Genomic Heterogeneity of ALK Fusion Breakpoints in Non-Small-Cell Lung
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Modern Pathology (2018) 31, 791–808 © 2018 USCAP, Inc All rights reserved 0893-3952/18 $32.00 791 Genomic heterogeneity of ALK fusion breakpoints in non-small-cell lung cancer Jason N Rosenbaum1, Ryan Bloom2, Jason T Forys3, Jeff Hiken3, Jon R Armstrong3, Julie Branson4, Samantha McNulty4, Priya D Velu1, Kymberlie Pepin5, Haley Abel6, Catherine E Cottrell7, John D Pfeifer4, Shashikant Kulkarni8, Ramaswamy Govindan5, Eric Q Konnick9, Christina M Lockwood9 and Eric J Duncavage4 1Department of Pathology and Laboratory Medicine, University of Pennsylvania, The University of Pennsylvania Center for Personalized Diagnostics, Philadelphia, PA, USA; 2Unum Therapeutics, Cambridge, MA, USA; 3Cofactor Genomics, St Louis, MO, USA; 4Department of Pathology and Immunology, Division of Anatomic and Molecular Pathology, Washington University School of Medicine, St Louis, MO, USA; 5Department of Internal Medicine, Division of Medical Oncology, Washington University School of Medicine, St Louis, MO, USA; 6McDonnell Genome Institute, Washington University School of Medicine, St Louis, MO, USA; 7Nationwide Children’s Hospital, Institute for Genomic Medicine, Columbus, OH, USA; 8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA and 9Department of Laboratory Medicine, University of Washington, Seattle, WA, USA In lung adenocarcinoma, canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALK rearrangement occurs in a minority (2–7%) of lung adenocarcinoma, and only ~ 60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALK genomic rearrangement detected by fluorescence in situ hybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALK rearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next- generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALK rearrangements, and those without (20.6 months vs 5.4 months, Po0.01). Together, these data demonstrate abundant genomic heterogeneity among ALK-rearranged lung adenocarci- noma, which may account for differences in treatment response with targeted ALK inhibitors. Modern Pathology (2018) 31, 791–808; doi:10.1038/modpathol.2017.181; published online 12 January 2018 Cancer accounted for 8.8 million deaths in 2015, five (1.69 million) of those deaths, making it the with lung cancer comprising approximately one in leading cause of cancer mortality worldwide.1 The anticipated number of new cases of lung and bronchial cancer in the United States for 2017 is 2 Correspondence: Dr JN Rosenbaum, MD, Department of Pathology 222 500 with 155 870 deaths. The 5-year survival and Laboratory Medicine, University of Pennsylvania, The rate of lung cancer from 2006 to 2012 in the United University of Pennsylvania Center for Personalized Diagnostics, States was 18% (15% for men and 21% for women). 3020 Market Street Ste. 220, Philadelphia, PA 19104, USA. E-mail: [email protected] The treatment and prognosis of lung cancer varies Received 12 June 2017; revised 24 October 2017; accepted 26 considerably based on the specific pathologic find- October 2017; published online 12 January 2018 ings and subtype. Broadly, lung cancer is divided www.modernpathology.org Genomic heterogeneity of ALK fusions 792 JN Rosenbaum et al into small-cell lung cancer that is aggressive (though however, the partner breakpoint within EML4 is sensitive to treatment with radiation), and non- quite variable, occurring in a variety of introns, most small-cell lung cancer that can show a range of commonly intron 13, 20, and 6 (reference sequence behaviors and treatments dependent on other prop- NM_019063.4).4 Other ALK translocation partners erties of the tumor, including molecular alterations. (the most common of which is KIF5B) constitute a Non-small-cell lung cancer itself is divided into small but significant proportion of ALK rearrange- squamous carcinoma and adenocarcinoma. In lung ments in lung adenocarcinoma, and new rearrange- adenocarcinoma, the canonical EML4-ALK inversion ment partners are reported regularly.7,21–31 The results in a fusion protein containing a dysregulated, tumorigenic properties of ALK fusion proteins derive constitutively active ALK kinase domain.3–8 from constitutive activity of the tyrosine kinase Although evidence of ALK rearrangement only domain of ALK. The 5′ fusion partner provides occurs in a minority (2–7%) of cases,9 ~ 60% will promoter and initiation sites, and permits constitu- respond to targeted inhibition of ALK by drugs such tive dimerization of the translated protein.3,7,32 The as crizotinib, ceritinib, alectinib, and brigatinib.10–17 canonical EML4-ALK rearrangement is well estab- ALK rearrangement is associated with specific lished as a driver of cancer, and limited data suggest clinical features, including higher incidence among that different rearrangements may vary in their women, younger patients, patients of Asian descent, pathogenic properties, even among the canonical and non-smokers. Moreover, there are a handful of fusions.33–35 In addition, single-nucleotide variants histo- and cyto-pathologic features associated with (SNVs) altering the coding sequence of ALK or other ALK rearrangement (acinar structure, higher histolo- genes (such as EGFR) can drive oncogenesis or gic grade).18–20 None of these features (alone or in impart resistance to targeted inhibition, despite the combination) provide a strong enough association to presence of an ALK rearrangement.36–46 diagnose the chromosomal rearrangement or select Clinically, targeted anti-ALK therapy is initiated patients for testing, however, and ancillary diagnos- based on evidence of ALK genomic rearrangement as tic tests remain critical for identifying the therapeu- detected by fluorescence in situ hybridization (FISH) tic target. performed on interphase cells in formalin-fixed, The canonical ALK rearrangement in lung adeno- paraffin-embedded tissue sections. Because of the carcinoma is an inversion of the short (p) arm of promiscuous nature of the translocation, a ‘break- chromosome 2, with breakpoints in the ALK and apart’ FISH probe is the preferred identification EML4 genes (Figure 1). The ALK breakpoint lies most strategy, forming the basis for the original ‘in vitro frequently within intron 19 (reference sequence device companion diagnostic’ (CDx) assay approved NM_004304.4), though there are rare exceptions; by the US-FDA.47,48 Although break-apart FISH Figure 1 Inversion of chromosome 2p results in constitutively active EML4-ALK fusion protein. Structural rearrangements involving the ALK gene drive a number of cancers. In non-small-cell lung cancer, the most common structural rearrangement involving the ALK locus is an inversion of a portion of chromosome 2p, juxtaposing the 5′ portion of EML4 to the 3′ portion of ALK (a). ALK is not normally transcribed in adult lung, but under the control of the EML4 promoter, the EML4-ALK fusion gene is transcribed. Notably, the reciprocal translocation juxtaposing the 5′ portion of ALK to the 3′ portion of EML4 also occurs, but as it retains the ALK promoter, it is not transcribed (indicated in gray). Wild-type ALK is a receptor tyrosine kinase known to drive developmental pathways, particularly in the nervous system (tyrosine kinase domain indicated by dark-green bars). In the presence of ALK ligand, wild-type ALK proteins homodimerize, driving downstream developmental processes such as cellular proliferation (b). Under transcriptional control of the EML4 promoter, the EML4-ALK fusion protein is expressed in lung tissue, and the dimerization domain of EML4 (dark purple bars) permits unregulated dimerization of the TK domain, constitutively activating downstream pathways. Modern Pathology (2018) 31, 791–808 Genomic heterogeneity of ALK fusions JN Rosenbaum et al 793 probes are generally considered sensitive, they do has recently become available in the clinical setting not permit differentiation or identification of ALK and can be performed on routine formalin-fixed, partner genes or non-canonical breakpoints, raising paraffin-embedded material. For fusion detection, a the possibility that not all aberrant ALK FISH major advantage of RNA-Seq over targeted DNA- patterns (among