the therapeutic implications of eml4/alk, ros-1 and other new biomarkers lyudmila bazhenova, md...
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The Therapeutic Implications of EML4/ALK, ROS-1 and Other New Biomarkers
Lyudmila Bazhenova, MDAssociate Clinical ProfessorLung Cancer Unit LeaderUC 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%
EGFR; 15%
EML4-ALK; 5%
HER 2; 2%BRAF; 2%
FGFR4; 2%
PIK3CA; 1%
MEK; 1%
ROS1; 1%
RET; 1%
Unkn; 40%
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
Amount of protein on the surface
of the cell
IHC expression
F
Break apart FISH
RT-PCR
ALK Fusion Gene
ALK Fusion Gene
Echinoderm microtubule associated protein-like 4
anaplastic lymphoma kinase
?
Adapted from Soda et al. Nature; 2007.
N 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; 2Kris MG. ASCO 2011; abstract CRA7506.3Rodig SJ, Clin Cancer Res; 2009;15Soda M, et al. Nature; 2007;448
Some degree of tumor shrinkage 90%ORR - 60.8%DCR - 82.5% at week 8Median time to response 7.9 weeks (2.1 - 39.6 weeks)Median response duration 49.1 weeksMedian PFS 9.7 months (95% CI 7∙7–12∙8)
N=149
Clinical Efficacy of Crizotinib
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 8010.2Gadgeel, World Lung 2013, O16.06.3Camidge. World Lung, MO0706
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 2013Bergethon 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: crizotinibShaw AT, JCO 2012;30:(suppl; abstr 7508)Ou, Exp revi. of anticancer therapy 2012,;12Gu TL, PLoS One. 2011; 6:e15640.Birch AH, PLoS One. 2011; 6:e28250
Lee, Cancer May 2013Davis Clin Cancer Res . Sep 2012Bergeron, 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
Bergeron, JCO, 30, 2012.
Baseline 12 weeks
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. 2012, 30 (suppl; abstr 7508.)
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 Drilon, Cancer Discover March 2013Wang R, J Clin Oncol 30: 2012 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 ALK ROS RET
Discovery 2007 2007 2012
Type of the product
Receptor tyrosine kinase
Frequency 4% 1.7% 1.4%
Histology AdenoCA, AdenoSCC,
SCC
AdenoCAPoorly differentiated
AdenoCAAdenoSCC
Poorly differentiated?
Other characteristics
Never smokers, younger
Approved agent Crizotinib None None
Agent in consideration
Crizotinib SunitinibSorafenib
CarbozatinibVandetanibPonatinibLenvatinib
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