Although chromosomal translocations are the most common. ALK Gene Rearrangements

GENEOFTHEMONTH A New Therapeutic Target in a Molecularly Defined Subset of Non-small Cell Lung Cancer Benjamin Solomon, MBBS, PhD,* Marileila Varella-Garcia, PhD, and D. Ross Camidge, MD, PhD Abstract: Transforming rearrangements of the ALK (anaplastic lymphoma kinase) gene have recently been described in non-small cell lung cancer (NSCLC). The most common rearrangement arises from an inversion in the short arm of chromosome 2 that creates a fusion between the 5 portion of the EML4 (echinoderm microtubule-associated protein-like 4) gene and the 3 portion of the ALK gene. At least seven ALK gene rearrangement variants have been described involving different EML4-ALK breakpoints or rarely other non-eml4 fusion partners. ALK rearrangements may be readily identified in tumor tissue by reverse transcription-polymerase chain reaction or fluorescent in situ hybridization. Although ALK gene rearrangements affect only about 4% of all lung cancers, they are more frequent in adenocarcinomas, in never or light smokers, and seem almost mutually exclusive with activating EGFR or KRAS mutations. Promising results seen in patients with NSCLC containing fluorescent in situ hybridization-detected ALK rearrangements treated on a phase I study with PF02341066, an oral ALK inhibitor, indicate that ALK represents a new therapeutic target in this molecularly defined subset of NSCLC. Key Words: ALK, Lung cancer. (J Thorac Oncol. 2009;4: 1450 1454) Although chromosomal translocations are the most common genetic change in cancer genomes 1 and are frequently described in hematologic malignancies, they have only recently been documented in epithelial malignancies such as non-small cell lung cancer (NSCLC) 2 and prostate cancer. 3 In 2007, EML4-ALK, a novel fusion gene arising as a result of an inversion in chromosome 2 that juxtaposed the 5 end of the EML4 gene with the 3 end of the ALK gene, was reported in a subset of patients with NSCLC. This gene *Department of Haematology and Medical Oncology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; and Division of Medical Oncology, University of Colorado Denver, Denver, Colorado. Disclosure: Dr. Varella-Garcia received honorarium as a speaker for Abbott Molecular, the manufacturer of the FISH protein listed in the manuscript. The other authors declare no conflicts of interest. Address for correspondence: D. Ross Camidge, MD, PhD, Mailstop F704, Room ACP 2256, 1665 Aurora Court, Aurora, CO 80045. E-mail: ross.camidge@ucdenver.edu Copyright 2009 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/09/0412-1450 arrangement was shown to be transforming both in vitro and in vivo. Since then, several variant ALK gene rearrangements have been identified, and the clinical/pathologic features associated with ALK-positive NSCLC have been described. Recently, evidence that the fusion proteins resulting from ALK gene rearrangements constitute a bona fide therapeutic target has been provided in a phase I trial of a specific ALK inhibitor in known ALK-positive NSCLC. IDENTIFICATION OF EML4-ALK IN NSCLC In 1997, Soda et al. 2 screened a cdna library derived from the tumor of a 62-year-old Japanese male smoker with adenocarcinoma of the lung for transforming activity. One of the cdnas with transforming activity was found to code for a novel fusion gene arising from an inversion on the short arm of chromosome 2 [Inv(2)(p21p23)] that joined exons 1 13 of EML4 to exons 20 29 of ALK. 2 The resulting chimeric protein, EML4-ALK, contained an N-terminus derived from EML4 fused to the C-terminal kinase domain of ALK, which permitted ligand-independent dimerization and constitutive activation of ALK. In addition to its transforming ability in 3T3 cell lines and xenografts, lung-specific expression of EML4-ALK in transgenic mice resulted in multiple lung adenocarcinomas. 4 Significantly, treatment with an ALK inhibitor was able to cause regression of tumors. 2,4 Multiple variants of EML4-ALK have since been reported. 2,5 9 All variants encode the same cytoplasmic portion of ALK but contain different truncations of EML4 (at exons 2, 6, 13, 14, 15, 18, and 20), and those tested to date have all demonstrated biologic gain of function (reviewed by Horn and Pao 10 ). Other rare fusion partners for ALK have also been described, including TRK-fused gene TFG 11 and KIF5B. 8 STRUCTURE AND FUNCTION OF ALK AND EML4 EML4 and ALK are closely located genes situated on the short arm of chromosome 2 (2p21 and 2p23, respectively) where they are separated by a distance of 12.7 megabases and are oriented in opposite directions. Full-length ALK was cloned in 1997 12,13 and is a member of the insulin receptor family of receptor tyrosine kinases, of which leukocyte tyrosine kinase is the closest homologue. ALK is a 1620 amino acid transmembrane protein, which has 1450 Journal of Thoracic Oncology Volume 4, Number 12, December 2009

Journal of Thoracic Oncology Volume 4, Number 12, December 2009 an extracellular domain with an N-terminal signal peptide, two MAM motifs (meprin, A5 protein, and receptor protein tyrosine phosphatase mu), a low-density lipoprotein class A motif, and a large glycine-rich region putative ligand binding site. It also contains a short transmembrane domain and an intracellular domain with a juxtamembranous segment with a binding site for insulin receptor substrate-1, a kinase domain with three tyrosine-containing motif (YxxxYY: tyrosines 1278, 1282, and 1283 within the activation loop) and a C-terminus with binding sites for Src homology-2 and phospholipase C-. Activation of ALK by binding of ligand leads to dimerization, autophosphorylation, and signaling through the RAS-ERK, JAK3-STAT3, and PI3-K/Akt pathways, which lead to effects on cell proliferation, survival, migration, and alterations in cytoskeletal rearrangement. 14 The normal physiological function of ALK in humans is poorly understood. Drosophila melanogaster ALK (dalk) 15 and its ligand Jeb (jelly belly) 16 are important for the formation of the visceral musculature of the gut 17,18 and for the assembly of the neuronal circuitry in the visual system. 19 No mammalian counterpart to Drosophila Jeb has been identified. Putative mammalian ligands are the heparin-binding proteins pleiotrophin 20 and midkine. 21 Mammalian ALK is expressed in the nervous system during embryogenesis and at reduced levels after birth. Immunohistochemical studies in adult human tissue reveal weak expression restricted to components of the central nervous system. 22 Interestingly, there are no gross abnormalities in ALK knockout mice, with the exception of subtle neurocognitive changes consistent with alterations in hippocampal function. 23 Several detailed reviews of the basic biology of ALK have recently been published. 14,24,25 Echinoderm microtubule-associated protein-like 4 (EML4) is a 120 KDa cytoplasmic protein that is a human homolog of echinoderm microtubule-associated protein, the major microtubule binding protein in dividing sea urchin eggs, and is essential for the formation of microtubules. 26,27 It is composed of a N-terminal basic region, a HELP domain, and four WD repeats in the C-terminus. ALK GENE REARRANGEMENTS IN CANCER ALK was originally identified in 1994 in anaplastic large-cell lymphomas (ALCL) 28,29 as a component of a chimeric protein NPM-ALK created as a result of a translocation between chromosomes 2 and 5 [t(2;5)(p23;q35)]. ALK rearrangements occur in 60 to 80% of ALCL, which are CD30 positive non-hodgkin lymphomas (NHL) that present more frequently in young patients and have a male preponderance. ALCL accounts for about 40% of childhood NHL but only 5% of adult NHL. 30,31 NPM-ALK is the most common fusion in ALK-positive ALCL and consists of the N-terminal portion of nucleophosmin (NPM) fused with the kinase domain containing C-terminus of ALK. The fusion gene is readily detected by reverse transcription-polymerase chain reaction (RT-PCR) or fluorescent in situ hybridization (FISH) and can also be detected by the ALK-1 (Dako, Carpinteria, CA) antibody with typical nuclear and cytoplasmic localization. Numerous other rearrangements involving ALK have been identified, including t(1;2)(q21;p23) (TPM3-ALK), inv(2)(p23q35) (ATIC-ALK), t(2;3)(p23;q21) (TFG-ALK), t(2;17)(p23;q23) (CLTC-ALK), t(2;19)(p23;q13.1) (TPM4-ALK), and t(x;2) (q11 12;p23) (MSN-ALK). 30,31 In addition to ALCL, ALK gene rearrangements have also been described in 50% of inflammatory myofibroblastic tumors 32,33 and rare ALK-positive diffuse large B-cell lymphomas. 34 Point mutations and amplifications of ALK have been identified in sporadic and familial neuroblastomas. 35 38 Proteomic studies have also reported on ALK proteins being associated with esophageal squamous cell carcinoma. 39,40 As has been observed in ALCL and IMT, with only rare exceptions, ALK gene rearrangements in NSCLC contain the complete tyrosine kinase domain of ALK (beginning from exon 20, residues 1058 1620). In the gene rearrangement, the promoter of the 5 partner gene controls the transcription of the resulting fusion gene. The fusion partner in the translated fusion protein typically contains an oligomerization domain that mediates constitutive dimerization and subsequent autophosphorylation and constitutive activation of the ALK kinase in the absence of ligand. The partner protein also determines cellular localization, which may affect the induced phenotype in different fusions. In the case of EML4- ALK, the location is cytoplasmic. 9,41,42 This is distinct from NPM-ALK where NPM drives nuclear and cytoplasmic expression, CTLC-ALK that has a granular cytoplasmic staining distribution, 43 and KIF5B-ALK that is associated with a perinuclear distribution. 8 METHODS OF DETECTION ALK gene rearrangements or the resulting fusion proteins may be detected in tumor specimens using immunohistochemistry, RT-PCR of cdna, and FISH. The ALK-1 antibody, which has proven useful in ALCL and inflammatory myofibroblastic tumors, has not been as reliable in lung carcinomas. 42 Nevertheless, several strategies to improve the accuracy of the immunohistochemistry ALK assays in lung cancer including amplification of the signal with a tyramide cascade 44 and intercalation of an antibody-enhanced polymer 8 have recently been described. RT-PCR of cdna has been the most commonly applied screening strategy for ALK gene rearrangements in archival tissue. Initially, only a few primer sets were tested, but currently, a number of multiplex assays have been designed to simultaneously capture all possible in-frame fusions between EML4 and ALK in which the kinase domain of ALK would be preserved. 2,5,7 However, although different breakpoints in EML4 or ALK may be detected with such techniques, novel ALK fusion partners will not. FISH has been performed with the ALK Dual Color Break-Apart probe (Abbott Molecular, Des Plaines, IL) 41,42,45 or homebrew reagents. 46 The commercial break-apart probe is comprised of a SpectrumOrange (red)-labeled 250 kb probe to the 3 end of ALK with a SpectrumGreen (green)- labeled 300 kb probe to the 5 end of ALK (Fig. 1). Because the EML4 and ALK loci are mapped relatively close in 2p (12.7 MB apart) and the chromosome inversion is frequently associated with deletion around the ALK 5 breakpoint, 7,46 the Copyright 2009 by the International Association for the Study of Lung Cancer 1451

Solomon et al. Journal of Thoracic Oncology Volume 4, Number 12, December 2009 the distance between them is larger than would be expected for stochastic separation of these signals. This distance is estimated using the signal size as a reference, and therefore analyses must be performed by experienced laboratory personnel. In such settings, break-apart FISH probes separated by at least 8 MB can be readily detected with high sensitivity and specificity in paraffin-embedded tissues. 41 More sophisticated strategies have also been proposed, such as a threecolor probe set with an additional fluorescent probe targeting the deleted portion of chromosome 2 in ALK-rearranged NSCLC 44 or a dual-fusion color probe directed against both fusion derivatives (Varella-Garcia, unpublished data). Although none of these FISH probe sets can comprehensively identify novel rearrangements, they can detect atypical patterns of breakpoints in ALK, leading to additional analyses to confirm the details of the gene rearrangements as needed. FIGURE 1. A, Chromosome 2 idiogram showing location and design of the dual-color ALK break-apart fluorescence in situ hybridization (FISH) probe that includes DNA contigs adjacent to the ALK gene, the 3 contig is labeled with red fluorochromes and the 5 is labeled with green fluorochromes. B, Non-small cell lung cancer (NSCLC) specimen showing fused red/green signals representing normal copies of the ALK (yellow arrows) and single red and green signals (red and green arrows) indicating that chromosomal break occurred between the 3 and the 5 contigs. interpretation of a positive rearrangement (through the introduction of a gap between the red and green probes) in formalin-fixed, paraffin-embedded tissue sections can be challenging. Internal and external controls must be used to estimate whether, in each nucleus showing separated signals, CLINICAL/PATHOLOGIC FEATURES The overall incidence of ALK gene rearrangements in NSCLC in the series reported to date is 3.8% (107 ALK rearrangements in 2835 tested tumors with a range from 0.4 to 13.4% see Table 1). 2,5,7 9,11,41,42,45 48 Most ALK gene rearrangements have been found in adenocarcinomas, in never or light smokers. Consequently, any differing proportions reported in the different series have to be considered in light of their underlying enrichment for factors such as specific histologies or patients with no or light smoking histories. Coexpression with EGFR or KRAS mutations seems to be extremely rare, suggesting that ALK is a distinct oncogenic driver. Patients with ALK-positive NSCLC have been reported as being younger than patients without the gene rearrangement in some series. 9,45 Shaw et al have described TABLE 1. Author Frequency of in NSCLC Method of Testing Population of Origin ALK Gene Rearrangements Identified Total Number of NSCLC Tumors Tested Percentage Soda et al. 2 RT-PCR Japan 5 75 6.7 Rikova et al. 11 RT-PCR China 4 a 103 3.9 Shinmura et al. 48 RT-PCR Japan 2 77 2.6 Perner et al. 46 FISH Switzerland, USA 16 603 2.7 Inamura et al. 47 RT-PCR Japan 5 200 2.5 Koivunen et al. 7 FISH USA (138), Korea (167) 8 305 2.6 Takeuchi et al. 5 RT-PCR Japan 11 253 4.3 Wong et al. 9 RT-PCR China 13 266 4.9 Boland et al. 41 IHC, FISH USA 6 335 1.9 Martelli et al. 42 b RT-PCR Italy, Spain 9 120 7.5 Rodig et al. 44 c IHC, FISH USA 1 227 0.4 Shaw et al. 45 d FISH USA 19 141 13.5 Takeuchi et al. 8 IHC, RT-PCR Japan 8 e 130 6.2 Total 107 2835 3.8 a Includes one tumor with TRG-ALK fusion. b 542 patients who had testing by IHC only are not included. c Patients in the original report that are described in another series 45 are excluded. d Patients tested were preselected for factors associated with EGFR mutation positivity, enriching the tested population for factors that included adenocarcinomas and low/never smoking status. e Includes one tumor with KIF5-ALK fusion. NSCLC, non-small cell lung cancer; RT-PCR, reverse transcription-polymerase chain reaction; FISH, fluorescent in situ hybridization; IHC, immunohistochemistry. 1452 Copyright 2009 by the International Association for the Study of Lung Cancer

Journal of Thoracic Oncology Volume 4, Number 12, December 2009 that ALK-positive patients typically do not derive benefit from treatment with EGFR inhibitors but have similar response rates to chemotherapy as patients who have EGFR wild-type tumors. Histopathologic associations have been made with acinar patterns 47 or solid patterns containing signet-ring features. 44,45 To date, the overall incidence in largely unselected series conducted in Asian (4.2%, 54 of 1271) and Western (3.4%, 54 of 15164) populations seems similar. Whether there will be significant geographical or racial variation in the frequency of ALK gene rearrangements when only more defined clinical populations, such as adenocarcinomas with no/light smoking history, and/or only those known to be EGFR and KRAS wild type are considered remains to be seen. TARGETING ALK GENE REARRANGEMENTS IN NSCLC Significant effort has been directed toward the generation of therapeutically useful ALK inhibitors. 49 In preclinical studies, several ALK inhibitors have shown activity against NPM-ALK and EML4-ALK containing cell lines. 2,4,7,50,51 TAE684, a small molecule ALK inhibitor, inhibited the growth of, and induced apoptosis in, H3122 an EML4- ALK containing NSCLC cell line in vitro and caused regression of xenografts in vivo. 7 Another small molecule tyrosine kinase inhibitor PF02341066, originally developed as an inhibitor of mesenchymal epithelial transition growth factor (c-met), was found to also be a very potent inhibitor of ALK. PF02341066 inhibited ALK phosphorylation and signal transduction with associated G 1 -S phase cell cycle arrest and induction of apoptosis in NPM-ALK positive ALCL cells in vitro and in vivo. 50 Preliminary results of the phase I study of PF02341066 in an expanded cohort of patients with NSCLC with ALK gene rearrangements (detected by break-apart FISH probe) have recently been presented. In the first 29 evaluable patients treated at the recommended dose of 250 mg twice daily, 17 of 29 patients (59%) had partial responses and 24 of 29 (83%) had disease control (partial response stable disease). 52 Based on these encouraging results, a randomized phase III trial comparing ALK inhibition to standard second-line cytotoxic chemotherapy in patients with ALK-positive NSCLC has now commenced. CONCLUSION Inversions in the short arm of chromosome 2 that result in one of several ALK gene rearrangements occur in approximately 4% of NSCLC and comprise a newly defined molecular subtype of NSCLC. The most common fusion protein resulting from the gene rearrangement is EML4-ALK. ALK gene rearrangements seem to be more common in adenocarcinomas from never or light smokers whose tumors are wild type for EGFR and KRAS. As is the case in other ALK driven cancers, ALK gene rearrangements in NSCLC result in constitutive ALK activity, which is important for both tumorigenesis and tumor maintenance. Activity of the ALK inhibitor PF02341066 in a subset of patients with ALK-positive NSCLC treated on a phase I clinical trial has provided proof of concept that ALK gene rearrangements and their associated fusion proteins are valid therapeutic targets in NSCLC. The results of a phase III trial comparing ALK inhibition to cytotoxic chemotherapy in patients with ALK-positive NSCLC are awaited. ACKNOWLEDGMENTS Supported by Young Investigator Awards from the International Association for the Study of Lung Cancer (to B.S. and D.R.C.). REFERENCES 1. Futreal PA, Coin L, Marshall M, et al. 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J Clin Oncol 2009;27:15s (suppl; abstr 3509). 1454 Copyright 2009 by the International Association for the Study of Lung Cancer