mutazione di BRAF nel melanoma e

immunoterapia nel melanoma e tumore

polmonare

Giulio Metro

S.C. Oncologia Medica – Ospedale Santa Maria della

Misericordia, Azienda Ospedaliera di Perugia

“Corso di laurea in tecniche di laboratorio biomedico”

5 Aprile 2016, Perugia

Screening

SEER Stat Fact Sheets:Melanoma of the Skin

GLOBOCAN 2012

Nessun consenso esiste sulla definizione di popolazionead alto rischio o sull’appropriato intervallo di screening

La sorveglianza della popolazione ad alto rischio nonriduce la mortalità da melanoma

Evidenza insufficiente di un impatto positivo delloscreening sulla mortalità

Screening sovradiagnosi e biopsie non necessarie

POPOLAZIONE AD ALTO RISCHIO1.Storia personale2.Storia familiare3.Numerosi e atipici nevi4.Carnagione chiara in uomini e donne >65 anni

Korn E. et al. J Clin. Oncol. 2008; 26:527-34

Sopravvivenza mediana: 6.2 months

Vivi ad 1 anno: 25.5%

Sopravvivenza libera da progressione: 1.7 months

Liberi da progressione a 6 mesi: 14.5%

Meta-analisi di studi di fase II di gruppi cooperativi: cosa si puòottenere nel melanoma metastatico con la chemioterapia?

Recettori

tirosin-

chinasici

Ligando

RAS

RAF

MEK 1

GTP

GDP

MEK 2

ERK 1

ERK 2

Proliferazione &

sopravvivenza

cellulare

PI3K

AKT

mTOR

La via RAS/RAF/MEK/ERK: BRAF

BRAFi (Vemurafenib,

Dabrafenib)

BRAF

1. Flaherty KT, et al. Cancer 2010;116:4902–13.

2. Garnett MJ, et al. Cancer Cell 2004;6:313–9.

3. Karreth FA, et al. Curr Opin Genet Dev 2009;19:4–11.

• Le chinasi RAF sono componenti della via MAP chinasi1

• Le chinasi RAF mediano la via del segnale RAS-RAF, che trasmette i segnali

proliferativi dalla superficie cellulare tramite I recettori dei fattori di crescita. In

condizioni normali tali segnali mediano il controllo fisiologico della proliferazione,

differenziazione e sopravvivenza1-3

• BRAF è espresso soprattutto nei tessuti neuronali e melanociti1

• Le MAP chinasi (MEK) sono l’unico substrato conosciuto di BRAF1

• La deregolazione del segnale RAS-RAF è stata implicata nello sviluppo

tumorale3

BRAFV600

cell

proliferation and

survival

Constitutive activation is independent of extracellular

factors and does not respond to biochemical signals

that would normally regulate activity

RAS–RAF

pathway

BRAF

Normal cell

proliferation and

survival

Normal

activation of

RAS via

extracellular

factors at

the inner cell

membrane via

RTK

BRAFV600

MEK

ERK

MEK

ERK

MEK

ERK

cell

proliferation and

survival

RAS–GTP

(activated RAS)

BRAF inhibitors

Growth

factors

PP

PP

PP

PP

RTK

Oncogenic

BRAF signaling

Oncogenic BRAF

signaling arrested with

BRAF inhibitors

Selective binding

of oncogenic

BRAF kinase by

vemurafenib,

blocks the

Constitutively

Activated pathway

and downstream

activity

Ascierto PA. et al. J Trans Med. 2012 May 3;10(1):85.

BRAF Mutations Affect

Kinase Activity• Ninety percent of BRAF mutations in melanoma result in substitution at

position V6002

– All tested mutations at V600 are classified as high-activity mutants

when compared with BRAFwild-type (WT)3,4

– These mutations constitutively stimulate ERK phosphorylation3

• A unifying feature of the high- and intermediate-activity BRAF mutants is

that they disrupt the hydrophobic interaction between the P loop and the

activation segment of the kinase domain4

– This results in destabilization of the inactive conformation of BRAF,

thus stimulating its kinase activity and leading to increased ERK

phosphorylation

BRAF = rapidly accelerated fibrosarcoma isoform B; ERK = extracellular signal―regulated kinase; WT = wild-type.1. Forbes SA, et al. Nucleic Acids Res 2010;38:D652–7 2. Garnett MJ, et al. Cancer Cell 2004;6:313–9.

BRAF Mutation

at Position V600

Melanomas (Total

2,651 V600X)1

Relative

Frequency (%)1

V600E 2,436 91.9

V600K 162 6.1

V600D 35 1.3

V600R 18 0.7

3. Wan PTC, et al. Cell 2004;116:855–67. 4. Pritchard C, et al. Biochem Soc Trans 2007;35:1329–33.

BRAF kinase domain structure. Position 600 is indicated in yellow. The BRAF activation segment is shown in purple; dashes indicate the region that was not resolved. The P loop is shown in green (adapted from Garnett MJ, et al).2

Types of BRAF mutation in melanoma

Clinicopathologic features

• Studies evaluating matched primary and metastatic melanomas

showed that BRAF mutations in primary tumor are maintained in

metastatic lesions1

• Patients with multiple metastasis showed, with few exceptions, the

same BRAF mutation in all tested lesions1

• BRAF mutations are early somatic events in the development of many

melanoma2

• Features of antecedent primary melanoma significantly associated with

BRAF mutation are histology (superficial and nodular), truncal

location, presence of mitoses, age at diagnosis ≤ 50 years2, 3

1. Omholt et al Clin Can res 20032. Platz A et al Mol Oncol 2008

3. Long G JCO 2011 Apr 1;29(10):1239-46

1. Long G JCO 2011 Apr 1;29(10):1239-46.2. Platz A et al Mol Oncol 2008

Prognostic and clinicopathologic associations of

oncogenic BRAF in metastatic melanoma

• The prognostic role of BRAF V600 mutation is still debated

• Some studies support a possible negative impact of BRAF mutations on

prognosis in metastatic melanoma1,2 .

• The presence of mutant BRAF had no impact on the disease-free interval from

diagnosis of first-ever melanoma to first distant metastasis; however, it may

have an impact on survival thereafter1

• BRAF mutations were significantly associated with diminished duration of

response to chemotherapy and the presence of BRAF mutations in melanoma

metastases was associated with shortened survival from the time point of

removal of the lesions or from the diagnosis of stage IV disease (Kumar 2003;

Houben 2004).

Testing Methods for BRAF Mutation Analysis

PCR Real-time Sequencing

BRAFV600E Kinase Mutation

Cellular

Proliferation

RTK

RAF

ATP

ATP

ERK

MEK

BRAFV600E

RAS

40-60% of melanomas

Chapman P. et al Abs LBA4 ASCO 2011

Vemurafenib inhibits BRAFV600E Kinase

Cellular

Proliferation

RTK

RAF

VEMURAFENIB(PLX4032, RO5185426)

ATP

ATP

ERK

MEK

BRAFV600E

RAS

40-60% of melanomas

Chapman P. et al Abs LBA4 ASCO 2011

Vemurafenib Is Selective for Oncogenic BRAF

• Vemurafenib is a small molecule inhibitor that binds potently to and selectively

inhibits the oncogenic BRAF kinase (BRAFV600)1

• Developed by a structure-guided discovery approach coupled with

optimization chemistry1,2

• Has a novel mode of action

Vemurafenib binds to the oncogenic BRAF

kinase (yellow) (adapted from Bollag G, et al)1

BRAF = rapidly accelerated fibrosarcoma isoform B.

1. Bollag G, et al. Nature 2010;467:596–9.2. Tsai J, et al. Proc Natl Acad Sci USA 2008;105:3041–6.

Vemurafenib1

Vemurafenib Inhibits Tumor Growth in

Vivo1

BID = twice daily; QD = once daily; SEM = standard error of the mean.

1. Yang H, et al. Cancer Res 2010;70:5518–27.

• Vemurafenib is reported to inhibit tumor growth and prolong survival in BRAF

mutation-positive melanoma xenograft models:

Mice implanted with Colo829 xenografts started treatment 17 days after implantation. Animals were treated with vehicle or Vemurafenib at a dose of 100 mg/kg BID for 21 days or with temozolomide at a dose of 100 mg/kg once daily for 5 days

Vehicle BID x 21 days

Vemurafenib 100 mg/kg BID x 21 days

Temozolomide 100 mg/kg QD x 5 days

Days after tumor cell implant

Vemurafenib: Phase I – Early FDG-PET Responses

FDG = F-18-fluorodeoxyglucose; PET = positron emission tomography.

1. Flaherty KT, et al. N Engl J Med 2010;363:809–19.

• Patients underwent FDG-PET at baseline and on Day 15 of the first 4 weeks of

therapy

• A marked decrease in tumor uptake of FDG was observed at Day 15 after

Vemurafenib treatment

BRIM-2 (NP22657): Phase II Study in Previously

Treated Patients with Metastatic Melanoma1,2

* Ascertained by the cobas® 4800 BRAF V600 Mutation Test.

AJCC = American Joint Committee on Cancer; BID = twice daily;

• Multicenter (USA and Australia), open-label, Phase II study

• Enrolment completed

• Primary endpoint:

– BORR (RECIST, version 1.1) assessed by IRC every 6 weeks

• Key secondary endpoints:

– BORR assessed by investigator

– Duration of response, PFS and OS

– Safety and tolerability

– Quality of life

– Validation of cobas® 4800 BRAF V600 Mutation Test

– PK and effect on QT interval and correlation on exposure using

electrocardiogram parameters

Vemurafenib (960 mg BID orally)

• 132 patients enrolled • Metastatic melanoma (stage IV, AJCC) • BRAFV600 positive*• Failed ≥ 1 prior therapy

1. Sosman J, et al. Oral presentation at Melanoma 2010, Sydney, Australia. 2. A Study of RO5185426 in previously treated patients with metastatic melanoma. NCT00949702. Available at www.clinicaltrials.gov (last accessed August 10, 2010).

0

10

20

30

40

50

60

70

CR+PR SD PD

Assessment of Primary Endpoint: Tumor

Responses by Independent Review Committee

(IRC)

• ORR 53% by IRC

• ORR 57% by investigator assessments (INV)

• RR, including unconfirmed, 69% (INV)

• PR in 4 of 10 BRAFV600K patients

Resp

on

se r

ate

(%

)

n=70 n=38 n=18

53% CR+PR

5% CR

29%

14%

Error bars represent 95% confidence intervals

Ribas A. et al Abs 8509 ASCO

2011

BRIM-3 (NO25026): Phase III Study in Previously

Untreated Patients with Metastatic Melanoma

• International, multicenter, randomized, open-label Phase III study

• This study is currently ongoing

• Co-primary endpoints:

– Overall survival

– PFS

• Key secondary endpoints:

– Efficacy: BORR, time to response, duration of response, time to

treatment failure

– Safety and tolerability

– Pharmacokinetics

– Contribute to validation of cobas® 4800 BRAF V600 Mutation Test

Vemurafenib(960 mg BID orally)

Dacarbazine(1000 mg/m2 IV q3w)

1:1

o Estimated enrollment: 680 patients o Unresectable metastatic melanoma (stage

IIIc/IV, AJCC) o BRAFV600 mutation positive, determined

by cobas® 4800 BRAF V600 Mutation Testo Chemo-naïve for advanced disease

N=680

NCT01006980. Available at www.clinicaltrials.gov (last accessed August 10, 2010).

Chapman P. et al Abs LBA4 ASCO 2011

100

90

80

70

60

50

40

30

20

10

0

Pro

gre

ssio

n-f

ree

su

rviv

al (

%)

0 6 12 18 24

338

337

63

186

22

77

3

16

0

0

100

269

37

113

14

49

0

3

No. at risk

1.6 6.9

Hazard ratio 0.38 (95% CI: 0.32–0.46)Log-rank p<0.001 (post-hoc)

Dacarbazine(n=338)

Vemurafenib (n=337)

Progression-free survival (February 01, 2012

cut-off) censored at crossover

Time (months)

Dacarbazine

Vemurafenib

Chapman P. et al. abs 8502

ASCO Ann. Meeting Chicago 2012

100

90

80

70

60

50

40

30

20

10

0

Ove

rall

surv

ival

(%

)

0 6 12 18 24

Vemurafenib (n=337)

Median f/u 12.5 months

Dacarbazine (n=338)

Median f/u 9.5 months

338

337

173

280

79

178

24

44

0

1

244

326

111

231

50

109

4

7

9.7 13.6

Overall survival (February 01, 2012 cut-off)

censored at crossover

Hazard ratio 0.70 (95% CI: 0.57–0.87)p<0.001 (post-hoc)

Time (months)

Dacarbazine

Vemurafenib

No. at risk

Chapman P. et al. abs 8502

ASCO Ann. Meeting Chicago 2012

Confirmed objective response rates

(RECIST 1.1) across vemurafenib clinical

trial programme

PLX 06-02

Phase I BRIM 2

BRIM-3 ORR(February 01, 2012

cut-off)

Vemurafenib

(95% CI)

56.0%

(38–74)

53.0%

(44–62)

57.0%

Dacarbazine

(95% CI)

– – 8,6%

Mc Arthur G et al ECCO/ESMO Abstract #28LBA

Chapman Annual ASCO Meeting Chicago 2012

Selected adverse events (% of patients)

Vemurafenib, n=337 Dacarbazine, n=287

Adverse events All Grade 3 Grade ≥4 All Grade 3 Grade ≥4

Arthralgia 56 6 – 4 1 –

Rash 41 9 – 2 – –

Fatigue 46 3 – 35 2 –

Photosensitivity 41 4 – 5 – –

LFTs 26 10 1 6 2 –

Cutaneous SCC 19 19 – <1 <1 –

Keratoacanthoma 11 10 – <1 <1 –

Skin papilloma 28 <1 – <1 <1 –

Nausea 38 2 – 45 2 –

Neutropenia <1 – <1 12 6 3

Discontinuations due to AE: 7% vemurafenib; 2% dacarbazine

8 patients reported new primary melanomas in the vemurafenib groupChapman P. et al. abs 8502

ASCO Ann. Meeting Chicago

2012

VEMURAFENIB

Approvazione EU, 17 February 2012

Vemurafenib è indicato come monoterapia per il

trattamento dei pazienti adulti con melanoma

avanzato e mutazione di BRAF V600.

http://ec.europa.eu/health/documents/community-register/html/h751.htm

ExperiencePatients

sample (n)

% of patients with a rapid

disease progression kinetics

BRIM-2 39 41%

BRIM-3 42 52%

Ascierto et al. 28 43%

Ackerman et al. 32 50%

Italian ipi EAP 54 41%

Fisher et al. 42 38%

Evidenza di rapida progressione di malattia all’instaurarsi della

resistenza all’inibitore di BRAF

Meccanismi di resistenza agli inibitori di

BRAF

Recettori

tirosin-

chinasici

Ligando

RAS

RAF

MEK 1

GTP

GDP

MEK 2

ERK 1

ERK 2

Proliferazione &

sopravvivenza

cellulare

PI3K

AKT

mTOR

Blocco di più bersagli contemoporaneamente: BRAF e MEK

BRAFi (Vemurafenib,

Dabrafenib)

MEKi (Selumetinib,

Trametinib)

Dabrafenib+trametinib vs Vemurafenib Vemurafenib+cobimetinib vs

vemurafenib+placebo

Sopravvivenza libera da progressione con inibitore di BRAF

+ inibitore di MEK

Dabrafenib/trametenib PFS = 11.4 months

vs HR = 0.56

Vemurafenib PFS = 7.3 months

ORR= 64% vs 51%

Vemurafenib/cobimetinib PFS = 9.9 months

vs HR = 0.51

vemurafenib/placebo PFS = 6.2 months

ORR= 68% vs 45%

Larkin J. et al N Engl J Med. 2014Robert C. et al N Engl J Med. 2015

HR= 0.69

(95% CI 0.53-0.89)

P=0.002

HR = 0.65

(95% CI 0.42-1.00)

P=0.046

Larkin J, et al N Engl J Med. 2014Robert C, et al N Engl J Med. 2015

Sopravvivenza globale con inibitore di BRAF

+ inibitore di MEK

Dabrafenib+trametinib vs Vemurafenib Vemurafenib+cobimetinib vs

vemurafenib+placebo

Eventi avversi della combinazione verso la monoterapia

Science, 2013Science 2013

I “checkpoint” immunitari

CTLA-4 = cytotoxic T-lymphocyte-associated antigen 4 ; MHC = major histocompatibility complex; PD-1 = programmed death-1;

PD-L1 = programmed death ligand 1; TCR = T-cell receptor.

Blocco CTLA-4 (ad esempio

ipilimumab)

Blocco PD-1 (ad es. nivolumab,

pembrolizumab)

Fase di iniziazione Fase di effettrice

D E C E M B E R 2 0 1 1 | VO L 4 8 0 | N AT U R E | 4 8 1

Recettori delle cellule T co-stimolatori

From Michot et al. Eur J Cancer 2016

Mel = melanoma; RCC = renal cell carcinoma; NSCLC = non-small cell lung cancer; HNSCC = head &

neck squamous cell cancer; B Cell NHL = B cell non-Hodgkin lymphoma; MSI High CRC = microsatellite

instability high colorectal carcinoma; TNBC; triple negative breast cancer; Mesoth = mesothelioma;

HCC = hepatocellular carcinoma; Oesophag = oesophageal; SCLC = small cell lung cancer

Lawrence et al, Nature 2013Lawrence et al, Nature 2013

Istituto Toscano Tumori – Livorno, Italy

Altered protein contain new epitopes for immune recognition, providing a common denominator for immunotherapy

High mutational rates may contribute to increased immunogenicity

ORR significantly higher with nivolumab vs dacarbazine

(40.0% vs 13.9%, respectively; P < .001)

Biological: Nivolumab

Biological: Placebo matching Nivolumab

Drug: Dacarbazine

Drug: Placebo matching Dacarbazine

R

418 pts

Primary End-Point Overall Survival

Survival End Points

HR for death: 0.42 (99.79% CI: 0.25-0.73; P < .001)

HR for death or disease progression: 0.43 (95% CI: 0.34-0.56; P < .001)

Robert C, et al. N Engl J Med. 2014

The future approach will be greatly informed by results of trials comparing BRAF/MEK inhibition followed by anti–PD-1/PD-L1 plus ipilimumab, vs the opposite strategy, and those evaluating the combination of anti–PD-1/PD-L1 plus ipilimumab.

Treatment paradigm in advanced melanoma poised for change… again

Symptom load may still matter

Toxicity may be an issue

Combination yields promising outcomes

Squamous cell lung cancer: Overall Survival

Symbols represent censored observations

Nivolumab

Docetaxel

135 113 86 69 52 31 15 7 0

137 103 68 45 30 14 7 2 0

Number of Patients at Risk

Time (months)

Nivolumab

Docetaxel

1-yr OS rate = 42%

1-yr OS rate = 24%

OS

(%

)Nivolumab

n = 135

Docetaxel

n = 137

mOS mo,

(95% CI)

9.2

(7.3, 13.3)

6.0

(5.1, 7.3)

# events 86 113

HR = 0.59 (95% CI: 0.44, 0.79), P = 0.00025

24211815129630

100

90

80

70

60

50

40

30

10

0

20

OS by PD-L1 Expression

mOS (mo)

Nivolumab Docetaxel

PD-L1 ≥1% 9.3 7.2

PD-L1 <1% 8.7 5.9

mOS (mo)

Nivolumab Docetaxel

PD-L1 ≥5% 10 6.4

PD-L1 <5% 8.5 6.1

mOS (mo)

Nivolumab Docetaxel

PD-L1 ≥10% 11 7.1

PD-L1 <10% 8.2 6.1

1% PD-L1 Expression level 5% PD-L1 Expression level 10% PD-L1 Expression level

Nivolumab PD-L1+

Nivolumab PD-L1–

Time (months)

24211815129630

Time (months)

24211815129630

Time (months)

24211815129630

100

90

80

70

60

50

40

30

10

0

20

OS

(%

)

24211815129630

100

90

80

70

60

50

40

30

10

0

20

Docetaxel PD-L1+

Docetaxel PD-L1–

Non-squamous cell lung cancer:

Overall Survival

Symbols represent censored observations.

Nivolum

ab

(n =

292)

Doceta

xel

(n =

290)

mOS, mo 12.2 9.4

HR = 0.73 (96% CI: 0.59, 0.89);

P = 0.0015

Nivolumab

Docetaxel

1-yr OS rate = 51%

1-yr OS rate = 39%

292 232 194 169 146 123 62 32 09

290 244 194 150 111 88 34 10 05

Nivolumab

Docetaxel

Number of Patients at Risk

OS

(%

)

Time (months)

100

90

80

70

60

50

40

30

10

0

20

27211815129630 24

Nivo

Doc

Nivo

Doc

100

90

80

70

60

50

40

30

10

0

20

Time (months)

100

90

80

70

60

50

40

30

10

0

20

24211815129630 27

Time (months)

24211815129630 27

Symbols represent censored observations.

OS by PD-L1 Expression

mOS (mo)

Nivo 10.4

Doc 10.1

mOS (mo)

Nivo

17.2

Doc

9.0

mOS (mo)

Nivo

9.9

Doc 10.3

mOS (mo)

Nivo

19.4

Doc

8.0

Time (months)

≥5% PD-L1 expression level

<5% PD-L1 expression level

mOS (mo)

Nivo

18.2

Doc

8.1

mOS (mo)

Nivo

9.7

Doc

10.1

≥1% PD-L1 expression level

HR (95% CI) = 0.59 (0.43, 0.82)

Time (months)

<1% PD-L1 expression level

OS

(%

)

HR (95% CI) = 0.90 (0.66, 1.24)

HR (95% CI) = 0.43 (0.30, 0.63)

HR (95% CI) = 1.01 (0.77, 1.34)

OS

(%

)

Time (months)

Time (months)

≥10% PD-L1 expression level

<10% PD-L1 expression level

HR (95% CI) = 0.40 (0.26, 0.59)

HR (95% CI) = 1.00 (0.76, 1.31)

24211815129630 27

100

90

80

70

60

50

40

30

10

0

20

100

90

80

70

60

50

40

30

10

0

20

24211815129630 27

24211815129630 27

100

90

80

70

60

50

40

30

10

0

20

24211815129630 27

100

90

80

70

60

50

40

30

10

0

20

Representative PD-L1 expression in NSCLC samples stained using Dako assay

PD-L1 expression measured:– using a sensitive and specific rabbit anti-human PD-L1 antibody (clone 28-8)– as a percentage of tumoural plasma membrane staining of any intensity (information on

immune cell expression recorded qualitatively)

NegativeControl

Antibody

Positive

% Staining 1% 5% 20% 65%

Antonia et al., WCLC, 2013

Nivolumab more effective in smokers

VariableORR, % (n/N) [95%

CI]P-value

Smoking exposure 0.018

≤5 pack-yrs 0 (0/14) [0, 23]

>5 pack-yrs 30 (20/66) [20, 43]

Time since quitting 0.22

Current smoker 27 (6/22) [11, 50]

1–5 yrs prior 46 (6/13) [19, 75]

6–15 yrs prior 17 (2/12) [2, 48]

>15 yrs prior 18 (6/33) [7, 36]

0

20

80

60

40

Months Since Treatment Initiation

100

PFS

(%

)

PFS by smoking exposure

Never/minimal smokers (mPFS 1.7 months)Former/current smokers (mPFS 2.2 months)HR (95% CI) = 0.41 (0.22, 0.74), P = 0.003

mPFS = median Progression-free survival

Hellmann et al et al. ASCO 2014

Kroemer et al. Oncoimmunology. 2012;1(5): 579–580. .

Matsushita et al. Nature.2012 Feb 8;482(7385):400-4.

Synder et al. NEJM. 2014; 371. Rizvi et al. Science. 2015; 348.

• These mutations lead to

tumor specific

neoepitopes which

serve as neoantigens.

• Those tumors with the

most highly antigenic

proteins are more likely

to lead to a T cell

response.

• Each patients tumor

mutanome is unique

and there appears to be

certain candidate

neoantigens or

neoantigen signatures

that lead to more

durable immune

responses.

Potenziale tossicità auto-immune: nessun organo è risparmiato

From Michot et al. Eur J Cancer 2016

Potenziale tossicità auto-immune: maggiore con gli anti-

CTLA4

Tossicità auto-immune grado 1 e 2 Tossicità auto-immune grado 3 e 5

From Michot et al. Eur J Cancer 2016

Conclusioni

• Vemurafenib ha cambiato la storia naturale dei pazienti con

melanoma avanzato e mutazione di BRAF

• L’immunooncologia rappresenta il presente e il futuro della

terapia oncologica e rappresenta una modalità trasversale

di terapia oncologica