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 surfaceof 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: crizotinib

Shaw 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 RETDiscovery 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