Post on 19-Jan-2016
Caratterizzazione molecolare dei tumori: impatto sulla pratica clinica
Nicola Normanno
ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUMORI
FONDAZIONE G. Pascale – NAPOLISC Biologia Cellulare e Bioterapie
CENTRO RICERCHE ONCOLOGICHE MERCOGLIANO (AV)
Laboratorio di Farmacogenomica
Genomic alterations affecting actionable signaling pathways
Garraway JCO 2013
Genomics-Driven Oncology
Garraway JCO 2013
The major classes of genomicalterations that give rise to cancer
Modified from McConaill JCO 2010
EGFRErbB-2BRAFKRASNRASPIK3CAAKT1MAP2K1
EML4-ALKROS-1RET
EGFRErbB-2MET
Sequencing,Real Time PCR etc.
FISH,Immunohistochemistry
Genotyping and genomic profilingin personalized medicine
Li JCO 2013
Clinical cancer genomics
Dienstmann JCO 2013
Colon and Lung Panel - a single workflow solution
Tumor Sample
Colon and Lung AmpliSeq ™ panelv2
DNA mutations
Ion PGM ™ Systemsemiconductor
sequencing
Ion Reporter ™ Software automated analysis & reporting
Sample to report in less than 36 hours
DNA analysis
RNA lung fusion research panelALK, ROS,RET, NTKR1 fusions & expression
RNA Analysis
KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2, PTEN, NRAS, STK11, MEK1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBXW7, FGFR3, NOTCH1, ERBB4, FGFR1, FGFR2
GeneNumber 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%)
22 multiple gene mutation analysis in mCRC treated with FOLFIRI +
cetuximab
*7 cases with double TP53 mutation; ^1 case with double KRAS mutation; §2 cases with double PIK3CA mutation
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%)
Ciardiello, Normanno et al Ann Oncol 2014
Scarpa A & Normanno N - PlosONE 2013
36/38 (95%) adequate libraries
EGFR
KRAS
PIK3CA
BRAF
TP53
STK11
6/36(16%)
10/36(28%)
7/36(18%)
3/36(8%)
2/36(5%)
1/36(3%)
24/36 (67%) at least one9/36 (25%) multiple
Molecular typing of lung adenocarcinoma on cytological samples using a multigene next generation sequencing panel
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 DM511880% tumor cellsHS 45 BRAF V600EHS 1,25 p.E746_A750Del EGFR
Normal
BRAF
EGFR
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§ %V600EMelanoma 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
Yang Lancet Oncol 2015
Exon 19 deletions L858R
BRAF mutations and prognosis in melanoma
Bucheit Cancer 2013
All BRAF mutant patients BRAF mutant treated with BRAFi or MEKi
BRAF mutant not treated with BRAFi or MEKi NRAS mutant patients
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 NSCLCSequencing vs Therascreen
Zhou JCO 2011
PFS according to mutant allele frequency (MAF) of p.L858R EGFR mutation
Ono Ann Onc 2014
ORR was significantly higher in the group with MAF >9% (79.1%) than inthe group with MAF ≤9% (20%) (P = 0.022, Fisher’s exact test).
The Allelic Frequencies of Baseline BRAFV600 Mutations Did Not Impact PFS
The median AF of BRAFV600 mutations is 33.9% in coBRIM
PFSBased on BRAF Allele Frequency
100
80
60
40
20
0.00 200 400 600 800
Days
Perc
enta
ge P
rogr
essi
on F
ree 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)
0 200 400 600 8000.0
20
40
60
80.
1001st Quartile2nd Quartile3rd Quartile4th Quartile
Days
PFS by BRAF AF Quantile (all patients)
26
Perc
enta
ge P
rogr
essi
on F
ree
AF, allelic frequency; PFS< progression-free survival; WT, wild type.
The heterogeneity score (HS) was
obtained by normalizing the
frequency of mutant alleles for the fraction
of neoplastic cells
Heterogeneity Score (HS) in mCRC patients enrolled in the CAPRI trial
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)70% tumor cellsHS 14,29 KRAS G13DHS 17,14 PIK3CA ex20HS 54,29 BRAF V600EHS FBXW7 R465H 48,6
Normal
BRAF
KRAS
PIK3CA
Case 118 (PR, PFS 3,9 mo)70% tumor cellsHS 22,86 KRAS G12DHS 74,29 PIK3CA ex9HS 57,14 TP53
Normal
TP53
KRAS
PIK3CA
FBXW7 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
Camidge Nat Rev Clin Oncol 2014
Mechanisms of acquired biological resistance to EGFR TKIs in NSCLC
The "three levels" system of resistance to anti-EGFR moAbs in mCRC
Normanno Nat Rev Clin Oncol 2009
Level 1: the antenna EGFR S492R mutation ERBB2 gene amplification MET gene amplification
ErbB-2MET
Level 2: the main switch KRAS mutations NRAS mutations
Level 3: the internal circuits BRAF? PIK3CA?
Melanoma escape pathways duringdisease progression on BRAF inhibitor therapy
Shi Cancer Discov 2015
Resistant cells are selected by treatment with target based agents
Sensitive Resistant
Before TargetTherapy
After TargetTherapy
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
Siravegna Nat Med 2015Bettegowda Sci Transl Med 2014
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
Meric-Bernstam JCO 2015
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
Genomics-Driven Oncology
Garraway JCO 2013
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