1. James R. Rigas Comprehensive Thoracic Oncology Program
Molecular Basis of Lung Cancer Therapy
James R. Rigas
Comprehensive Thoracic Oncology Program

2. Traditional View of Lung Cancer
Adenoca
Small
Cell
Large Cell
Squamous

3. Presentation EGFR mutations ALK gene rearrangements BRAF mutations
RADIANT adjuvant results in EGFR mutations
Uncommon EGFR mutations
Resistance in EGFR mutations (+) patients
ALK gene rearrangements
Resistance in ALK gene rearrangement (+) patients
BRAF mutations
Other mutations, fusions and amplifications

4. Locations and Types of the 134 EGFR Gene Mutations Detected in Lung Cancers
Del 19
T790M
Locations and types of the 134 epidermal growth factor receptor (EGFR) gene mutations detected in lung cancers. The structure of the EGFR gene is shown at left, and the locations and types of the mutations in the tyrosine kinase (TK) domain are shown at right. All mutations were located within exons 18–21, which encode the N lobe and part of the C lobe of EGFR (shaded area of the gene on the left, which is presented in magnified form on the right). Three major types of mutations (shown in bold) formed 94% of the 134 mutations detected and consisted of deletions in exon 19 immediately 5′ of the αC-helix (11 types, labeled Δ1–Δ11), duplications and/or insertions in exon 20 immediately 3′ of the αC-helix (eight types labeled D1–D8), and a single-point mutation, L858R (labeled M1), in the A-loop. The remaining 6% of mutations consisted of missense mutations in the P-loop in exon 18 (six types labeled M2–M7), in the αC-helix in exon 20 (a single type labeled M8), or in the A-loop in exon 21 (a single type labeled M9).
L858R
Shigematsu H et al. JNCI J Natl Cancer Inst 2005;97:
Journal of the National Cancer Institute, Vol. 97, No. 5, © Oxford University Press 2005, all rights reserved.

5. Platinum-based doublet chemotherapy q3wks x 4 cycles*
EURTAC Study Design
Stage IIIB/IV NSCLC
EGFR exon 19 deletion or exon 21 L858R mutation (DNA sequencing/Genescan and Taqman)
Chemonaive
ECOG PS 0–2
Measurable or evaluable disease
Erlotinib 150 mg/day PD
Stratification
Mutation type
ECOG PS (0 vs 1 vs 2) R
Platinum-based doublet chemotherapy q3wks x 4 cycles* PD
Secondary endpoints
Objective response rate
Overall survival (OS)
Location of progression
Safety
EGFR mutation analysis in serum
Quality of life
Primary endpoint
Progression-free survival (PFS)
US FDA approval May 14, 2013
Cobas® EGFR Mutation Test
41 mutations in Exons 18, 19, 20 and 21
ECOG = Eastern Cooperative Oncology Group; PS = performance status; PD = progressive disease
*Cisplatin 75mg/m2 d1 / docetaxel 75mg/m2 d1; cisplatin 75mg/m2 d1 / gemcitabine 1250mg/m2 d1,8; carboplatin AUC6 d1 / docetaxel 75mg/m2 d1; carboplatin AUC5 d1 / gemcitabine 1000mg/m2 d1,8 5 5

6. Primary endpoint: PFS in ITT population (updated analysis 26 Jan 2011)
1.0
0.8
0.6
0.4
0.2
Erlotinib (n=86)
Chemotherapy (n=87)
HR=0.37 (0.25–0.54)
Log-rank p<0.0001
PFS probability
5.2
9.7
Time (months)
Patients at risk
Erlotinib Chemo
Data cut-off: 26 Jan 2011
US FDA approval May 14, 2013

7. Study design US FDA approval July 12, 2013
Stage IIIB (wet)/IV lung adenocarcinoma (AJCC version 6)
EGFR mutation in tumor
(central lab testing; Therascreen EGFR29* RGQ PCR)
Randomization 2:1
Stratified by:
EGFR mutation (Del19/L858R/other)
Race (Asian/non-Asian)
Afatinib 40 mg/day†
Cisplatin + Pemetrexed
75 mg/m mg/m2
i.v. q21 days, up to 6 cycles
Primary endpoint: PFS (RECIST 1.1, independent review)‡
Secondary endpoints: ORR, DCR, DoR, tumor shrinkage, OS, PRO§, safety, PK
*EGFR29:19 deletions in exon 19, 3 insertions in exon 20, L858R, L861Q, T790M, G719S, G719A and G719C (or G719X), S768I.
†Dose escalated to 50 mg if limited AE observed in cycle 1. Dose reduced by 10 mg decrements in case of related G3 or prolonged G2 AE. ‡Tumor assessments: q6 weeks until Week 48 and q12 weeks thereafter until progression/start of new therapy.
§Patient-reported outcomes: Q-5D, EORTC QLQ-C30 and QLQ-LC13 at randomization and q3 weeks until progression or new anti-cancer therapy.
US FDA approval July 12, 2013
Yang JC, et al.

8. EGFR Gene Mutations in Adenocarcinoma Lung Cancer
Therascreen ®
EGFR (29) RGQ PCR Kit

9. PFS: Common mutations (Del19/L858R) Independent review – patients with Del19/L858R (n=308)
1.0
Afatinib n=204
Cis/pem n=104
PFS event, n (%)
130 (64)
61 (59)
Median PFS (months)
13.6
6.9
Hazard ratio (95% confidence interval)
0.47 (0.34–0.65) p<0.0001
0.8
0.6
Progression-free survival (probability)
51%
0.4
0.2
21%
0.0
Progression-free survival (months)
Number at risk Afatinib Cis/Pem
US FDA approval July 12, 2013
Yang JC, et al.

10. RADIANT Study Schematic

11. RADIANT Study Results Hierarchical testing rendered all
secondary endpoints non-significant.

12. LUX-Lung clinical trials and eligibility
Treatment
Line of treatment
Mutation test
LUX-Lung 2
Phase II
N=129
Afatinib
First- and second-line (after chemo)
Direct sequencing (central)
LUX-Lung 3
Phase III
N=345
Afatinib vs. Pemetrexed/ cisplatin
First-line
EGFR29* (central)
LUX-Lung 6
N=364
Afatinib vs. Gemcitabine/ cisplatin
*EGFR mutations detected by TheraScreen EGFR29 test:
Common: 19 deletions in exon 19 and L858R in exon 21
Uncommon: 3 insertions in exon 20, L861Q, T790M, G719S, G719A and G719C, S768I

13. EGFR mutation-positive patients in LUX-Lung trials
Del19 n=408
L858R n=330
Uncommon n=100
LUX-Lung 2
Phase II
N=129
n=52
n=54
n=23
LUX-Lung 3
Phase III
N=345
n=170
n=138
n=37
LUX-Lung 6
N=364
n=186
n=40
Patients with uncommon mutations treated with afatinib
Uncommon n=75
n=23
n=26
n=26

14. Subgroups of patients with uncommon mutations
Categories
De novo
T790M
Exon 20 insertions
Other
(exon 18, 19, 20, 21) n= 14 23 38
Mutations
(n)
T790M alone (3)
T790M+Del19 (3)
T790M+L858R (6)
T790M+G719X (1)
T790M+L858R+G719X (1)
n/a
L861Q alone (12)
G719X alone (8)
G719X+S768I (5)
G719X+L861Q (3)
E709G or V+L858R (2)
S768I+L858R (2)
S768I alone (1)
L861P alone (1)
P848L alone (1)
R776H+L858R (1)
L861Q+Del19 (1) K739_1744dup6 (1)

15. Tumour shrinkage in patients with uncommon mutations
Independent review (n=67†) *
De novo T790M (n=14):
T790M alone(*), T790M+Del19, T790M+L858R, T790M+G719X, T790M+L858R+G719X
Exon 20 insertions (n=20)
Other (n=33):
L861Q, G719X, G719X+S768I, G719X+L861Q, E709G or V+L858R, S768I+L858R,
S768I, L861P, P848L, R776H+L858R, L861Q+Del19, K739_1744dup6 * *
†8 patients were not included due to insufficient data

16. EGFR TKI resistance mechanisms
T790M+ EGFR- TKI failures 2nd/3rd line NSCLC
An area of high unmet medical need
There are no current proven treatment options for these patients. Chemotherapy or EGFR-TKIs are used, but their benefit isn’t proven
EGFR TKI resistance mechanisms
AZD9291 or CO1686
“Patients with EGFR-mutant lung adenocarcinoma develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) after a median of months. In half of these cases, a second EGFR mutation, T790M, underlies acquired resistance”
Oxnard, et al. Clin Cancer Res Dec 6, 2010

17. Clinical activity AZD9291 in T790M+
Fast Track status granted by FDA in October 2013
Breakthrough Therapy Designation submission early February 2014
Best % change from baseline in target lesions in central testing T790M+
T790M+ Response Rate* = 64% (95% CI 53%-74%)
For the 89 T790M+ evaluable patients there are
57 PRs (35 confirmed), 28 SDs, 4 PDs
Population: T790M+ patients with observed or imputed target lesion data (n=89)
*Confirmed+ unconfirmed response
Imputations and exclusions
Two patients with a BOR of PD are imputed as +20% increase as no follow up assessments for target lesions (E , E , both died prior to first scan).
No patients are excluded
Response ongoing for 34/35 T790M+ patients with confirmed PR
D Discontinued treatment
* Imputed
T790M status is assigned by central testing of a recent tumour sample
Independent review of scans is underway as per FDA request 41/43 confirmed so far
Preliminary data, data cut off 16th January 2014

18. CO1686

19. FISH Assay for ALK Rearrangement*
Telomere
2p23 region
Centromere
p25.2
p25.2
ALK 29.3
p24.3
p24.3
t(2;5) ALK gene
breakpoint region
p24.1
p24.1
p23.2
p23.2
p22.3
p22.3
p22.1
p22.1
EML4 42.3
p16.3
p16.3
p16.1
p16.1 3’ 5’
p14
p14
p13.2
p13.2
p12
p12
~250 kb
~300 kb
q12.1
q12.1
q12.3
q12.3
Break-apart FISH assay for ALK-fusion genes1
q14.1
q14.1
q14.3
q14.3
q21.2
q21.2
q22.1
q22.1
q23.2
q22.2
q22.2
q23.2
q24.1
q24.1
q24.3
q24.3
q31.3
q31.3
q32.1
q32.3
q33.2
q32.1
q32.3
q33.2
Non-split signal
q34
q34
Split signal
q36.1
q36.3 q37.2
q36.1
q36.3 q37.2
ALK break-apart FISH assay [Courtesy John Iafrate, Massachusetts General Hospital]
*Assay is positive if rearrangements can be detected in ≥15% of cells FISH = fluorescence in situ hybridization
1Shaw AT et al. J Clin Oncol 2009;27:4247–4253

20. Crizotinib: First-in-human/Patient Trial
Cohort 5 (n=6)
300 mg BID
2 DLTs: grade 3 fatigue
Part 1:
Dose escalation
Cohort 6 (n=9)
250 mg BID MTD/RP2D
Cohort 4 (n=7)
200 mg BID
Cohort 3 (n=8)
200 mg QD
1 DLT: grade 3 ALT elevation
Part 2:
Molecularly enriched cohorts
(ALK and c-MET)
Cohort 2 (n=4)
100 mg QD
Enrolling patients with ALK-positive NSCLC after preliminary observation of impressive activity in a few patients
Data from database April 7, 2010
Data presented for 82 patients, study ongoing
Cohort 1 (n=3)
50 mg QD
ALT = alanine aminotransferase

21. Tumor Responses to Crizotinib for Patients with ALK-positive NSCLC60 40 20
–20
–40
–60
–80
–100
Progressive disease
Stable disease
Confirmed partial response
Confirmed complete response
Maximum change in tumor size (%)
–30%
US FDA accelerated approval Aug 26, 2011
Full approval Nov 21, 2013 *
*Partial response patients with 100% change have non-target disease present

25. Study Design – LDK378 (Ceritinib)
Dose-Escalation Phase
Advanced ALK-rearranged malignancies
NCT
Completed
n=59
Dose escalation started at 50 mg/day
Escalate to MTD (750 mg/day)
Primary objective: determination of MTD
Secondary objectives: characterize safety, PK, and antitumor activity
Patients received treatment until disease progression, unacceptable toxicity, or withdrawal of consent
Continuous oral dosing 21-day cycles
Expansion
Phase
ALK-rearranged NSCLC
Other ALK-activated tumors
Additional n=71 enrolled
Crizotinib naive
Crizotinib pretreated
Shaw AT et al. N Engl J Med. 2014;370:

26. Best Change (%) in Tumor Response from Baseline in NSCLC Patients Treated with Ceritinib
100
Crizotinib Pretreated
Crizotinib Naive 80
PFS Event 60 40 20
Best Change (%) from Baseline
-20
-40
-60
-80
US FDA accelerated approval Apr 30, 2014
-100
Based on investigator assessment of response.
PFS, progression-free survival.
Shaw AT et al. N Engl J Med. 2014;370:
From N Engl J Med, Shaw AT, Kim DW, Mehra R, et al, Ceritinib in ALK-Rearranged Non–Small-Cell Lung Cancer, Vol 370, Page Copyright © 2014 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

27. BRAF-Mutations in NSCLC harbor BRAF V600E-mutations
W. Pao, N. Girard
Lancet Oncology,
February 2011
A. Marchetti et al., J. Clin. Oncol. 29, 1, 2011
1,046 NSCLC samples
Frequency of BRAF-mutations 3.5%
BRAF-mut. in 4.9% adeno- and 0.3% SCC
57% BRAFV600E-mutations
43 % non-activating BRAF mutations
697 NSCLC-adeno. ca. Samples
Frequency of BRAF-mutations 3%
50% BRAFV600E-mutations
50% non-activating BRAF mutations
P. Paik et al., J. Clin. Oncol. 29, 2046, 2011
% of NSCLC
harbor BRAF V600E-mutations

28. BRF113928: Initial Study Design
Single arm, Phase II, open label
Green-Dahlberg 2-stage: H(0): ORR ≤ 10% versus H(1): ORR ≥30%
NSCLC (Adenocarcinoama)
BRAFV600E-mutation
≥ 2nd Line
dabrafenib
150 mg twice daily
N = 40
Primary objective: Investigator-assessed ORR
Secondary objectives: PFS, duration of response, OS, safety and tolerability, population PK

29. Interim Analysis (ASCO 2013)
US FDA granted Breakthrough
Therapy Jan 13, 2014
Investigator based response assessments demonstrated:
7PRs (5 confirmed—duration 29 and 49 weeks for 2 pts and remaining 3 patient 6+ to 24+ weeks)
1 stable disease
4 progressive disease
Planchard et. al ASCO Annual Meeting Proceedings

30. Rationale for the Combination
Sustained target inhibition to observe more prolonged and durable anti-tumor effect
Delay and potentially prevent the development of resistance
Prevent/delay hyperproliferative lesions and secondary malignancies (Cu SCC)
RAS
BRAF V600
BRAF WT
mutBRAF
BRAFi +
MEKi
MEK
pERK
Proliferation, survival
Invasion , Metastasis

31. Molecular Subsets Adenocarcinoma Lung Cancer
MAP2K1
NRAS
ROS1 fusions
KIF5B-RET
AKT1
PIK3CA
BRAF
HER2
ALK
Fusions
EGFR
KRAS
Unknown
1. Mascaux C et al. Br J Cancer 2005;92:131–9; 2. Winton et al. N Engl J Med 2005;352:2589–97; 3. Eberhard et al JCO 2005;23:5900–9; 4. Pao et al. PLOS Medicine (1): e17; 5. Pao et al. Nat Med 2012;18:349–51; 6. Kris et al. ASCO 2011

32. Copy number alterations Point mutations & indels Gene expression
Structural variants
Translocations
Fusions
Inversion
Copy number alterations
Amplifications
Deletions
LOH
Point mutations & indels
Missense
Nonsense
Splice site
Frameshift
Gene expression
Outlier expression
Isoform usage
Pathways & signatures
Wild type AGTGA
Mutant AGAGA
Adapted from: Roychowdhury et al. Sci Transl Med; 20122

33. Conclusion Future Directions
Molecular selection of patients improves therapy outcomes for patients with advanced adenocarcinoma of the lung
Future Directions
Extent patient selection and targeted therapies to squamous cell and earlier stages of NSCLC (i.e. RADIANT)
Next generation sequencing needed
Molecular characterization of resistance
Development of a structure to conduct studies in uncommon molecular selected populations (BRAF, HER2, RET, etc)
Clinical integration of tumor genomic and proteomic testing to better direct therapy for NSCLC (Gerber)