New high throughput technologies

in Molecular Diagnostics
LAquila, 29 ottobre 2013

Precision Molecular Diagnostics

toward Personalized
Cancer Treatment and Prevention

Enrico Ricevuto
Medical Oncology
San Salvatore Hospital
University of LAquila

Cancer
Biological characterization: Why?

to justify differential prognosis (clinical outcome)

different aggressiveness
different sensitivity/resistance
to predict differential pharmacologic effect
different sensitivity/resistance
to identify new druggable or actionable
targets
pathways

Cancer Genome Landscapes

Genomic alterations and affected signal transduction pathways

B Vogelstein et al, Science 13: 339; 1546-58

Precision Molecular Diagnostics toward Personalized

Cancer Treatment and Prevention
Outline

Precision Molecular diagnostics: identification of

functionally relevant genetic alterations
Personalized Oncology: individual treatment/prevention
strategies, by actionable/druggable genetic alterations
Cancer predisposition and Prevention
Breast/Ovarian Cancer predisposition
HNPCC
Cancer Treatment
Metastatic Colorectal cancer
Advanced Breast cancer
4

Precision and Personalized Oncology

Key steps

Identification of molecular targets (biomarkers)

Drivers/Passengers
Druggable/actionable
Prognostic and Predictive relevance
Retrospective analysis
Prospective study
Treatment with targeted agents
mCRC
KRAS wild-type/anti-EGFR
aNSCLC
EGFR mutations/anti-EGFR
ALK/EML4 rearr/anti-ALK
aMelanoma
BRAF mutations/anti-BRAF
5

Precision and Personalized Oncology

Key Organization needs

Clinical strategies approved by Multidisciplinar Institutional

Boards and Ethic Committee: patients written informed
consent
Clinical practice protocols
Clinical Trials
Centralized Collection of patients samples: blood, primary
tumors and metastases
Detection of biomarkers: multigene platforms
Innovative treatment and prevention strategies:
Actionable/druggable biomarkers dynamically characterizing
individual tumours and metastases

Innovative targeted drugs

6

Predisposizione genetica BRCA1 e BRCA2

Diagnostica molecolare

Analisi mutazionale1,2,3
Analisi di riarrangiamenti del locus cromosomico
(delezioni, duplicazioni)4,5,6
Analisi delle varianti alleliche (VUS)7: alterazioni dello
splicing
Mutazioni missense di significato sconosciuto (UV)
Varianti alleliche, anche potenzialmente silenti (SNPs)
Mutazioni introniche

Ricevuto et al, Clin Canc Res 01,7,1638-46

3
Aretini P, BCRes Treat 03,81,71-9
Giannini, G et al, BCRes Treat 06,100,834
91
Casilli F et al, Hum Mut 02,20,218-26
5
6
Casilli F et al, J Med Gen 06,43, e49
Buffone A et al, BCRes Treat 07,106,2897
96
Gaildrat P et al, J Med Gen 12, 49,609-17
1

Diagnosi di Predisposizione BRCA1/BRCA2

in base ai criteri di rischio
FC

Total
+
N % BRCA %

N.

M>4

37 (20) 11

30

M3

48 (26) 7

15

M2

41 (22) 4
17 (9) 0

EO-BC
+
BRCA

Male BC
+
BRCA
%

BRCA1

BRCA2

100

50

83

6
-

25

1
0

0
0

15

29

14

14

32

20

62%

38%

N.

43

10

40

10

16

19

11

40 (22) 29

72

O1

1
1

1
0

100
0

Totale

185

52

28

51

M1
M/O
O>2

Attivit 2002-2008
Universit di LAquila, Universit La Sapienza di Roma

Giannini, G et al, B C Res Treat 06,100,83-91

Res Treat 07, 106,289-96

Buffone
A et al, B C8
8

BRCA1 germline rearrangements

Dupl17kbEx3-8

6 7

Dupl5.6kbEx18-20

18 19

20

Dupl6kbEx13
13

BRCA1

1 2

1 2

3
DelEx3
Complex
(no Alu seq)

Del?5kbEx1-2
(? Alu seq)

Del14kbEx1-2

Del37kbEx1-2
Del37kbEx1-2

6 7

9 10

11

13

12

14 15

16

17

18 19

Del?kbEx1-2

Del?kbEx1-2

21 22 23 24

Del?4kbEx20

Del7kbEx8-9

20

Del11.6kbEx13-15

Del23.8kbEx8-13

Del3.8kbEx13

Del3kbEx17
Del3kbEx15

Del14kbEx13-16

Del0.5kbEx22
Del1kbEx17

Del?kbEx17-19

Del5.6kbEx15-16

Del3.4kbEx21-22
DelEx17-20
complex

Published rearrangements
Unpublished rearrangements

Del?kbEx1-22

Rearrangements detected by Multiplex PCR

9
Casilli et al. Human Mutation
(2002)

Delezione BRCA1: Esoni 1-22

QMPSF 1

QMPSF 2
1-2

C2

NBR2-1

10
6

C1

17

16

IC 557

24

IC 557

control

QMPSF 3

control

QMPSF 4
13

C2

22

23

C2

9
11a

20

5 19

12

15

21

14

11b

18

IC 557

control

IC 557

control

10
Casilli et al. Human Mutation
(2002)

10

Riarrangiamenti cromosomici BRCA1/BRCA2

in relazione ai criteri di rischio
Totale
+
%
1
100

BCW
N.
+
1
1

EO-BCW
N.
+
0
0

BCM
N.
+
0
0

M+C
N.
+
0
0

NA
N.
+
0
0

10 CM

N.
1

8 CM

7 CM

50

6 CM

50

5 CM

20

4 CM

10

50

3 CM

34

18

24

2 CM

29

14

12

13

1 CM

11

TOTALE 96

19

20

52

31

10

15

26

20

100

Attivit 2002-2008
Universit di LAquila, Universit La Sapienza di Roma
11

11

Rischio genetico
Gestione clinica: fasi

Selezione delle famiglie a rischio

Consulenza Genetica Oncologica
Analisi genetica
Segregazione della predisposizione
Sorveglianza diagnostica
Strategie terapeutiche preventive
Gestione clinica dei carriers affetti

12

12

Predisposizione genetica tumori mammella ed ovaio

Indirizzi di gestione clinica

Integrazione nei programmi di prevenzione

Distinzione livelli di sorveglianza diagnostica
Strategie di prevenzione chirurgica
(mastectomia bilaterale, ovariectomia)

Integrazione nella gestione clinica delle pazienti affette

Implicazioni prognostiche
Implicazioni predittive
Strategie terapeutiche specifiche

Strategie concomitanti di Cura e Prevenzione chirurgica

Trattamento con chemioterapia e inibitori PARP
13

13

HNPCC
Molecular Diagnostics of MMR genes

Point Mutations

MLH1
MSH2
25%
MSH6
5%

25%

Genomic rearrangements
MLH1

Splicing Variants

Methylation MLH1, 5alteration (EpCAM)

MSH2
<4%

10%

10%
2%

80%
14

14

Metastatic Colorectal Cancer

Evolution of medical treatment

Parameters
None
One fit (unfit) all
Clinical
One fit some (10-20%)
Patient fitness (age, comorbidities)
Tumor
metastatic extension
Biomarker One fit few (1-10%)
KRAS

MCRC patients
Biomarkers driving medical treatment

KRAS gene?
Wild-type (55-60%)

Integrated medical and surgical treatment strategy

Sequential lines of medical treatment
More targeted agents to use (anti-EGFR)

Mutant

(40-45%)

Reduced possibility to integrate medical and surgical

treatment
Reduced lines of medical treatment and targeted agents to
use

16

Patient Fitness

MCRC

Fit (44%)
I Line

Triplet + Targeted agents

Doublet + Targeted agents

Patient Fitness

MCRC

Fit (44%)
I Line

FIr-B/FOx + Liver Surgery

PFS
12 months

OS
31 months

Bruera G et al, BMC Cancer 2010;10:567

Patient Fitness

I Line

MCRC

Fit (44%)

Unfit (56%)

FIr-B/FOx + Liver Surgery

Conventional Treatments

PFS
12 months

7 months

OS
31 months

Bruera G et al, BMC Cancer 2010;10:567

13 months

Bruera G et al, Submitted 2013

Metastatic extension

MCRC

Fit (44%)
I Line

Unfit (56%)

FIr-B/FOx + Liver Surgery

Other/Multiple Mets (56%)

Liver-Limited (44%)

PFS
12 months

17 months

OS
21 months

47 months

Bruera G et al, Clin Colorectal Cancer 2012; 11(2):119-126

Bruera G et al, BMC Medicine 2012, 10(1):135

MCRC: Intensive chemotherapy,

Liver metastasectomies
Overall Liver metastases Liver only
(%)
(%)
(%)
Triplet

4Drugs

100

66

44-53

15

36

Cet-FOLFOX6

38

Cet-FOLFIRI

30

FOLFOXIRI-Bev

32

40

FIr-B/FOx

26

39

54

60

FOLFOXIRI

Chrono/IFLO-Cet

Abbreviation: Bev, Bevacizumab;

Cet, Cetuximab.
Bruera and Ricevuto,
Exper Opin Biol Ther 2011; 11(6):821-4
Ficorella C, Bruera G et al, Clin Colorectal Cancer 2012;11(4):229-37
21

II Line Treatments

MCRC

Fit (44%)
I Line

FIr-B/FOx + Liver Surgery

II Line

Treated pts (74%)

PFS
10 months

OS
14 months

Bruera G et al, Int J Oncology, in press 2013

Frequency of KRAS mutations in CRC

Normanno N, et al. Nat Rev Clin Onc 2009, 6:519-527

23

Influence of KRAS status on efficacy of cetuximab

plus FOLFOX

Bokemeyer C, et al. Ann Oncol 2011, 22:1535-1546

24

Metastatic Colorectal Cancer

Evolution of medical treatment

Parameters
None
One fit (unfit) all
Clinical
One fit some (10-20%)
Patient fitness (age, comorbidities)
Tumor
metastatic extension
Biomarker One fit few (1-10%)
KRAS

C. 35 G>A (G12D)

KRAS genotype

MCRC

Fit (44%)
I Line

FIr-B/FOx + Liver Surgery

Mutant (47%)

Wild-type (53%)

XELOX-BEV

PFS

OS

Bruera G et al, BMC Medicine 2012, 10(1):13526

Diaz-Rubio E et al, PLoS ONE 2012; 7(10): e47345

c.35 G>A KRAS mutant

genotype
I Line

MCRC

Fit (44%)

Unfit (56%)

FIr-B/FOx + Liver Surgery

Conventional Treatments

c.35 G>A mutant (25%)

Wild-type (53%)

PFS

OS

Bruera G et al, BMC Medicine, 2013, 11 (1):59

27

c.35 G>A KRAS mutant

genotype
I Line

MCRC

Fit (44%)

Unfit (56%)

FIr-B/FOx + Liver Surgery

Conventional Treatments

c.35 G>A mutant (25%)

Other mutant (22%)

PFS

OS

Bruera G et al, BMC Medicine, 2013, 11 (1):59

28

c.35 G>A KRAS

mutant

MCRC

Fit (44%)

Unfit (56%)

I Line

FIr-B/FOx + Liver Surgery

Conventional Treatments

II Line

Treated patients (74%)

c.35 G>A mutant (20%)

Wild-type (55%)

PFS

OS
Bruera G et al, Int J Oncology, in press 2013
29

c.35 G>A KRAS

mutant

MCRC

Fit (44%)

Unfit (56%)

I Line

FIr-B/FOx + Liver Surgery

Conventional Treatments

II Line

Treated patients (74%)

c.35 G>A mutant (20%)

Other mutant (25%)

PFS

OS
Bruera G et al, Int J Oncology, in press 2013
30

Molecular modeling of human KRAS

Chen CC, et al. PLoS ONE 2013, 8(2):e55793

31

Colorectal cancer
Prevalent DNA genetic alterations
Mutation rate
(%)
KRAS

35-45

MSI-H

15

MIN: Microsatellite Instability

p53

40

CIN: Chromosomal Instability

BRAF
PI3KCA

4.7-8.7
14
De Roock W et al, Lancet Oncol 2010;11:753-62
Tol J, N Engl J Med. 2009;361:98-9.
Ince WL et al, J Natl Cancer Inst 2005, 97:981-9.
Baldus SE et al, Clin Cancer Res. 2010;16:790-9
Laurent-Puig P et al, J Clin Oncol. 2009;27:5924-30
Ogino S et al, J Clin Oncol. 2009;27:1477-84
Velho S et al. Eur J Cancer 2005; 41:1649-1654
32

Metastatic Colorectal Cancer

Evolution of medical treatment

Parameters
None
One fit (unfit) all
Clinical
One fit some (10-20%)
Patient fitness (age, comorbidities)
Tumor metastatic extension
Biomarker
One fit few
(1-10%)
Multigene
One fit very few (<1%)
K/H/NRAS 50%
BRAF
4.7-8.7%
PIK3CA
14%
PTEN
17%
MSI
15%
P53
40%

Biological factors (major drivers) and targeted

agents

Perspectives

34

Metastatic Colorectal Cancer

Evolution of medical treatment

Parameters
None
One fit (unfit) all
Clinical
One fit some (10-20%)
Patient fitness (age, comorbidities)
Tumor metastatic extension
Biomarker
One fit few
(1-10%)
Multigene
One fit very few (<1%)
K/H/NRAS secondary mutations
BRAF
4.7-8.7%
PIK3CA
14%
PTEN
17%
MSI
15%
P53
40%

Metastatic Colorectal Cancer

Evolution of medical treatment

Parameters
None
One fit (unfit) all
Clinical
One fit some (10-20%)
Patient fitness (age, comorbidities)
Tumor metastatic extension
Biomarker
One fit few
(1-10%)
Multigene
One fit very few (1/103-1/102)
Multi-molecular
One fit1 (1/104-5-1/103)
Cytogenetics
Genetics
Epigenetics
Genomics

LAquila Personalized Cancer Treatment and Prevention Program

Colorectal cancer
Tissue Marker
T/M
KRAS
HRAS
NRAS
BRAF
PIK3CA
P53
MI
PTEN
EGFR
HER2
MET
IGF2
Blood
MLH1
MSH2
MSH6

Methods
M
M
M
M
M

MCRC
23/18
x
x
x
x
x

M
M

x
x
IHC/M
IHC/CG/M
IHC/CG
IHC/CG
x
CG
x
M
M
M

x
x
x

x
x
x
10 (15)

2013 Support by Amgen, COMPEL, Merck, Sanofi

37

Patient Fitness

MCRC

Fit (44%)
I Line

FIr-B/FOx + Liver Surgery

PFS
12 months

OS
31 months

Bruera G et al, BMC Cancer 2010;10:567

Heinemann V al, ASCO 2013, LBA3506 38

mCRC
Precision program in Molecular Diagnostics
Samples
all
Patients
KRAS wild-type
pre 7
post 6
pre/post
mutant
pre 6
post 6
pre/post

23
11

2
12

I line treatment
antiantiVEGF EGFR CT
15 4 4
6 4 1
4
3 0
3 2 1
1 1 0
9 0 3
3 0 3
6 0 0
0 0 0

39

Breast Cancer
Prevalence according to ER/HER2 status
ER

Total

+++

Negative

Negative

35-40

15-20

15-20

Positive

60-65

10-15

50-55

100

30

70

HER2

Total

40

Breast Cancer
Prevalence according to ER/HER2 status
ER

Total

+++

Negative

Negative

35-40

15-20

15-20

Positive

60-65

10-15

50-55

100

30

70

HER2

Total

41

TCGA Network: Luminal-BC

PIK3 pathway mutations and correlations with
genomic and clinical features

42

ER+-BC
PI3K/AKT/mTOR signaling pathway

43

HR+/ABC, NSAI-refractory
BOLERO-2 trial: NGS population
Biomarker analysis
Samples (FFPE) 309 (80% primary)
analysed 227 (73.5%)
Samples/pts
31.4%
Genes
182
Exons 3230
NGS Platform Agilent Illumina
Genetic alteration
mut/rearr/CNV

modified from Hortobagyi, ASCO13 CSS: LBA

509

44

BOLERO-2 trial
Genetic alterations in NGS population

modified from Hortobagyi, ASCO13 CSS: LBA

509

45

BOLERO-2 trial
Frequency of genetic alterations in key pathways

modified from Hortobagyi, ASCO13 CSS: LBA

509

46

BOLERO-2 trial
Predictive effect by genetic status
in single genes and pathways

modified from Hortobagyi, ASCO13 presentation

47

BOLERO-2 trial
Mutation clusters across key pathways in ER+-BC

modified from Hortobagyi, ASCO13 CSS: LBA

509

48

Breast Cancer
Prevalence according to ER/HER2 status
ER

Total

+++

Negative

Negative

35-40

15-20

15-20

Positive

60-65

10-15

50-55

100

30

70

HER2

Total

49

HER2+ MBC
PIK3CA and anti-HER2 drugs

Baselga J et al, NEJM 2012

50

Breast Cancer
Prevalence according to ER/HER2 status
ER

Total

+++

Negative

Negative

35-40

15-20

15-20

Positive

60-65

10-15

50-55

100

30

70

HER2

Total

51

Breast Cancer
Prevalence according to ER/HER2 status
ER

Total

+++

Negative

Negative

35-40

15-20

BRCA1+ 11%
BRCA1-ness >50%

Positive

60-65

10-15

50-55

100

30

70

HER2

Total

52

Personalized Advanced BC
French program: SAFIR 01

Detection of Genomic alterations in MBC

Enrich phase I-II trials with actionable genetic alterations
Patients
423
Biopsies
404
CGH array: 287 (71%)
Targettable
Genetic alteration
(67%)
Treatment driven
(25%)
Clinical benefit

modified from F Andre, ASCO13 CSS, 511

53

194
48

12 (3%)

Personalized Advanced BC
French program: SAFIR 01
Targetable alterations
that led to treatment proposition

Therapies matched
to the genomic alterations

modified from F Andre, ASCO13 CSS, 511

54

Personalized Advanced BC
French program: SAFIR 02

modified from F Andre, ASCO13 CSS, 511

55

LAquila Personalized Cancer

Treatment and Prevention Program
Tissue

Marker

Methods
23

T/M

Blood

EGFR
IHC/CG
HER2
IHC/CG
KRAS
M
HRAS
M
NRAS
M
BRAF
M
PTEN
IHC/M
PIK3CA
M
AKT1
M
P53
M
x
CCND1
CG
FGFR 1
CG
MET
IHC/CG
Microsatellite instability
M
BRCA1
BRCA2

M
M
7 (11)

MCRC
13
x
x
x
x
x
x

ABC
36

Common

x
x
x
x
x
x
x

x
x
x
x
x
x
x

x
x

x
x
6 (11)

4 (6)

2013 Support by Amgen, COMPEL, Merck, Sanofi

56

LAquila Personalized Cancer Treatment and Prevention Program

Tissue
T/M

Blood

Marker
EGFR
HER2
HER3
ALK/EML4
MET
PTEN
MGMT
1p
19q
FGFR1
ROS
KRAS
HRAS
NRAS
BRAF
PIK3CA
P53
CDK4inh
MI
KIT
PDGFR2A
VHL
PBMRM1
BAP 1
BRCA11
BRCA2
MLH1
MSH2
MSH6
29

Methods
MCRC
IHC/CG/M
x
IHC/CG
x
x
CG/M
x
IHC/CG
IHC/CG
x
IHC/M
x
x
IHC/Meth
CG
CG
CG
x
CG
M
x
M
x
M
x
M
x
M
x
M
x
x x
Meth
x
M
x
x
M
M
M
M
M
M
x x
M
x x
M
x
x
M
x
x
M
x
x
14 10 7

BC OC UC NSCLC HCC Mel GIST RCC SNC PDA GC

x
x
3
x
3
1
x
1
x
x
3
x
x
4
x
1
x
1
x
1
x
2
x
1
x
x
3
1
x
2
x
x
3
x
x
x
x
5
x
x
x
6
1
x
3
x
x
2
x
1
x
1
x
1
x
1
2
2
x
3
x
3
x
3
6
9
2
4
2
3
3
3 1

12

57

Precision and Personalized Oncology

Major challenge: Genetic heterogeneity

58

Metastatic ccRCC
Heterogeneity

Gerlinger M et al, N Engl J Med 2012;366:883-92

Renal cell carcinoma

Evolution of medical treatment

Markers
None
One fit (unfit) all
Clinical
One fit some (>10%)
Monogene One fit few (1-10%)
Multigenes One fit one (<1%)
Multiple biopsies
Genetic and epigenetic alterations

LAquila Personalized Cancer Treatment Program

Clinical Strategy

Selection, Identification and Clinical management of cancer predisposition

PB/OCP syndrome
HNPCC syndrome

Observational Studies in clinical practice

mCRC
Free-choice clinical practice protocols (FCCPP)

KRAS wtI line anti-VEGF/anti-EGFR

KRAS mutant
BC

ABC
ER-positive/AI+mTOR-inhibitors
HER2-positive/HER2 and pan-HER inhibitors

PST
Free-choice clinical practice protocol (FCCPP)
mRCC
Free-choice clinical practice protocol (FCCPP)

I/II/III line of treatment

Clinical trials

ABC
PIK3CA inhibitors
61

Biotechnological and
Applied Clinical Sciences
University of LAquila
LAquila, Italy
E. Alesse
A. Tessitore

Medical Oncology
S. Salvatore Hospital
C. Ficorella
E. Ricevuto
K. Cannita
G. Bruera
D. Di Giacomo
V. Cocciolone
E. Palluzzi

Pathology, S. Salvatore Hospital

G. Calvisi
G. Coletti
A. Dal Mas

Medical Genetic
S. Salvatore Hospital
E. DAlessandro
C. Ligas

University of Medicine and Pharmacy.

Rouen, France
Dept. Human Genetics and
Institute for Biomedical Research
(INSERM U1079)
T. Frebourg
M. Tosi

Lab. Somatic Genetic

J.C. Sabourin
A. Lamy
62