1. Istituto Toscano Tumori
New targets in NSCLC
Federico Cappuzzo
Istituto Toscano Tumori
Ospedale Civile
Livorno-Italy
Istituto Toscano Tumori –Livorno, Italy

2. Molecular events in lung cancer
Adenocarcinoma
Squamous-cell carcinoma
EGFR resistance mutations
0.8%
HER2
0.9%
EGFR
9.5%
KRAS
27%
Unknown
53.8%
BRAF
1.7%
PI3K
2.6%
ALK
3.7%
Barlesi F, ASCO 2013
Paik PK et al, ASCO 2012
Istituto Toscano Tumori – Livorno, Italy

3. ROS1 Translocations in NSCLC
Patients with ROS1 rearrangements share many features in common with ALK-positive patients (adenocarcinoma histology, younger age at diagnosis, never or light smokers)
Mutually exclusive with EGFR, HER2, KRAS, BRAF mutations and with ALK or translocation
Prognostic role is not defined
SDC4 exon 2
ROS1 exon 32
SDC4 exon 2
ROS1 exon 34
Istituto Toscano Tumori – Livorno, Italy

4. ROS1 fusion partners in NSCLC
Stumpfova and Janne PA, CCR 2013
Istituto Toscano Tumori – Livorno, Italy

5. Crizotinib: selective inhibitor of ALK, MET and ROS
Upstate 102 kinase panel
Cellular selectivity on 10 of 13 relevant hits
13 ‘hits’ <100X selective for Met
Kinase
IC50 (nM) mean*
Selectivity ratio
Met 8 –
ALK
40–60
5–8X
ROS 55 7X
RON 80
10X
Axl
294
34X
322
37X
Tie2
448
52X
Abl
1,159
166X
IRK
2,887
334X
Lck
2,741
283X
Sky
>10,000
>1000X
VEGFR2
PDGFRβ
High probability of ALK, MET and ROS inhibition at clinically relevant doses
Bang Y, et al. ASCO 2010
*Measured using ELISA capture method
Istituto Toscano Tumori – Livorno, Italy

6. Activity of crizotinib in ROS1+ NSCLC
ORR 72%; DCR 90%
Shaw AT, et al. NEJM 2014
Istituto Toscano Tumori – Livorno, Italy

7. Median DOR of Crizotinib in ROS1+ NSCLC
Median DOR 17.6 mos (95%CI,14.5 – NR)
Shaw AT, et al. NEJM 2014
Istituto Toscano Tumori – Livorno, Italy

8. Crizotinib in ROS1+ NSCLC: PFS
Median PFS: 19.2 months
Shaw A et al, NEJM 2014
Istituto Toscano Tumori – Livorno, Italy

9. Acquired resistance to crizotinib in ROS1 NSCLC
Awad MM, et al. NEJM 2013
Istituto Toscano Tumori – Livorno, Italy

10. Second generation ROS1 inhibitors
Istituto Toscano Tumori – Livorno, Italy

11. Crizotinib in MET amplified or ROS1 translocated NSCLC: The METROS trial
Istituto Toscano Tumori – Livorno, Italy

12. Clinical characteristics of MET amplified NSCLC%
Total amplified (ratio ≥2.2) 16
100
Squamous 5
31.2
Non-squamous 11
68.8
Never smokers
Current/former 15
93.7
Smoking unknown 1
6.3
Cappuzzo F et al., J Clin Oncol 2009
Istituto Toscano Tumori – Livorno, Italy

13. Survival of Resected NSCLC According to MET Copy Number
1,0
1,0
p=0.0045 ,8 ,8
≥4 - <5 copies/cell ,6 ,6
<2 copies/cell
MET <5 copies/cell(N=383)
CUMULATIVE SURVIVAL
CUMULATIVE SURVIVAL
≥3 - <4 copies/cell ,4 ,4
≥2 - <3 copies/cell
MET ≥5 copies/cell (N=48)
≥6 copies/cell
≥5 - <6 copies/cell ,2 ,2
Median survival:
MET FISH-:47.5 months
MET FISH+: 25.8 months
Un importante meccanismo di resistenza è l’amplificazione di MET. In uno studio condotto su 450 pts resecati chirurgicamente abbia o osservato come l’aumento di numero di copie del gene si associa ad una prognosi peggiore
0,0
0,0 20 40 60 80
100 20 40 60 80
100
120
MONTHS
MONTHS
Cappuzzo et al., JCO 2009
Istituto Toscano Tumori – Livorno, Italy

14. High levels of MET amplification drive resistance to EGFR-TKIs
Gefitinib Resistant
Gefitinib Sensitive
To be a driver MET should be high amplified. In our previuos experience, no amplification was detected in cell lines with EGFR mutations before gefitinib exposure, while very high levels were detected in the same cell line that become resistant to EGFR-TKI, and these high MET levels are extremely rare in patients
MET amplification in HCC827 GR6
Ratio MET/centromere >5
NO MET amplification in HCC827
Ratio MET/centromere <2
Modified from Cappuzzo F, et al. Ann Oncol 2008
Istituto Toscano Tumori – Livorno, Italy

15. Sensitivity to anti-Met agents only in presence of high levels of MET amplification
In this preclinical model, crizotinib induced apoptosis only in the two cell lines with high levels of MET amplification (EBC-1 and H1993)
Smolen GA et al., PNAS 2006, Tanizaki J et al., JTO 2011
Garcia L, University of Colorado, personal communication
Istituto Toscano Tumori – Livorno, Italy

16. Tumor Shrinkage Seen in Intermediate and High MET Cohorts
Best percent change from baseline in target tumor lesionsa by patient
Low MET n=2
Intermediate MET n=6
High MET n=6
100 80 60 40 20
–20
–40
–60
–80
–100
Disease progression
Stable disease
Partial responseb
Complete responseb
% Change From Baseline
Threshold for partial response c
Intermediate MET patients (n=6):
Patient :  Data in the database support an unconfirmed PR (so best response SD).  Patient discontinued due to an adverse event prior to response confirmation
Patient :  Confirmed response PR
Patient :  Best response SD.  Patient progressed at second post-baseline assessment
Patient :  Best response PD.  Patient progressed at first post-baseline assessment
Patient :  Best response SD.  At snapshot only one post-baseline assessment of unconfirmed PR; therefore best response documented as SD.
Patient :  Best response SD at both post-baseline visits
aConfirmed objective responses.
bBased on investigator assessment.
cTwo patients in the intermediate MET group had an unconfirmed PR that was not confirmed in a second assessment.
Garcia L, University of Colorado, personal communication
Istituto Toscano Tumori – Livorno, Italy

17. RET rearrangements in lung adenocarcinoma
11,294,741-bp pericentric inversion on chromosome 10 generating a new gene fusion joining exons 1-15 of KIF5B to exons of RET
Mutually exclusive with EGFR, HER2, KRAS, BRAF mutations and with ALK or ROS1 translocation
Exclusively identified in moderately to poorly differentiated adenocarcinomas with solid predominant subtype, younger age never or light smokers and with a particular pattern of metastatic spread (lymph nodes)
Istituto Toscano Tumori – Livorno, Italy

18. RET translocation and sensitivity to anti-RET drugs
Lipson et al. Nature Med. 2012
Istituto Toscano Tumori – Livorno, Italy

19. RET FISH+ve NSCLC succesfully treated with vandetanib
58 y-o man, past smoker (5 pck/yr)
ADC, stage IV for supraclavicular, mediastinal, retroperitoneal and inguinal nodes abdominal,
Pre-treated with standard carboplatin-pemetrexed for 2 cycles with evidence of progression
Second-line* Vandetanib 300 mg daily
* Patient unsuitable for standard chemotherapy due to recent myocardial infarction
Gautschi O, J Thor Oncol 2013
Istituto Toscano Tumori – Livorno, Italy

20. Activity of cabozantinib in RET + NSCLC
Drilon et al, Cancer Discov 2013
Istituto Toscano Tumori – Livorno, Italy

21. HER2 dysregulation in lung cancer
Overexpression
<10%
Amplification
<10%
Mutation
<3%
Istituto Toscano Tumori – Livorno, Italy

22. HER2 amplification is not prognostic in resected NSCLC
Cappuzzo et al., JTO 2012
Istituto Toscano Tumori – Livorno, Italy

23. High levels of HER2 amplification are responsible for acquired resistance to EGFR-TKIs in absence of T790M
Takezawa et al., Cancer Discovery 2012
Istituto Toscano Tumori – Livorno, Italy

24. High levels of HER2 amplification are responsible for acquired resistance to cetuximab in colorectal cancer
Yonesaka et al Science Transl Med 2011
Istituto Toscano Tumori – Livorno, Italy

25. HER2 mutation exon19 exon20 exon21 YVMA776-779ins GSP781-783ins
The vast majority of HER2 mutations, 92% (24/26) are in-frame insertions in exon 20
YVMA ins
GSP ins
E A Y V M A G V G S P Y V S R L I A K
770
785
831
G776V,Cins
Istituto Toscano Tumori – Livorno, Italy

26. HER2 Mutations in NSCLC Reference N Race % Never Smoker (%) Female (%)
Sasaki 95
Japan
1.0
2.7
3.3
Marchetti
403
Caucasian
2.2
3.1
4.1
Shigematsu
671
All
1.6
3.2
3.6
Stephens
120
4.0 -
Arcila
560
5.0*
5.0
2.0
* In EGFR and KRAS wild-type population
Istituto Toscano Tumori – Livorno, Italy

27. Trastuzumab efficacy in pretreatred NSCLC patients harboring HER2 mutation
Therapy
Best Response 1
Vinorelbine-trastuzumab
Partial response 2
Carboplatin-paclitaxel-trastuzumab
Stable disease 3
Docetaxel-masatinib
Progression 4 5 6 7 8
Lapatinib 9 10 11 12
Docetaxel-trastuzumab 13 14 15 16
Trastuzumab
RR: 56.2%
Modified from Mazieres et al. ESMO 2012
Istituto Toscano Tumori – Livorno, Italy

28. BRAF mutations in NSCLC
Detectable in up to 5% of lung adenocarcinomas using high sensitive methods
V600E is the most frequent mutation (up to 60% of all BRAF mutations)
V600E more frequent in female and in micropapillary features
Non-V600E mutations associate with smoking exposure with no prognostic effect
Effetto prognostico solo per la V600E
Marchetti et al, JCO 2011
Istituto Toscano Tumori – Livorno, Italy

29. Dabrafenib inhibits BRAF V600E Kinase
RTKs
SOS
Grb2 P P
SHC
Mode of Action
Reversible, small molecule
BRAF inhibitor
ATP competitive
Molecular Activity:
BRAF V600E: IC nM
BRAF WT: IC nM
Selectivity:
IC50 of nM against 8 of
282 human kinases P P P P
RAS
PI3K/AKT/mTOR
pathway
BRAF
V600
BRAF
CRAF
MEK
ERK1/2
p90RSK
MSK1
Proliferation, Growth, Survival
Davies H, et al. Nature. 2002;417: ; Platz A, et al. Mol Oncol. 2008;1: ; Karasarides M, et al. Oncogene. 2004;23: ; Curtin JA, et al. N Engl J Med. 2005;353: ; Flaherty K, et al. J Clin Oncol. 2009;27 [abstract 9000]; Kefford R, et al. J Clin Oncol. 2010;28 [abstract 8503].
Data not registered for darafenib
Istituto Toscano Tumori – Livorno, Italy

30. Dabrafenib in V600E BRAF mutated NSCLC: results of a phase II study
*** 50 40 30
*** 20
*** 10 *
−10
−20 ** ** **
Maximum Percent Reduction at Time of Best Disease Assessment
−30 ** *
−40
−50 ** *
Best Confirmed Response
Smoking History **
−60 * ** *
−70
Partial response
Nonsmoker
Smoker, ≤ 40 pack years
Smoker, > 40 pack years ** ** *
−80
Stable disease
***
−90
Progressive disease
−100
Planchard et al. ASCO 2013
Istituto Toscano Tumori – Livorno, Italy

31. Dabrafenib in BRAF mutated NSCLC: 2014 update
BRAF V600E mutations 1.5% of NSCLC, mutually exclusive to other driver alterations
Phase II dabrafenib in NSCLC p harboring V600E mut
78 previously treated p; median age 66 yrs, 50% female, 15% ECOG 2, 37% never-smoker
32% PR / 24% SD > 12 weeks / 29% PD / 14% NE
Disease control rate: 51% independent review vs 56% investigator
Median duration of response 11.8 mo
PFS 5.5 mo
Safety profile manageable
Planchard et alesmo 2014
Istituto Toscano Tumori – Livorno, Italy

32. Conclusions
Crizotinib is an emerging effective treatment in ROS1+ or MET amplified NSCLC
MET amplification could be detected in NSCLC generally not considered for biomarker assessment
RET translocation is a rare event but already druggable with available agents
Strategies against HER2 mutations should be extensively investigated
Drugs available for metastatic melanoma could represent a new option for BRAF mutated NSCLC
Istituto Toscano Tumori – Livorno, Italy