
SC Biologia Cellulare e Bioterapie
CENTRO RICERCHE ONCOLOGICHE
MERCOGLIANO (AV)
Laboratorio di Farmacogenomica
Caratterizzazione molecolare dei
tumori: impatto sulla pratica clinica
Nicola Normanno
Genomic alterations affecting actionable
signaling pathways
Garraway JCO 2013
Genomics-Driven Oncology
Garraway JCO 2013
The major classes of genomic
alterations that give rise to cancer
FISH,
Immunohistochemistry
Sequencing,
Real Time PCR
etc.
EGFR
ErbB-2
MET
EGFR
ErbB-2
BRAF
KRAS
NRAS
PIK3CA
AKT1
MAP2K1
EML4-ALK
ROS-1
RET
Modified from McConaill JCO 2010
Genotyping and genomic profiling
in personalized medicine
Li JCO 2013
Clinical cancer genomics
Dienstmann JCO 2013
Colon and Lung Panel - a single workflow
solution
DNA analysis
KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2,
PTEN, NRAS, STK11, MEK1, ALK, DDR2,
CTNNB1, MET, TP53, SMAD4, FBXW7, FGFR3,
NOTCH1, ERBB4, FGFR1, FGFR2
Colon and Lung AmpliSeq ™
panelv2
DNA mutations
Tumor
Sample
Ion Reporter ™ Software
Ion PGM ™ System
RNA lung fusion research panel
semiconductor
sequencing
ALK, ROS,RET, NTKR1 fusions & expression
RNA Analysis
Sample to report in less than 36 hours
automated analysis & reporting
22 multiple gene mutation analysis in mCRC
treated with FOLFIRI + cetuximab
Gene
Number of cases (>2%) with mutations, n (%)
(N=182 analyzed)
TP53
72* (39.5%)
KRAS
45^ (24.7%)
30 at codon 12 or 13 (16.5%); 16 at other (8.8%)
PIK3CA
24§ (13.2%)
16 at exon 9 (8.8%); 10 at exon 20 (5.5%)
BRAF
15 (8.2%)
10 at codon 600 (5.5%); 5 at other (2.7%)
NRAS
13 (7.1%)
FBXW7
9 (4.9%)
MET
7 (3.8%)
Mutations in genes EGFR, CTNNB1, FGFR3, SMAD4 occurred in 2 cases each (1.1%); mutations
in genes ERBB2, FGFR2, PTEN occurred in 1 case each (0.55%)
*7 cases with double TP53 mutation; ^1 case with double KRAS mutation; §2 cases with double PIK3CA mutation
Ciardiello, Normanno et al Ann Oncol 2014
Molecular typing of lung
adenocarcinoma on cytological
samples using a multigene next
generation sequencing panel
36/38 (95%)
adequate libraries
EGFR
6/36
KRAS
10/36
TP53
7/36
PIK3CA
3/36
BRAF
2/36
STK11
1/36
(16%)
(28%)
(18%)
(8%)
(5%)
(3%)
24/36 (67%) at least one
9/36 (25%) multiple
Scarpa A & Normanno N - PlosONE 2013
A patient story in 2014
• A 39-year-old woman, was referred to S. Orsola Malpighi oncology unit
in November 2013 for stage IV lung cancer.
• Total body CT scan showed: massive expansive process that occupies
much of the middle lobe and basal portion; enlarged lymphnodes both
on mediastinum and mesentere; neoformations of both adrenals;
presence of two nodular brain.
• 18 FDG-PET additionally showed intense metabolic activity of bone (D7,
L4, 3 and 10 right rib), liver (segment 7) and peritoneum.
• She underwent to a needle biopsy of the lung with a diagnosis of
undifferentiated carcinoma (immunohistochemistry negative for CD
117, synaptophysin, CD30, S100, calretinin, TTF1, smooth muscle actin;
partially positive for vimentine ed EMA; positive per citokeratin 8). A
second histological revision suggested for a high grade sarcomatoid
carcinoma.
• EGFR and ALK negative
A patient story in 2014
• Between November and December 2013 she underwent to a first line of
chemotherapy with cisplatin (60 mg/mq), epirubicin (60 mg/mq) and
ifosfamide (5000 mg/mq) but after only two cycles has been progressing
on all sites of disease
• Subsequently she was subjected to a second line of treatment with
carboplatin (AUC 5) and paclitaxel (175 mg/mq). After three doses, there
was a rapid clinical deterioration for worsening of intestinal
subocclusion due to carcinomatosis.
• Request of wide molecular screening at the Laboratory of
Pharmacogenomic of CROM with the Ion AmpliSeq™ Colon and Lung
Cancer Panel
BRAF mutations and response to BRAF
inhibitors in non-small-cell lung cancer
Case DM5118
80% tumor cells
HS 45 BRAF V600E
HS 1,25 p.E746_A750Del EGFR
Normal
EGFR
BRAF
Unknown
A patient story in 2014
• Off-label vemurafenib was started in March 2014 at a dose of 720 mg
administered twice per day
• After 10 days of administration she had an admission to emergency
room for an epileptic episode with aphasia associated to hyperpyrexia
(38°C). CT scan of the brain showed perilesional edema and she
underwent to whole brain radiotherapy
• After one month of treatment a total body CT scan showed: dramatic
reduction of the process of the right middle lobe (8x6.7 cm vs 12x7.7 cm)
and reduction in number and dimension of limphnodes and other
metastatic sites
• The patient suddenly died on May 2014 due to progression of cerebral
lesions
Challenges in genomics-driven oncology
• Alterations of “actionable” oncogenic pathways are not
always predictive of response to targeted agents
• Different molecular alterations of the same oncogene may
not be equivalent
• Intra-tumor heterogeneity may affect response to targeted
agents
• The molecular profile of solid tumors may significantly
change following treatment with target based agents
• Genomic testing programs should be strongly linked to
matched clinical trials
Challenges in genomics-driven oncology
• Alterations of “actionable” oncogenic pathways are not
always predictive of response to targeted agents
• Different molecular alterations of the same oncogene may
not be equivalent
• Intra-tumor heterogeneity may affect response to targeted
agents
• The molecular profile of solid tumors may significantly
change following treatment with target based agents
• Genomic testing programs should be strongly linked to
matched clinical trials
Survival of patients with driver mutations
and matched therapy
Kris JAMA 2014
Tsimberidou CCR 2014
Molecularly targeted therapy vs
conventional therapy: the SHIVA trial
Le Tourneau Lancet Oncol 2015
BRAF mutations in melanoma, CRC and
NSCLC
Cancer
Frequency Prognostic Predictive§ %V600E
Melanoma
50%
Y/N
Y
90%
CRC
10%
Y
N
90%
NSCLC
3%
Y*
Y
50%
*only V600E
§for response to cIass I RAF kinase inhibitors
Challenges in genomics-driven oncology
• Alterations of “actionable” oncogenic pathways are not
always predictive of response to targeted agents
• Different molecular alterations of the same oncogene may
not be equivalent
• Intra-tumor heterogeneity may affect response to targeted
agents
• The molecular profile of solid tumors may significantly
change following treatment with target based agents
• Genomic testing programs should be strongly linked to
matched clinical trials
OS of EGFR mutant NSCLC patients
treated with afatinib
Exon 19 deletions
L858R
Yang Lancet Oncol 2015
BRAF mutations and prognosis in melanoma
All BRAF mutant patients
BRAF mutant treated with BRAFi or MEKi
BRAF mutant not treated with BRAFi or MEKi
NRAS mutant patients
Bucheit Cancer 2013
Challenges in genomics-driven oncology
• Alterations of “actionable” oncogenic pathways are not
always predictive of response to targeted agents
• Different molecular alterations of the same oncogene may
not be equivalent
• Intra-tumor heterogeneity may affect response to targeted
agents
• The molecular profile of solid tumors may significantly
change following treatment with target based agents
• Genomic testing programs should be strongly linked to
matched clinical trials
Heterogeneity of EGFR mutations
• 16/42 (38%) EGFR mutant
samples consisted of only
EGFR-mutated cells, whereas
the other 26 samples consisted
of cells with both wild type
and mutated EGFR, with the
proportion of EGFR-mutant
cells ranging from 30% to 90%
• Among the 37 wild type cases,
four (10.8%) showed low
mutant frequency ranging
from 7.69% to 20.83%
Bai JCO 2012
Detection of EGFR mutations in NSCLC
Sequencing vs Therascreen
Zhou JCO 2011
PFS according to mutant allele frequency
(MAF) of p.L858R EGFR mutation
ORR was significantly higher in the
group with MAF >9% (79.1%) than in
the group with MAF ≤9% (20%) (P =
0.022, Fisher’s exact test).
Ono Ann Onc 2014
The Allelic Frequencies of Baseline
BRAFV600 Mutations Did Not Impact PFS
PFS by BRAF AF Quantile
(all patients)
Vem AF ≤33.9% (n = 101)
Vem AF >33.9% (n = 105)
Vem + cobi AF ≤33.9% (n = 99)
Vem + cobi AF >33.9% (n = 95)
100
80
60
40
20
0.0
0
200
400
Days
600
800
Percentage Progression Free
Percentage Progression Free
PFS
Based on BRAF Allele Frequency
1st Quartile
2nd Quartile
3rd Quartile
4th Quartile
100
80.
60
40
20
0.0
0
200
400
Days
600
800
The median AF of BRAFV600 mutations is 33.9% in coBRIM
AF, allelic frequency; PFS< progression-free survival; WT, wild type.
26
Heterogeneity Score (HS) in mCRC
patients enrolled in the CAPRI trial
The heterogeneity
score (HS) was
obtained by
normalizing the
frequency of mutant
alleles for the fraction
of neoplastic cells
Normanno N, et al. Ann Oncol 2015;26:1710–1714
Genotype of low (<33) KRAS HS tumors
ID
Patient
KRAS HS
Score
Additional Mutations
4553
12,00
NONE
4516
14,29
PIK3CA ex 20, BRAF V600E, FBXW7
4137
17,14
PIK3CA ex 9 and 20, ERBB2, TP53
3964
20,33
NONE
4139
22,86
PIK3CA ex9, TP53 (2 different mutations)
4141
25,71
PIK3CA ex9, BRAF ex11, TP53
4123
28,57
NONE
4124
30,00
PIK3CA ex9, TP53
4374
32,00
FGFR3
4166
32,00
TP53
Normanno et al Ann Oncol 2015
Heterogeneity Score (HS) and efficacy of
treatment in the CAPRI trial
Case 177 (SD, PFS 5,9 mo)
Case 118 (PR, PFS 3,9 mo)
70% tumor cells
HS 14,29 KRAS G13D
HS 17,14 PIK3CA ex20
HS 54,29 BRAF V600E
HS FBXW7 R465H 48,6
70% tumor cells
HS 22,86 KRAS G12D
HS 74,29 PIK3CA ex9
HS 57,14 TP53
Normal
BRAF
KRAS
PIK3CA
FBXW7
Normal
KRAS
TP53
PIK3CA
Normanno et al Ann Oncol 2015
Challenges in genomics-driven oncology
• Alterations of “actionable” oncogenic pathways are not
always predictive of response to targeted agents
• Different molecular alterations of the same oncogene may
not be equivalent
• Intra-tumor heterogeneity may affect response to targeted
agents
• The molecular profile of solid tumors may significantly
change following treatment with target based agents
• Genomic testing programs should be strongly linked to
matched clinical trials
Mechanisms of acquired biological
resistance to EGFR TKIs in NSCLC
Camidge Nat Rev Clin Oncol 2014
The "three levels" system of resistance to
anti-EGFR moAbs in mCRC
ErbB-2
MET
Level 1: the antenna
 EGFR S492R mutation
 ERBB2 gene amplification
 MET gene amplification
Level 2: the main switch
 KRAS mutations
 NRAS mutations
Level 3: the internal
circuits
 BRAF?
 PIK3CA?
Normanno Nat Rev Clin Oncol 2009
Melanoma escape pathways during
disease progression on BRAF inhibitor therapy
Shi Cancer Discov 2015
Resistant cells are selected by
treatment with target based agents
Before Target
Therapy
Sensitive
After Target
Therapy
Resistant
Multiple resistance mechanisms to EGFR TKIs
Blakely ASCO 2013
Melanoma heterogeneity and branched evolution
during the acquisition of BRAF inhibitor resistance
Shi Cancer Discov 2015
Detection of different mechanismsm of resistance
to anti-EGFR moAbs in plasma of CRC patients
Bettegowda Sci Transl Med 2014
Siravegna Nat Med 2015
Challenges in genomics-driven oncology
• Alterations of “actionable” oncogenic pathways are not
always predictive of response to targeted agents
• Different molecular alterations of the same oncogene may
not be equivalent
• Intra-tumor heterogeneity may affect response to targeted
agents
• The molecular profile of solid tumors may significantly
change following treatment with target based agents
• Genomic testing programs should be strongly linked to
matched clinical trials
Clinical trial enrollment after genomic
testing
Challenges to trial accrual
included:
 patient preference of
noninvestigational treatment
or local treatment
 poor performance status or
other reasons for trial
ineligibility
 lack of trials/slots
 insurance denial
Meric-Bernstam JCO 2015
Genomics-Driven Oncology
Surgeon
Endoscopist
Radiologist
Medical
Oncologist
Surgeon
Endoscopist
Radiologist
Medical
Oncologist
Medical
Oncologist
Surgeon
Radiotherapist
Pathologist, Molecular Biologist, Geneticist
Garraway JCO 2013