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

SurgeonEndoscopistRadiologist

SurgeonEndoscopistRadiologist

MedicalOncologist

MedicalOncologist

Pathologist, Molecular Biologist, Geneticist

MedicalOncologist

SurgeonRadiotherapist