Ramon Salazar, MD, PhD, MSc - UB · Ramon Salazar, MD, PhD, MSc Institut Català d’Onologia...

“Avances tecnológicos en la

terapéutica del cáncer de

colón”

Ramon Salazar, MD, PhD, MSc Institut Català d’Oncologia

Barcelona, Spain

1 de marzo 2012

Tertúlias con el Profesor Ramon Salazar

Quimioterapia antitumoral

tumoral

Diana

Cancer is a group of genetic diseases

Dr. Dulbecco accepts his Nobel Prize

in 1975.

Dr. Dulbecco in the lab at the Salk

Institute for Biological Studies.

Dr. Dulbecco and colleagues described

how a tumor virus could insert its own

genes into the chromosome of the cell it

infects, and so "turn on" the uncontrolled

growth that is the hallmark of cancer

"genes can be used by cancer, as well as

used against the disease, led Dulbecco to

challenge the scientific community to

systematically sequence and catalog all

human genes," and how this gave

"intellectual birth" to the worldwide Human

Genome Project

Cómo nos ayuda la tecnología

• Generar fármacos de gran potencia y especificidad, con buenas PK/PD

• Determinar y caracterizar diferentes subgrupos de cáncer con alteraciones genéticas o moleculares específicas…

– modulables farmacológicamente

(inhibir oncogenes és más fácil que restablecer genes supresores)

– informativas sobre decisiones terapéuticas (e.g. pronóstico)

Strong correlations between cancer genotype and drug response

Personalized medicine 1.01

• HER2 expression (breast) Trastuzumab Powder Sol 150 mg (2mg/Kg/wk)

• BCR-ABL translocation (CML) Imatinib Comp 100/400 mg (400-800 mg/od)

•C-KIT mutation (GIST) Imatinib Comp 100/400 mg (400-800 mg/od)

• KRAS mutation (colon Resistance) Cetuximab Sol 2 mg/mL (400/250 mg/m2/bwk)

• EGFR mutation (lung) Erlotinib Comp 25/100/150 mg (150 mg/od)

• BRAF mutation (melanoma, colon) Vemurafenib Comp 240 mg (960 mg/bd)

• ALK mutation/translocation Crizotinib Caps 200/250 mg (250 mg/bd)

(neuroblastoma, lung, breast)

BUT Mono(or pauci)genetic cancers are rare

…and they also develop resistance

The Genomic Landscapes of Human Breast and Colorectal Cancers

Wood, et al. Science 318, 1108 (2007)

~80 mutations in an individual tumor

< 15 likely to be responsible for driving the initiation, progression,

or maintenance of the tumor, acting on limited pathways

Low frequency mutations are the most prevalent

Hills more influential than mountains

Cómo gestionamos esta complejidad

en el cáncer colorectal: 3 ejemplos

1 Optimización ttos anti EGFR por selección de pacientes con tumores sin mutaciones en KRAS y otros oncogenes relacionados

2 Aplicación de alta tecnología para identificación de mecanismos de resistencia y su evasión en modelos BRAF mutados

3 Aplicación de firmas genéticas de RNAm

Some genotype - drug response

relationships are readily explained

Grb

RAF

EGFR

KRAS

MEK

ERK

Cetuximab

Wild type

Clinical response

Grb

BRAF

RTK

RAS

MEK

ERK

PLX4270

Clinical response when mutant

Downstream

pathway activation

Oncogene

addiction

Mutant

No clinical response

KRAS

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Pro

po

rtio

n E

ven

t-F

ree

Pmab + BSC

BSC Alone

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

Weeks

36 38 40 42 44 46 48 50 52

Patients at risk

78 76 72 26 10 8 6 5 5 5 5 4 4 4 4 2 2 2 2 2 2 2 1 1 1

91 77 61 37 22 19 10 9 8 6 5 5 4 4 4 4 4 4 3 3 3 2 2 2 2

84

100

Pmab + BSC

BSC Alone

HR = 1.00 (95% CI: 0.74–1.37)

76 /84 (90)

95 /100 (95)

7.4

7.3

Events/N (%)

Median

In Weeks

No hay beneficio de ttos anti EGFR en CCR pacientes no seleccionados

El beneficio está restringido a la ausencia de mutación en el gen KRAS

0 . 0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

0 . 6

0 . 7

0 . 8

0 . 9

1 . 0

0 . 0

0 . 1

0 . 2

0 . 3

0 . 4

0 . 5

0 . 6

0 . 7

0 . 8

0 . 9

1 . 0

0 2 4 6 8 10 12 14 16 18 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4

Weeks

3 6 3 8 4 0 4 2 4 4 4 6 4 8 5 0 5 2

7 7 6 5 5

10 9 9 6 6 6 5 4 3 3 2 2 2 2 1

124

115/124 (93) 12.3 19.0

114/119 (96) 7.3 9.3

Pmab + BSC

BSC Alone

Events/N (%)

Median

In Weeks

Mean

In Weeks

HR = 0.45 (95% CI: 0.34–0.59)

Stratified log-rank test, p < 0.0001

Pmab + BSC

BSC Alone

Patients at Risk

119 112 106 80 69 63 58 50 45 44 44 33 25 21 20 17 13 13 13 10

119 109 91 81 38 20 15 15 14 11 6

Pro

po

rtio

n w

ith

PF

S

p < 0.0001 for quantitative-interaction test comparing PFS log-HR

(pmab/BSC) between KRAS groups

•Estudio abierto fase II de ultra-selección de pacientes mediante tecnología de genotipado de nueva

generación para el esquema Folfiri + Panitumumab en pacientes con cáncer colorrectal estadio IV resistentes

a irinotecán sin mutaciones detectables utilizando técnicas de alta sensibilidad para la detección de

mutaciones en los genes KRAS, PIK3Ca BRAF y NRAS

•Coordinadores: Dr. Ramón Salazar y Gabriel Capellá




Segundo ejemplo

Some genotype - drug response

relationships are readily explained

Grb

BRAF

RTK

RAS

MEK

ERK

Vemurafenib (PLX4270)

Clinical response when mutant

Oncogene

addiction

85-90% of BRAF mutations are V600E (valine to glutamine)

Oncogene (2007) 26, 3291–3310

Ligand

BRAF is mutated in many tumor types

BRAF*

Benefit of BRAF inhibition in melanoma

Before

Vemurafenib

15 weeks of

Vemurafenib

81%

5,20%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Melanoma Colon cancer

PLX4032 response rate in B-RAFV600E-positive tumours

N Engl J Med. 2010 363:809-19 Kopetz et al., ASCO 2010

Vemurafenib (PLX4032) - A selective BRAFV600E kinase inhibitor

Differential response of BRAF inhibition in BRAF mutant melanoma versus colon cancer

Personalized medicine

Alterations in pathways

Cancer genome analyses

Cross talk between pathways

Functional genetic analyses

BRAFV600E mutant CRC cell lines are also less

responsive to PLX4032 than melanomas having the

same mutation

CRC

Melanoma

Short-term cell viability assay Long-term colony formation assay

A Prahallad, C Sun, S Huang, F Di Nicolantonio, R Salazar,

R. Bernards. Nature (2012)

infect with

shRNA kinome library

PCR amplify

bar codes

WiDr

(BRAFV600E)

Deep

sequence Quantify shRNAs

vemurafenib culture to

allow selection control

Synthetic lethal shRNA screen: Inhibition of which

kinase synergizes with PLX in BRAF mutant CRC?



Inhibition of EGFR makes BRAF mutant CRC cells

vulnerable to BRAF inhibition

Grb

RAF

EGFR

KRAS

MEK

ERK

Cetuximab

Mutant



Synergistic response of BRAFV600E CRC

to EGFR and BRAF inhibition

Cetuximab 1.25 mg/ml

Gefitinib 0.125 M

VACO (BRAFV600E)

Also seen in WiDr and

KM20 (BRAFV600E) - x - = +



BRAFV600E inhibition causes feedback activation

of EGFR

VACO (BRAFV600E)

Also seen in WiDr and

KM20 (BRAFV600E) A Prahallad, C Sun, S Huang, F Di Nicolantonio, R Salazar,


WiDr xenografts (PLX+Cetuximab)

VACO423 xenografts (PLX+Cetuximab)

EGFR and BRAFV600E inhibitors synergize to

suppress colon cancer tumour growth in a xenograft

model



Ectopic expression of EGFR in melanoma is sufficient to confer resistance to BRAF inhibition

A375 (BRAFV600E)



Tercer ejemplo




Firmas genéticas de expresión

RNAm

– información sobre el comportamiento clínico

• Predicción de respuesta

• Pronóstico

ColoPrint

HR = 3.41 (P> 0.0001)

5-year DMFS

82%(95CI, 76-89%) low-risk

50%(95CI, 38-66%) high-risk

30%

DM

FS

Whole Genome analysis on 44K Agilent microarrays

70%

N= 188 (I:24; II:100; III:56; IV:8)

18 gene signature

CD: 92, CI: 74, Rect: 17

No QT: 145; QT: 31

18 genes identified that correlate Distant Metastasis-

free Survival, 18 most stable genes selected

Ramon Salazar et al, J Clin Oncol 2011: 29:17-24

Technical Validation of ColoPrint a roproducible and standardized test

ColoPrint uses the same

technology, methods and QC

as FDA-cleared MammaPrint

assay

Repeated runs of three samples over 20 days

performed by different operators

= less than 5% variation

ClinicalValidation of ColoPrint PARSC Study:

• Objective:

– validate the performance of ColoPrint in estimating the 3-year relapse rate in patients with stage II colon cancer.

– compare the risk assessment in stage II patients using ColoPrint profile vs a clinical risk assessment based on Investigator’s assessment of risk and ASCO high-risk recommendations.

– investigate therapy as a potential confounding factor for ColoPrint results

– assess the performance of ColoPrint in estimating the 3-year relapse rate in patients with stage III colon cancer.

• Principal Investigators:

– Europe: Dr. R. Salazar (ICO Barcelona)

– US: Dr. J. Marshall (Georgetown)

PARSC: Recent Status

• Aim 575 eligible stage 2 patients

• Status January 2012: – 32 sites open (EU 15, Asia 2, US 15)

– 340 eligible stage 2

– 300 eligible stage 3

• Expected last patient enrollement: Dec’12

Resumen: Avances tecnológicos • Identificar cuales son las principales dianas i vías oncogénicas en cada

tumor con relevancia terapéutica (tto y pronóstico) – Identificar las vías de resistencia intrínseca y adquiridas para desarrollar combinaciones

sinérgicas de fármacos contra varias dianas moleculares

• Hacerlo con plataformas tecnológicas aplicables en clínica

– Secuenciación por hotspots de Oncogens (e.g Fluidigm, Sequenome Oncocarta, Covax etc)

• KRAS, NRAS; BRAF, PIK 3Ca – Librerias genomicas (e.g. shRNA kinoma) – Secuenciación profunda de nueva generación de Genes supresores MLH1, MSH2, MSH6, PMS2 APC, p53 MYH – Inestabilidad de microsatèlites – Microarrays d´expressió RNAm (e.g. Colonprint ®) – Microarrays de metilación CpG – Plataformas Proteómicas

• Validación técnica y clínica de cualquier plataforma tecnológica son dos requisitos indispensables para aceptar su utilidad clínica

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Ramon Salazar, MD, PhD, MSc - UB · Ramon Salazar, MD, PhD, MSc Institut Català d’Onologia...

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