R4 고원진 / pf. 박태성 Current perspective Takaaki Sasaki, Scott J. Rodig, Lucian R. Chirieac, el al. EUROPEAN JOURNAL OF CANCER 4 6 ( ) – The biology and treatment of EML4-ALK non-small cell lung cancer

Introduction NSCLC : Major cause of death worldwide Most of the patients being diagnosed in advanced stage  Treatment is only palliative Chemotherapy : But conventional cytotoxic agents has reached a plateau efficacy  New compounds which target proteins : Somatic mutations in EGFR : EGFR kinase inhibitor (gefitinib) N engl j med 361;10 september 3, 2009

Introduction The fusion of ALK with EML4 : 2007 in Japanese NSCLCs Additional studies : 3% - 13% of lung tumours EML4-ALK fusions  Suggest that approximately 5%, worldwide ALK kinase inhibitors,  ALK inhibitors lead to apoptosis in vitro, tumour shrinkage in vivo The phenomenon of ‘oncogene addiction’  In the phase I trial of PF , a remarkable 60% radiographic response rate in EML4-ALK NSCLC patients

This review’s focus The clinical, biological and molecular feature of EML4-ALK NSCLC patients The use of ALK inhibitors as therapies for this patient

2. Clinical and molecular features of EML4-ALK NSCLC Most commonly in a unique clinical subgroup of NSCLC patients In younger patients More advanced NSCLC More common in never/former light smokers

2.1. Smoking history Frequency of mutations in never or lightNever or light 9.4% VS 2.9% in current smokers

2.2. Outcomes with current NSCLC therapies Clinical Features and Outcome of Patients With Non–Small-Cell Lung Cancer Who Harbor EML4-ALK  In the EML4-ALK cohort vs WT/WT cohort, Similar RRs to platinum-based CTx No difference in overall survival  EML4-ALK positivity, Resistance to EGFR TKIs EGFR TKI platinum-based J Clin Oncol 27:

2.3. Morphologic profile of ALK-rearranged NSCLC A variety of histological features with ALK-rearranged lung adenocarcinomas : acinar to signet-ring cell nests with mucin production The acinar pattern in Asian populations The signet-ring cell histology in the Western patients  Tumour cells with a solid or sheet-like pattern With abundant intracellular mucin and small, marginalised nuclei A solid growth pattern with >10% signet-ring cells

2.4. Variants of EML4-ALK and non-EML4 translocation partners The inversion on chromosome 2 p  Most commonly found in lung cancer cell lines  Leading to the formation of the EML4-ALK fusion oncogene  Multiple EML4-ALK variants The intracellular tyrosine kinase domain of ALK beginning at the portion encoded by exon 20 EML4 : variably truncated  Various variants of EML4-ALK

At least 11 variants have been reported to date E13;A20 (variant 1) 33% vs E6a/b;A20 (variant 3a/b) 29% The NSCLC cell lines, H3122 and DFCI032 vs H2228 The clinical significance of the different variants is not defined Two other fusions, TFG and KIF5B, as an other ALK-fusion partner  Non-EML4 fusion partners has implications for the method used for clinical detection of ALK translocated NSCLC

3. Diagnosis of EML4-ALK NSCLC Currently no standard method for detecting EML4-ALK NSCLC Polymerase chain reaction (PCR) Immunohistochemistry (IHC) Fluorescence in situ hybridisation (FISH)

3.1. PCR-based identification of EML4-ALK Reverse transcriptase (RT)-PCR Rapid diagnostic method, a theoretically Extreme sensitivity In practice, the technique faces several challenges  At least 11 variant EML4-ALK fusions, and non-EML4 translocation partners, primer pairs for all known ALK fusions  Store as formalin-fixed paraffin embedded (FFPE) tissues, RNA extracted from FFPE is difficult  False positive results RT-PCR based screening methods No implement in a routine clinical diagnostic laboratory

3.2. FISH-based methods I More specific detection of ALK-rearrangements  By probes flanking the ALK breakpoint with tumour cell nuclei A commercially available probe set,  For the diagnosis of ALK-rearranged ALCLs One probe 5’ of the ALK locus yield a merged signal,  ‘split’ (green–orange fluorescent)

3.2. FISH-based methods II ; Weakness The ‘split’ signal characteristic of an EML4-ALK fusion can be subtle,  The loss and inversion of only a small amount of genetic material on chromosome 2  The 5’ probe occasionally fails to hybridise, [presumably due a loss of the target locus in the tumour] An additional complicating factor is the destruction of tissue morphology [when formalin-fixed, paraffin embedded (FFPE) biopsy specimens]

3.2. FISH-based methods III Thus, Although FISH is a sensitive and specific means, it is not infallible In a recent study, an ALK-rearranged lung adenocarcinoma by immunohistochemical testing that was, initially, mistakenly diagnosed as ALK wild type by prior FISH analysis Furthermore, unlike PCR, FISH cannot distinguish between the different EML4-ALK fusion variants FISH, the diagnostic method in the current clinical trials of PF ≥15% split nuclei as indicative of an ALK rearrangement  The frequency of split signals and the number of nuclei evaluated remains to be determined

3.3. IHC detection methods I Immunohistochemical (IHC) analysis of FFPE tissue specimens Ability to assay for tumour-specific antigen expression without loss of the cytologic and architectural features Several antibodies specific for the human ALK protein have been developed and are used to diagnose ALK-rearranged ALCLs However, inadequate for the detection of the majority of ALK-rearranged lung adenocarcinomas The lower level of ALK expression in ALK-rearranged NSCLCs compared with ALK-rearranged ALCLs In an initial survey of 10 FISH-confirmed, ALK-rearranged lung adenocarcinomas, only 4 stained positively

 On novel antibodies, increased sensitivity and specificity Rapidly screen possibility Confirmatory FISH studies D5F3 (A,D,G,J), ALK1 (B,E,H,K), FISH (C,F,I,L) Clin Cancer Res 2010;16:

4. ALK-targeted therapy in NSCLC 4.1. Pre-clinical studies ALK inhibitors (TAE-684) effective in vivo The MET/ALK inhibitor, PF , inhibit the growth of ALK translocated cancer cell lines

4.2. Clinical studies PF (crizotinib), Orally bioavailable ALK inhibitor Clinical activity observed in a phase I dose escalation trial of an oral c-met and ALK inhibitor, PF (PF) The initial findings were presented at ASCO 2009, Conclusions:  The MTD of PF is 250 mg BID  53% RR (10/19) and DCR (PR, SD) of 79% (15/19)  The major AEs were fatigue or GI-related, and manageable Additional responses continue to be reported Dramatic findings have led to two subsequent clinical trials of PF

5. Conclusions EML4-ALK NSCLC represents a unique subset of NSCLC patients  ALK inhibitors may represent a very effective therapeutic strategy The challenge remains of diagnostic testing for EML4-ALK to identify this patient subset Continue to.. uncover unique biological and molecular features of this patient encounter drug resistance to ALK targeted therapies