Sandra Ortiz-Cuaran*, Laura Mezquita*, Aurélie Swalduz, Mihaela Aldea, Julien Mazières, Cecile Jovelet, Washington René Chumbi Flores, Ludovic Lacroix, Yohan Loriot, Luc Friboulet, Virginie Westeel, Maud Ngocamus, Anne Pradines, Claire Tissot, Christelle Clement Duchene, Christine Raynaud, Xavier Quantin, Radj Gervais, Etienne Brain, Isabelle Monnet, Etienne Giroux Leprieur, Virginie Avrillon,

Céline Mahier-Aït Oukhatar, Natalie Hoog-Labouret, Frank de Kievit, Karen Howarth, Jean-François Guichou, Clive Morris, Emma Green, Maurice Pérol, Benjamin Besse, Jean-Yves Blay, Pierre Saintigny*, David Planchard*.

Centre Léon Bérard and Cancer Research Center of Lyon, Lyon, France - Institute Gustave Roussy, Villejuif, France - Hôpital Larrey, Centre Hospitalier Universitaire, Toulouse, France - Hôpital de la Croix-Rousse, Lyon, France - Jean Minjoz University Hospital, Besançon - Centre de Recherche de Cancérologie de Toulouse, CRCT, Toulouse, France - Hôpital Nord, CHU de Saint-Étienne, France - Institut de Cancérologie de Lorraine Alexis-Vautrin, Vandoeuvre-lès-Nancy, France - Centre Hospitalier Victor-Dupouy, Argenteuil, France - Centre Hospitalier Universitaire Montpellier, Montepllier, France - Hospital Center University Caen, Caen, France - CLCC rene Huguenin Institut Curie, Sant

Cloud, France - Centre Hospitalier Intercommunal de Créteil, Créteil, France - APHP – Ambroise Paré Hospital, Boulogne-Billancourt, France - INCa, Boulogne Billancourt , France - Inivata Ltd. Cambridge, United Kingdom

INTRODUCTION BRAF mutations occur in 2 to 3% of patients with lung adenocarcinoma. In these patients vemurafenib, a selective oral BRAF inhibitor, is associated with a response rate of 42%, rising to 64% for combination treatment with dabrafenib and trametinib. Despite initial

responses, most patients ultimately develop resistance to therapy. The limited knowledge on the molecular profile of NSCLC patients who progress or respond poorly to BRAF-targeted therapies has undermine the development of subsequent targeted therapeutic strategies. Here, our objective was to assess the molecular mechanisms of resistance to BRAF-targeted therapies and to monitor disease response to treatment using liquid biopsies in lung adenocarcinoma patients treated with BRAF inhibitors.

STRATEGY

We performed genomic profiling of serial ctDNA in 79 metastatic BRAF-mutant NSCLC pts (96% V600E, 4% non-V600). BRAF status was ascertained based on local testing. Plasma samples were collected, from 2014-2018 in 27 hospitals, from pts treated with V (n=34), D (n=2) or D+T (n=23). We collected 41 plasma samples at baseline (BL) to BRAF-TT, 40 at progressive disease (PD) and ~200 samples during treatment follow-up, concurrent to routine radiological evaluation. Inivata InVisionFirst®-Lung assay was used to detect the presence of genomic alterations in 36-cancer related genes.

1.  Circulating tumor DNA analysis of BRAF mutations and co-occurring alterations at baseline

CONCLUSION ctDNA genotyping is an informative tool for monitoring disease response and resistance in lung adenocarcinoma patients treated with BRAF - targeted therapies. Reactivation of the MAPK pathway remains an important resistance mechanism to BRAFi-monotherapy or to BRAFi and MEKi combination therapy.

Circulating tumor DNA analysis reveals potential mechanisms of resistance to BRAF-targeted therapies in BRAF-mutant non-small cell lung cancer

RESULTS

FUNDING

2. Early clearance and dynamics of BRAF mutations in ctDNA as a predictor of response to BRAF targeted therapies

At disease progression, a consistent rebound in BRAFV600E plasma levels was observed in 63% of patients patients. Molecular progression observed in 3 patients with a median of 57 days before confirmation of radiographic progression. Potential mechanisms of acquired resistance were detected in in 16/37 patients, an included oncogenic or likely-oncogenic alterations in effectors of the MAPK pathway with, at disease progression on either BRAF inhibitor alone or in combination with MEK inhibition: Alterations in PIK3CA, IDH1and CTNNB1 were also observed.

DO NOT POST DO NOT POST

3. Genomic ctDNA profiling of disease progression on BRAF-targeted therapies

INCa_INSERM_DGOS_12563

InVisionFirst®-Lung (Amplicon-based, 36-gene panel, Inivata)

27 Hospitals From 2014 to 2018

79 patients, 232 plasma samples

Pre-BRAFi treatment

Partial response to TKI No.1

TKI No.1 TKI No.1 TKI No. 2

Progression to TKI No.1

Progression to TKI No.2

PATIENTS CHARACTERISTICS Median age at diagnosis was 66 years, gender was balanced, 34% of patients were never-smokers and the majority presented lung adenocarcinoma histology. Thirty-two patients had malignant effusion, and 9 had brain metastases at diagnosis. First-line treatment consisted on platinum-based chemotherapy in 47/78 (60.2%) patients. A total of 82 lines of BRAF-targeted therapy were administered.

n %Treatment overview Vemurafenib 36 46.2 Dabrafenib 1 1.3 Dabrafenib + Trametinib 26 33.3 BRAFi monotherapy, then Dabrafenib + Trametinib 5 6.4 Other regimens 4 5.1 Not treated with BRAF-TT 6 7.7BRAF targeted therapy - treatment line (n=72) * First line 17 23.6 Second line 45 62.5 Further lines 10 13.9

024681030405060

Mutation frequency (%)

AKT1

NTRK3

FGFR2

ERBB2

IDH1

CCTNB1

U2AF1

TP53

CDKN2A

Ong

oing

?D

: Dec

isio

n du

pat

ient

T: T

OX

?: M

issi

ng in

form

atio

nD T

X: n

o fu

rthe

r tre

atm

ent

0

5

10

15

20

25

30

Tim

e on

BR

AF-

TT (m

onth

s)

7027179

1783837

5150

43

341840

314749

35

1141727223484676356473755868

Ongoing

?D: Decision du patientT: TOX

?: Missing informationDT

X: no further treatment

XTOX

5

X

IDBest responseMedian total AFBRAF AFBRAFKRASNRASPIK3CAAKT1PTENNTRK3FGFR2ERBB2IDH1CCTNB1U2AF1TP53CDKN2ASTK11

5

22

19

14

70

12

2

71

79

17

8

38

37

51

50

57

43

34

18

40

31

47

49

35

11

41

3

72

72

23

48

15

46

76

35

64

73

75

58

68

68

45

44

42

36

9

39

Vemurafenib Chemotherapy Dabrafenib+Trametinib

SD PR PD NA Non V600E BRAF V600E

39.7 0

39.7 0 Mutation

frequency (%)

MAPK pathway PI3K pathway

Protein kinases Signal transducers RNA splicing

Surv

ival

pro

babi

lity

Median (95%CI) NE (0.6-NE) 12.1 (5.1-NE)

Logrank P-value: 0.1537

Months from treatment initiation

BRAF ctDNA undetectable

BRAF ctDNA positive

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

0 0 3 6 9 12 15 18 21 24 27 30 33 36

UND 8 7 5 4 1 0

POS 29 19 14 9 2 0

Circulating BRAFV600E mutation was detected in 31/42 (74%) of patients, whereas non-V600E mutations were detected plasma of in 3/5 patients. Patients with a detectable circulating BRAF mutation tended to have worse overall survival than patients with undetectable BRAF-mutant levels in plasma (Logrank P = 0.153). Co-occurring alterations included oncogenic PIK3CA mutations, ERBB2 amplification, a likely activating FGFR2 mutation as well as oncogenic AKT1 and KRAS mutations and a likely activating NTRK3 mutation.

We observed clearance of the BRAFV600E mutation in plasma, at the first-radiological evaluation, in 12/20 patients. In these patients, circulating levels of BRAFV600E were associated with response to treatment. Similar results were observed when we analyzed >100 serial longitudinal samples. In these cases too, the levels of BRAFV600E in plasma were associated with response to treatment.

TreatmentBest responseBRAFBRAF AFKRASNRASMAP2K1GNASGNA11PIK3CAPPP2R1AMETMYCNFE2L2U2AF1IDH1CTNNB1TP53STK11ID

72

*

68

67

Yes-nodriver?

59

* *

Yes-nodriver?

53524441

35

33

32

31

3027

Yes-notdriver?2625

24

20

1817

1615

1110

74

3

1 64 696540 55 57 58024681030405060

Mutation frequency (%)

GNAS

PIK3CA

PPP2R1A

MET

MYC

NFE2L2

U2AF1

IDH1

CCTNB1

TP53

Ong

oing

?D

: Dec

isio

n du

pat

ient

T: T

OX

?: M

issi

ng in

form

atio

nD T

X: n

o fu

rthe

r tre

atm

ent

MAPK pathway

PI3K pathway

RTKs

Alternative pathways

Mutation frequency (%)

Dabrafenib + Trametinib Dabrafenib Vemurafenib

Non-V600E V600E ND No baseline sample available Present before BRAF-TT Undetected before BRAF-TT

SD PR PD NA 24.6 0 ** *High-dose KRAS Q61R + G12V