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
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.
Genomic Evolution of Lung Cancer
KRAS, 30%
Unkn, 40%
RET, 1%
ROS1, 1%
MEK, 1%
PIK3CA, 1%
FGFR4, 2%
EGFR, 15%
EML4-ALK, 5%
HER 2, 2%
BRAF, 2%
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)
Testing for Fusion Oncogenes
IHC expression
Break apart FISH
Amount of protein on the surface
of the cell
F
RT-PCR
ALK Fusion Gene
ALK Fusion Gene
Echinoderm microtubule
associated protein-like 4
?
N
anaplastic lymphoma kinase
Adapted from Soda et al. Nature; 2007.
C
ALK Fusion Variants
Sasaki, European Journal of Cancer; 2010.
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%
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
1Shaw
AT, ASCO; 2010;
MG. ASCO 2011; abstract CRA7506.
3Rodig SJ, Clin Cancer Res; 2009;15
Soda M, et al. Nature; 2007;448
2Kris
Clinical Efficacy of Crizotinib
Some degree of tumor shrinkage 90%
ORR - 60.8%
DCR - 82.5% at week 8
Median time to response 7.9 weeks (2.1 - 39.6 weeks)
Median response duration 49.1 weeks
Median PFS 9.7 months (95% CI 7∙7–12∙8)
N=149
Unknown…
How crizotinib compares to chemotherapy 1st line
• QOL
• OS
• TTP
Camidge, Lancet oncology 13, 2012
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
PFS
Shaw NEJM; June 2013.
Patient Related Outcomes
Shaw NEJM; June 2013
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 0.1-136.8)
• 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.
Duration of Initial Response and Post
Progression Therapy
Crizotinib Resistance
•
•
•
•
L1196M
L1152R
C1156Y
F1174L
Sasaki Clinical Cancer Research. Epub 2011.
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
– CH5424802 (RG7853)
• HSP 90 inhibitors
Responses to Second Generation Inhibitors
in crizotinib Resistant Tumors
• LDK378( phase I)
58% ORR1
– CNS penetration
• CH5424802 ( phase I/II)
– 48% ORR2
– CNS penetration
• AP26113 ( phase I/II)
– 76% ORR3
– CNS penetration
1Shaw.
ASCO 2013 abstr 8010.
World Lung 2013, O16.06.
3Camidge. World Lung, MO0706
2Gadgeel,
ROS1 Fusion
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.
ROS 1 Fusion Gene
Arai, PLOS ONE; February 2013.
ROS 1 Fusion Gene Variants
ROS1 Fusion
• Patients: Younger, never smokers, adenocarcinoma, high grade
histology
• Frequency: 1.2 -1.7% 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
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
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
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
Bergeron, JCO, 30, 2012.
12 weeks
Clinical Validation of ROS1 as a
Therapeutic Target
Shaw et al. JCO. 2012, 30 (suppl; abstr 7508.)
•
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%)
RET FUSION
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
RET Fusion Gene
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
Ju YS, Genome Res, 2012
Wang R, J Clin Oncol 30: 2012
Drilon, Cancer Discover March 2013
Kohno, Cancer Science Aug 2013
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
Clinical Activity of Carbozatinib in RET
Fused Patients
4 weeks
4 weeks
4 weeks
Drilon, Cancer Discover March 2013.
Summary
Discovery
ALK
ROS
RET
2007
2007
2012
Type of the
product
Receptor tyrosine kinase
Frequency
4%
1.7%
1.4%
Histology
AdenoCA,
AdenoSCC,
SCC
AdenoCA
Poorly differentiated
AdenoCA
AdenoSCC
Poorly differentiated?
Other
characteristics
Approved agent
Agent in
consideration
Never smokers, younger
Crizotinib
None
None
Crizotinib
Sunitinib
Sorafenib
Carbozatinib
Vandetanib
Ponatinib
Lenvatinib
HER 2 Insertions
• Patients: Adenocarcinomas, never smokers
• Frequency: Incidence 2.8-4.2%
• 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
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
Q&A