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

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

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

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

ALK Fusion Gene

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

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%

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

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 Best % change. 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 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.

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 AP26113 ( phase I/II) 76% ORR3 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 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 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

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 12 weeks Bergeron, JCO, 30, 2012.

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 Was originally described in thyroid carcinomas.

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 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, 2012 Drilon, Cancer Discover March 2013 Wang 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 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.

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

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