ALK Gene Rearrangements a New Therapeutic Target in a Molecularly Defined Subset of Non-Small Cell Lung Cancer

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ALK Gene Rearrangements 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†

arrangement was shown to be transforming both in vitro and Abstract: Transforming rearrangements of the ALK (anaplastic in vivo. Since then, several variant ALK gene rearrangements lymphoma kinase) gene have recently been described in non-small have been identified, and the clinical/pathologic features cell lung cancer (NSCLC). The most common rearrangement arises associated with “ALK-positive” NSCLC have been described. from an inversion in the short arm of chromosome 2 that creates a Recently, evidence that the fusion proteins resulting from fusion between the 5Ј portion of the EML4 (echinoderm microtu- ALK gene rearrangements constitute a bona fide therapeutic bule-associated protein-like 4) gene and the 3Ј portion of the ALK target has been provided in a phase I trial of a specific ALK gene. At least seven ALK gene rearrangement variants have been inhibitor in known ALK-positive NSCLC. 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 IDENTIFICATION OF EML4-ALK IN NSCLC reaction or fluorescent in situ hybridization. Although ALK gene In 1997, Soda et al.2 screened a cDNA library derived rearrangements affect only about 4% of all lung cancers, they are from the tumor of a 62-year-old Japanese male smoker with more frequent in adenocarcinomas, in never or light smokers, and adenocarcinoma of the lung for transforming activity. One of seem almost mutually exclusive with activating EGFR or KRAS the cDNAs with transforming activity was found to code for mutations. Promising results seen in patients with NSCLC contain- a novel fusion gene arising from an inversion on the short arm ing fluorescent in situ hybridization-detected ALK rearrangements of chromosome 2 [Inv(2)(p21p23)] that joined exons 1–13 of treated on a phase I study with PF02341066, an oral ALK inhibitor, EML4 to exons 20–29 of ALK.2 The resulting chimeric indicate that ALK represents a new therapeutic target in this molec- protein, EML4-ALK, contained an N-terminus derived from ularly defined subset of NSCLC. EML4 fused to the C-terminal kinase domain of ALK, which permitted ligand-independent dimerization and constitutive Key Words: ALK, Lung cancer. activation of ALK. In addition to its transforming ability in (J Thorac Oncol. 2009;4: 1450–1454) 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 in- lthough chromosomal translocations are the most com- hibitor was able to cause regression of tumors.2,4 Amon genetic change in cancer genomes1 and are fre- Multiple variants of EML4-ALK have since been quently described in hematologic malignancies, they have reported.2,5–9 All variants encode the same cytoplasmic por- only recently been documented in epithelial malignancies tion of ALK but contain different truncations of EML4 (at such as non-small cell lung cancer (NSCLC)2 and prostate exons 2, 6, 13, 14, 15, 18, and 20), and those tested to date cancer.3 In 2007, EML4-ALK, a novel fusion gene arising as have all demonstrated biologic gain of function (reviewed by a result of an inversion in chromosome 2 that juxtaposed the Horn and Pao10). Other rare fusion partners for ALK have also 5Ј end of the EML4 gene with the 3Ј end of the ALK gene, been described, including TRK-fused gene TFG11 and was reported in a subset of patients with NSCLC. This gene KIF5B.8

*Department of Haematology and Medical Oncology, Peter MacCallum STRUCTURE AND FUNCTION OF ALK Cancer Centre, East Melbourne, Victoria, Australia; and †Division of AND EML4 Medical Oncology, University of Colorado Denver, Denver, Colorado. EML4 and ALK are closely located genes situated on Disclosure: Dr. Varella-Garcia received honorarium as a speaker for Abbott Molecular, the manufacturer of the FISH protein listed in the manuscript. the short arm of chromosome 2 (2p21 and 2p23, respectively) The other authors declare no conﬂicts of interest. where they are separated by a distance of 12.7 megabases and Address for correspondence: D. Ross Camidge, MD, PhD, Mailstop F704, are oriented in opposite directions. Room ACP 2256, 1665 Aurora Court, Aurora, CO 80045. E-mail: Full-length ALK was cloned in 199712,13 and is a mem- [email protected] Copyright © 2009 by the International Association for the Study of Lung ber of the insulin receptor family of receptor tyrosine kinases, Cancer of which leukocyte tyrosine kinase is the closest homologue. ISSN: 1556-0864/09/0412-1450 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 ALK Gene Rearrangements an extracellular domain with an N-terminal signal peptide, fied, including t(1;2)(q21;p23) (TPM3-ALK), inv(2)(p23q35) two MAM motifs (meprin, A5 protein, and receptor protein (ATIC-ALK), t(2;3)(p23;q21) (TFG-ALK), t(2;17)(p23;q23) tyrosine phosphatase mu), a low-density lipoprotein class A (CLTC-ALK), t(2;19)(p23;q13.1) (TPM4-ALK), and t(X;2) motif, and a large glycine-rich region putative ligand binding (q11–12;p23) (MSN-ALK).30,31 site. It also contains a short transmembrane domain and an In addition to ALCL, ALK gene rearrangements have intracellular domain with a juxtamembranous segment with a also been described in 50% of inflammatory myofibroblastic binding site for insulin receptor substrate-1, a kinase domain tumors32,33 and rare ALK-positive diffuse large B-cell lym- with three tyrosine-containing motif (YxxxYY: tyrosines phomas.34 Point mutations and amplifications of ALK have 1278, 1282, and 1283 within the activation loop) and a been identified in sporadic and familial neuroblastomas.35–38 C-terminus with binding sites for Src homology-2 and phos- Proteomic studies have also reported on ALK proteins being pholipase C-␥. Activation of ALK by binding of ligand leads associated with esophageal squamous cell carcinoma.39,40 to dimerization, autophosphorylation, and signaling through As has been observed in ALCL and IMT, with only rare the RAS-ERK, JAK3-STAT3, and PI3-K/Akt pathways, exceptions, ALK gene rearrangements in NSCLC contain the which lead to effects on cell proliferation, survival, migra- complete tyrosine kinase domain of ALK (beginning from tion, and alterations in cytoskeletal rearrangement.14 exon 20, residues 1058–1620). In the gene rearrangement, The normal physiological function of ALK in humans is the promoter of the 5Ј partner gene controls the transcription poorly understood. Drosophila melanogaster ALK (dALK)15 of the resulting fusion gene. The fusion partner in the trans- and its ligand Jeb (jelly belly)16 are important for the formation lated fusion protein typically contains an oligomerization of the visceral musculature of the gut17,18 and for the assem- domain that mediates constitutive dimerization and subse- bly of the neuronal circuitry in the visual system.19 No quent autophosphorylation and constitutive activation of the mammalian counterpart to Drosophila Jeb has been identi- ALK kinase in the absence of ligand. The partner protein also fied. Putative mammalian ligands are the heparin-binding determines cellular localization, which may affect the in- proteins pleiotrophin20 and midkine.21 Mammalian ALK is duced phenotype in different fusions. In the case of EML4- expressed in the nervous system during embryogenesis and at ALK, the location is cytoplasmic.9,41,42 This is distinct from reduced levels after birth. Immunohistochemical studies in NPM-ALK where NPM drives nuclear and cytoplasmic ex- adult human tissue reveal weak expression restricted to com- pression, CTLC-ALK that has a granular cytoplasmic stain- ponents of the central nervous system.22 Interestingly, there ing distribution,43and KIF5B-ALK that is associated with a are no gross abnormalities in ALK knockout mice, with the perinuclear distribution.8 exception of subtle neurocognitive changes consistent with alterations in hippocampal function.23 Several detailed re- views of the basic biology of ALK have recently been METHODS OF DETECTION published.14,24,25 ALK gene rearrangements or the resulting fusion pro- Echinoderm microtubule-associated protein-like 4 teins may be detected in tumor specimens using immunohis- (EML4) is a 120 KDa cytoplasmic protein that is a human tochemistry, RT-PCR of cDNA, and FISH. The ALK-1 homolog of echinoderm microtubule-associated protein, the antibody, which has proven useful in ALCL and inflamma- major microtubule binding protein in dividing sea urchin tory myofibroblastic tumors, has not been as reliable in lung eggs, and is essential for the formation of microtubules.26,27 It carcinomas.42 Nevertheless, several strategies to improve the is composed of a N-terminal basic region, a HELP domain, accuracy of the immunohistochemistry ALK assays in lung and four WD repeats in the C-terminus. cancer including amplification of the signal with a tyramide cascade44and intercalation of an antibody-enhanced polymer8 have recently been described. ALK GENE REARRANGEMENTS IN CANCER RT-PCR of cDNA has been the most commonly ap- ALK was originally identified in 1994 in anaplastic plied screening strategy for ALK gene rearrangements in large-cell lymphomas (ALCL)28,29 as a component of a chi- archival tissue. Initially, only a few primer sets were tested, meric protein NPM-ALK created as a result of a translocation but currently, a number of multiplex assays have been de- between chromosomes 2 and 5 [t(2;5)(p23;q35)]. ALK rear- signed to simultaneously capture all possible in-frame fusions rangements occur in 60 to 80% of ALCL, which are CD30 between EML4 and ALK in which the kinase domain of ALK positive non-Hodgkin lymphomas (NHL) that present more would be preserved.2,5,7 However, although different break- frequently in young patients and have a male preponderance. points in EML4 or ALK may be detected with such tech- ALCL accounts for about 40% of childhood NHL but only niques, novel ALK fusion partners will not. 5% of adult NHL.30,31 NPM-ALK is the most common fusion FISH has been performed with the ALK Dual Color in ALK-positive ALCL and consists of the N-terminal por- Break-Apart probe (Abbott Molecular, Des Plaines, IL)41,42,45 tion of nucleophosmin (NPM) fused with the kinase domain or “homebrew” reagents.46 The commercial break-apart containing C-terminus of ALK. The fusion gene is readily probe is comprised of a SpectrumOrange (red)-labeled 250 kb detected by reverse transcription-polymerase chain reaction probe to the 3Ј end of ALK with a SpectrumGreen (green)- (RT-PCR) or fluorescent in situ hybridization (FISH) and can labeled 300 kb probe to the 5Ј end of ALK (Fig. 1). Because also be detected by the ALK-1 (Dako, Carpinteria, CA) the EML4 and ALK loci are mapped relatively close in 2p antibody with typical nuclear and cytoplasmic localization. Nu- (12.7 MB apart) and the chromosome inversion is frequently merous other rearrangements involving ALK have been identi- associated with deletion around the ALK 5Ј breakpoint,7,46 the

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 per- sonnel. 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 sophis- ticated strategies have also been proposed, such as a three- color probe set with an additional fluorescent probe targeting the deleted portion of chromosome 2 in ALK-rearranged NSCLC44 or a dual-fusion color probe directed against both fusion derivatives (Varella-Garcia, unpublished data). Al- though 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 CLINICAL/PATHOLOGIC FEATURES situ hybridization (FISH) probe that includes DNA contigs The overall incidence of ALK gene rearrangements in adjacent to the ALK gene, the 3Ј contig is labeled with red NSCLC in the series reported to date is 3.8% (107 ALK fluorochromes and the 5Ј is labeled with green fluoro- rearrangements in 2835 tested tumors—with a range from 0.4 chromes. B, Non-small cell lung cancer (NSCLC) specimen to 13.4%—see Table 1).2,5,7–9,11,41,42,45–48 Most ALK gene showing fused red/green signals representing normal copies rearrangements have been found in adenocarcinomas, in of the ALK (yellow arrows) and single red and green signals never or light smokers. Consequently, any differing propor- (red and green arrows) indicating that chromosomal break tions reported in the different series have to be considered in Ј Ј occurred between the 3 and the 5 contigs. light of their underlying enrichment for factors such as specific histologies or patients with no or light smoking interpretation of a positive rearrangement (through the intro- histories. Coexpression with EGFR or KRAS mutations seems duction of a gap between the red and green probes) in to be extremely rare, suggesting that ALK is a distinct formalin-fixed, paraffin-embedded tissue sections can be oncogenic driver. Patients with ALK-positive NSCLC have challenging. Internal and external controls must be used to been reported as being younger than patients without the gene estimate whether, in each nucleus showing separated signals, rearrangement in some series.9,45 Shaw et al have described

TABLE 1. Frequency of ALK Gene Rearrangements in NSCLC ALK Gene Method of Rearrangements Total Number of Author Testing Population of Origin Identiﬁed NSCLC Tumors Tested Percentage Soda et al.2 RT-PCR Japan 5 75 6.7 Rikova et al.11 RT-PCR China 4a 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.42b RT-PCR Italy, Spain 9 120 7.5 Rodig et al.44c IHC, FISH USA 1 227 0.4 Shaw et al.45d FISH USA 19 141 13.5 Takeuchi et al.8 IHC, RT-PCR Japan 8e 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 series45 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 ALK Gene Rearrangements that ALK-positive patients typically do not derive benefit fusion proteins are valid therapeutic targets in NSCLC. The from treatment with EGFR inhibitors but have similar re- results of a phase III trial comparing ALK inhibition to sponse rates to chemotherapy as patients who have EGFR cytotoxic chemotherapy in patients with ALK-positive wild-type tumors. Histopathologic associations have been made NSCLC are awaited. with acinar patterns47or solid patterns containing signet-ring features.44,45 To date, the overall incidence in largely uns- elected series conducted in Asian (4.2%, 54 of 1271) and ACKNOWLEDGMENTS Western (3.4%, 54 of 15164) populations seems similar. Supported by Young Investigator Awards from the Whether there will be significant geographical or racial vari- International Association for the Study of Lung Cancer (to ation in the frequency of ALK gene rearrangements when B.S. and D.R.C.). only more defined clinical populations, such as adenocarcinomas with no/light smoking history, and/or only those REFERENCES known to be EGFR and KRAS wild type are considered 1. Futreal PA, Coin L, Marshall M, et al. A census of human cancer genes. remains to be seen. Nat Rev Cancer 2004;4:177–183. 2. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007; TARGETING ALK GENE REARRANGEMENTS IN 448:561–566. NSCLC 3. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. 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