1. DOM Grand Rounds--2013
The Therapeutic Implications of EML4/ALK, ROS-1 and Other New Biomarkers
Lyudmila Bazhenova, MD
Associate Clinical Professor
Lung Cancer Unit Leader
UC San Diego Moores Cancer Center

2. Objectives Review current state of targetable lung cancer biomarkers
Review and contrast clinical characteristics of patients with EML4-ALK, ROS 1, and KIFB5-RET fusion protein, testing strategies and agents with clinical activity.

3. Genomic Evolution of Lung Cancer

4. Mechanism of Action of ALK, ROS1 and RET Fusion Oncogenes
All three are receptor tyrosine kinases (RTK)
ALK and RET are capable of homodimerization and self (ligant independend) activation
Mechanism of self activation of ROS1 is being debated
Downstream signaling via RAS/ERK (proliferation), and PI3K/AKT and JAK/STAT( resistance to apoptosis)
Some prefer one pathways over another

5. Testing for Fusion Oncogenes
IHC expression
Break apart FISH
Amount of protein on the surface of the cell
RT-PCR

6. ALK Fusion Gene

7. ALK Fusion Gene Echinoderm microtubule associated protein-like 4 ? N C
anaplastic lymphoma kinase
Adapted from Soda et al. Nature; 2007.

8. ALK Fusion Variants
Sasaki, European Journal of Cancer; 2010.

9. Methods of ALK Detection
FISH break apart
Pros: independent of FPE
Cons: if inversion involves a small locus of 2p it could be false negative; can not distinguish variants; cut of is 15% of nuclei with split signal; low throughput
RT-PCR
Pros: Rapid detection and identification of each unique variant
Cons: False negatives; Loss of RNA during de parafinization; has to be multiplexed, i.e probes to all known variants. Unknown variants will not be detected.
IHC
Pros: easy
Cons: several antibodies have been developed which look promising as a screening tool. No commercially available IHC in the US.
VENTANA just received an approval in China with 93% concordance with FISH, sensitivity 100%, specificity 98%

10. Frequency: 4% in all, 33% in EGFR negative never smokers
EML4-ALK Fusion
Patients: younger, non smokers, with adenocarcinoma, adenosquamous carcinoma and rarely SCC
Frequency: 4% in all, 33% in EGFR negative never smokers
Biology: 16 EML4-ALK variants have been identified in NSCLC. Clinical significance of each variant is unknown.
Testing: Visys break apart FISH (> 15% cells with split signal in 50 nuclei scored).
ALK PCR, IHC
Therapy: crizotinib
b, Fusion of the N-terminal portion of EML4 (comprising the basic region, the HELP domain and part of the WD-repeat region) to the intracellular region of ALK (containing the tyrosine kinase domain). TM, trans membrane domain.
c, Both the ALK gene and the EML4 gene map to chromosome 2p, but have opposite orientations. In the NSCLC patient 33, EML4 is disrupted at a position 3.6 kb downstream of exon 13 and is ligated to a position 297 bp upstream of exon 21 of ALK, giving rise to the EML4–ALK (variant 1) fusion gene (left panel). Filled and open horizontal arrows indicate the direction of transcription and the positions of the Fusion-genome primers, respectively
Identification of EML4–ALK
To isolate novel transforming genes in NSCLC, we generated a retroviral cDNA expression library
In vitro difeerent variants of alk has different stability and responsiveness to ALK inhibitor and HSP 9- inhibitors.
1Shaw AT, ASCO; 2010;
2Kris MG. ASCO 2011; abstract CRA7506.
3Rodig SJ, Clin Cancer Res; 2009;15
Soda M, et al. Nature; 2007;448

11. Clinical Efficacy of Crizotinib
Some degree of tumor shrinkage 90%
ORR %
DCR % at week 8
Median time to response 7.9 weeks ( weeks)
Median response duration 49.1 weeks
Median PFS 9.7 months (95% CI 7∙7–12∙8)
N=149
Best % change.
Unknown…
How crizotinib compares to chemotherapy 1st line
QOL OS
TTP
Camidge, Lancet oncology 13, 2012

12. 1st Line or Second Line
No studies examining the best placement of the drug.
FDA approved the drug without mentioning the line of therapy.
One can make a leap of faith from EGFR inhibitors and use it in the first line.
Profile 1007 compared crizotinib to 2nd line chemotherapy
PFS 7.7 m vs. 3 m in favor of crizotinib (HR 0.49; 95% CI, 0.37 to 0.64; P<0.001)
RR 65% vs. 20 % in favor of crizotinib ( p<0.001)
OS not different, 64% of patients in chemotherapy arm received crizotinib
QOL: greater reduction of symptoms and delay in new symptoms on crizotinib arm.
Profile 1014 will compare crizotinib to 1st line chemotherapy.
Shaw NEJM ;June 2013

13. PFS
Shaw NEJM; June 2013.

14. Patient Related Outcomes
Shaw NEJM; June 2013

15. Characteristics of Progression
Patients were allowed to stay on the study post progression if they continued to derive clinical benefi
Median duration of treatment 43.1 m (Range )
69/149 patients had disease progression at the data cut off.
39 continued to receive crizotinib for at least 2 weeks post progression
12 of them did that for 6 months
Range of post progression treatment is 21 to 591 days.
Most common new sites of progression were brain ( N=10), lung (n=5), liver ( N=3)
Camidge, Lancet oncology 13, 2012.

16. Duration of Initial Response and Post Progression Therapy
Patients are ordered by initial best response before progression and duration of crizotinib treatment after
progression (n=39). *Defi ned as the time (in weeks) from the fi rst documentation of objective tumour response
(complete response or partial response) that was subsequently confi rmed, to the fi rst documentation of
progressive disease or death. Stable disease duration was calculated from the date of the fi rst dose to the date of
fi rst documented disease progression. †Defi ned as time from investigator-documented progressive disease to the
last date of crizotinib dose or censor at the time of analysis. Disease progression and best objective response were
derived according to Response Evaluation Criteria in Solid Tumors. ‡Treatment ongoing at the time of analysis.
ÅòReceived crizotinib as fi rst-line treatment.

17. Crizotinib Resistance
L1196M
L1152R
C1156Y
F1174L
Sasaki Clinical Cancer Research. Epub 2011.

18. Management of Crizotinib Resistance
Local treatment with radiation for locally progressing disease
Clonal evolution
Platinum based doublet or triplet
Second generation ALK inhibitors
AP26114
LDK378
CH (RG7853)
HSP 90 inhibitors

19. Responses to Second Generation Inhibitors in crizotinib Resistant Tumors
LDK378( phase I)
58% ORR1
CNS penetration
CH ( phase I/II)
48% ORR2
AP26113 ( phase I/II)
76% ORR3
1Shaw. ASCO 2013 abstr 8010.
2Gadgeel, World Lung 2013, O16.06.
3Camidge. World Lung, MO0706

20. ROS1 Fusion

21. Oncogenic ROS1
First described fusion gene FIG-ROS1 was found in glioblastoma
240kb deletion on 6p21q resulting in a fusion gene coding for oncogenic fusion protein.
Short and long isoforms
Induce tumorigenesis in xenograft mouse models
Also expressed in cholangiocarcinoma in 8.7% and ovarian cancer in 0.5%, gastric and colon, myofibroblastic tumors and angiosarcoma
EZR–ROS1 fusion gene has been shown to promote lung adenocarcinoma when ectopically expressed in lung epithelium
Gu TL, PLoS One; 2011.
Birch AH, PLoS One; 2011;
Lee, Cancer; May 2013
Bergethon et al. JCO; 2012 (30)8.

22. ROS 1 Fusion Gene
Arai, PLOS ONE; February 2013.

23. ROS 1 Fusion Gene Variants

24. ROS1 Fusion
Patients: Younger, never smokers, adenocarcinoma, high grade histology
Frequency: % in all
Biology: 9 variants have been identified in NSCLC so far
Clinical significance is unknown. Mechanism of activation is different.
FIG-, CD74-, SCL34A2-, TPM3-, SDC4-, EZR-, LRIG3, KDELR2–, and CCDC6–
Testing: Visys break apart FISH (> 15% cells with split signal in 50 nuclei scored)
ROS PCR, IHC
Therapy: crizotinib
Some of the
Shaw AT, JCO 2012;30:(suppl; abstr 7508)
Ou, Exp revi. of anticancer therapy 2012,;12
Gu TL, PLoS One. 2011; 6:e15640.
Birch AH, PLoS One. 2011; 6:e28250
Lee, Cancer May 2013
Davis Clin Cancer Res . Sep 2012
Bergeron, JCO, 30, 2012

25. Methods of ROS1 Detection
RT-PCR
Cons: False negatives; 9 variants have been described in a matter of 12 months. Has to be multiplexed, i.e., probes to all known variants. Unknown variants will not be detected.
FISH break apart
Cons: if inversion involves a small locus it could be false negative; can not distinguish variants; cut of is 15% of nuclei with split signal; low throughput
IHC
Cons: not commercially available, several antibodies appear promising

26. Response of a ROS1 Positive Patient to Crizotinib
49% homology in the TK domain and ATP binding site
Crizotinib is active in ROS1 fused cell cultures
Baseline
12 weeks
Bergeron, JCO, 30, 2012.

27. Clinical Validation of ROS1 as a Therapeutic Target
14 patients enrolled in phase I study
Safety/efficacy of crizotinib 250mg bid
ROS1 rearrangement by FISH
Negative for ALK rearrangement
Average 54 yo, 13/14 never smokers
80% received prior therapy
8/14 responded (57%)
Shaw et al. JCO , 30 (suppl; abstr 7508.)

28. RET FUSION

29. Methods of RET Detection
RT-PCR
Cons: False negatives; 3 variants have been described in a matter of 12 months. Has to be multiplexed, i.e probes to all known variants. Unknown variants will not be detected.
FISH break apart
Cons: if inversion involves a small locus it could be false negative; can not distinguish variants; cut of is 15% of nuclei with split signal; not widely available; low throughput
IHC
Current IHC antibodies do not correlate with RET fusion
Was originally described in thyroid carcinomas.

30. RET Fusion Gene

31. RET Fusion
Patients: AdenoCA and adenoSCC carcinoma, never or former smokers, poor differentiation ?, earlier LN metastases
Frequency:
1.4% in all,
5.6 % in “triple negative”( EGFR, ALK, KRAS)
6.3% in non smokers negative for EGFR, KRAS, ALK, HER2, BRAF, and ROS1
16% in non smokers negative for EGFR, KRAS, ALK, ROS1, NRAS, BRAF, HER2, PIK3CA, MEK1, and AKT
Biology: 4 variants have been identified in NSCLC so far
Clinical significance is unknown.
KIF5B-, CCDC6-, NCOA4-. TRIM33
kinesin family 5B gene
Some studies did not show that differentiation is different.
Ponatinib is also a RET inhibitor
Drilon A, Wang L, Hasanovic A et al. Response to cabozantinib in patients
with RET fusion-positive lung adenocarcinomas. Cancer Discov 2013 (Jun);
Ju YS, Genome Res, Drilon, Cancer Discover March 2013
Wang R, J Clin Oncol 30: Kohno, Cancer Science Aug 2013

32. RET Fusion Gene
Testing: Visys break apart FISH (> 15% cells with split signal in 50 nuclei scored)
RET PCR
Therapy: Unknown
Sunitinib, Sorafenib, Vandetanib, Carbozatinib, Ponatinib, and Lenvatinib all have potential for activity
All active in KIF5B-RET–transformed cell lines
Last 4 are in formal clinical trials

33. Clinical Activity of Carbozatinib in RET Fused Patients
Drilon A, Wang L, Hasanovic A et al. Response to cabozantinib in patients
with RET fusion-positive lung adenocarcinomas. Cancer Discov 2013 (Jun);
4 weeks
4 weeks
4 weeks
Drilon, Cancer Discover March 2013.

34. Summary ALK ROS RET Discovery 2007 2012 Type of the product
Receptor tyrosine kinase
Frequency 4%
1.7%
1.4%
Histology
AdenoCA, AdenoSCC,
SCC
AdenoCA
Poorly differentiated
AdenoSCC
Poorly differentiated?
Other characteristics
Never smokers, younger
Approved agent
Crizotinib
None
Agent in consideration
Sunitinib
Sorafenib
Carbozatinib
Vandetanib
Ponatinib
Lenvatinib

35. HER 2 Insertions Patients: Adenocarcinomas, never smokers
Frequency: Incidence %
Biology:
In-frame insertions into exon 20. Transgenic mouse models confirm oncogenicity
Therapy:
Drugs of interest: neratinib, afatinib, dacomitinib
Preclinical models show synergy with mTOR inhibitors.
Clinical trial of neratinib + temsirolimus ongoing, several PR are reported
Both afatinib and dacomitinib have case reports of responses

36. BRAF Mutations Patients: smokers and non smokers Frequency: 1.6-3%
Biology: majority of the mutations are non V600E (more likely in smokers), V600E ( more likely in never smokers)
Therapy:
One case report or a NSCLC patient with V600E patient responding to vemurafenib
Dabrafenib is being tested in patients with V600E NSCLC
MEK inhibitors are being considered for non V600E patients

37. Q&A