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CLIP2 As Radiation Biomarker in Papillary Thyroid Carcinoma

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Oncogene (2015) 34, 3917–3925 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE CLIP2 as radiation biomarker in papillary thyroid carcinoma M Selmansberger1, A Feuchtinger2, L Zurnadzhy3, A Michna1, JC Kaiser4, M Abend5, A Brenner6, T Bogdanova3, A Walch2, K Unger1,7, H Zitzelsberger1,7 and J Hess1,7 A substantial increase in papillary thyroid carcinoma (PTC) among children exposed to the radioiodine fallout has been one of the main consequences of the Chernobyl reactor accident. Recently, the investigation of PTCs from a cohort of young patients exposed to the post-Chernobyl radioiodine fallout at very young age and a matched nonexposed control group revealed a radiation-specific DNA copy number gain on chromosomal band 7q11.23 and the radiation-associated mRNA overexpression of CLIP2. In this study, we investigated the potential role of CLIP2 as a radiation marker to be used for the individual classification of PTCs into CLIP2- positive and -negative cases—a prerequisite for the integration of CLIP2 into epidemiological modelling of the risk of radiation- induced PTC. We were able to validate the radiation-associated CLIP2 overexpression at the protein level by immunohistochemistry (IHC) followed by relative quantification using digital image analysis software (P = 0.0149). Furthermore, we developed a standardized workflow for the determination of CLIP2-positive and -negative cases that combines visual CLIP2 IHC scoring and CLIP2 genomic copy number status. In addition to the discovery cohort (n = 33), two independent validation cohorts of PTCs (n = 115) were investigated. High sensitivity and specificity rates for all three investigated cohorts were obtained, demonstrating robustness of the developed workflow. To analyse the function of CLIP2 in radiation-associated PTC, the CLIP2 gene regulatory network was reconstructed using global mRNA expression data from PTC patient samples. The genes comprising the first neighbourhood of CLIP2 (BAG2, CHST3, KIF3C, NEURL1, PPIL3 and RGS4) suggest the involvement of CLIP2 in the fundamental carcinogenic processes including apoptosis, mitogen-activated protein kinase signalling and genomic instability. In our study, we successfully developed and independently validated a workflow for the typing of PTC clinical samples into CLIP2-positive and CLIP2-negative and provided first insights into the CLIP2 interactome in the context of radiation-associated PTC. Oncogene (2015) 34, 3917–3925; doi:10.1038/onc.2014.311; published online 6 October 2014 INTRODUCTION alterations in PTC are point mutations of the BRAF gene (V600E) One of the major consequences of the Chernobyl nuclear accident and various variants of RET gene rearrangements, all of which lead in 1986 has been a significant increase in the incidence of to a constitutive activation of the mitogen-activated protein 8 papillary thyroid carcinomas (PTCs) among children exposed to kinase (MAPK) pathway. The frequency of RET/PTC rearrange- the radioiodine fallout, particularly to iodine-131.1 To date, PTC ments (that is, RET/PTC1 and RET/PTC3) was associated with 4 radiation exposure levels in a study of the atomic bomb survivors has developed in 4000 individuals who were children or 9–11 adolescents at the time of exposure.2 Thus, young age at exposure and some but not all studies of post-Chernobyl PTC. However, is a significant risk factor for the development of radiation- RET/PTC3 rearrangements have also been observed with similar frequencies in sporadic PTCs from young patients, indicating a induced PTC.3 In order to delineate radiation-associated effects, it relation with young age of PTC onset.11,12 A more recent is crucial that the tumour cohorts of exposed and nonexposed 13 4 publication by Ricarte-Filho et al. reported a higher frequency cases are matched on age and other factors as closely as possible. of fusion oncogenes in radiation-induced PTCs, including rare TRK This approach was enabled by the Chernobyl Tissue Bank and BRAF rearrangements and the recently discovered ETV6– (CTB, www.chernobyltissuebank.com) that systematically collects NTRK3 kinase fusion oncogene. These promising findings require thyroid tumour tissue samples from residents who lived in the further validation in independent cohorts. We recently reported a contaminated regions of Ukraine and the Russian Federation at radiation-specific DNA copy number gain on the chromosomal the time of the accident. The CTB collection also includes a band 7q11.23 and a radiation-associated mRNA overexpression of substantial number of thyroid tumours from nonexposed patients. the gene CLIP2, located on chromosome 7q11.23.4 Based on these Several studies evidence that radiation exposure can induce copy findings, we aimed to investigate CLIP2 as a radiation biomarker in number alterations and deregulation of gene expressions with the PTC. However, one of the crucial requirements for a biomarker to potential of triggering carcinogenic processes, both of which was be used in epidemiological studies and risk modelling is its validity observed even at low doses of radiation.5–7 Common genetic in terms of sensitivity, specificity, reproducibility and biological 1Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Neuherberg, Germany; 2Research Unit Analytical Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Neuherberg, Germany; 3Institute of Endocrinology and Metabolism, National Academy of Medical Sciences of the Ukraine, Kiev, Ukraine; 4Institute of Radiation Protection, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Neuherberg, Germany; 5Bundeswehr Institute of Radiobiology, Munich, Germany and 6Radiation Epidemiology Branch, Division of Cancer Epidemiology and Genetics, NCI, Bethesda, MD, USA. Correspondence: Dr J Hess, Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstr. 1, Neuherberg 85764, Germany. E-mail: [email protected] 7Clinical Cooperation Group ‘Personalized Radiotherapy of Head and Neck Cancer’, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, Neuherberg, Germany. Received 21 March 2014; revised 16 July 2014; accepted 9 August 2014; published online 6 October 2014 CLIP2 as radiation marker in PTC M Selmansberger et al 3918 plausibility. A further aspect for the integration into molecular sections were scanned and the generated images imported into epidemiology is the suitability of both a highly specific marker and the image viewer software Panoramic Viewer (3DHISTECH, an appropriate assay for its detection.14 In addition, a biomarker Budapest, Hungary). The staining intensities were visually scored may provide insights into the mechanisms of the disease it is a as follows: negative staining (score 0), weak staining (score 1), surrogate for. Therefore, this study also aimed to test whether a intermediate staining (score 2) and strong staining (score 3), as CLIP2 radiation-specific mRNA overexpression persists at the demonstrated in Figure 2. Detailed scoring criteria are outlined in protein level and to establish a reproducible workflow that allows the Materials and methods section. The entire classification a classification of PTCs from patients with unknown radiation workflow is illustrated in Figure 3. The resulting visual scores for history. Moreover, we intended to reconstruct the CLIP2 the 33 cases of the Genrisk-T cohort are shown in Table 1. A interactome from global transcriptome data that were derived statistically significant difference (two-sided Fisher’s exact test, from radiation-induced PTCs in order to clarify the role of CLIP2 in P-value = 0.005) between the exposed and nonexposed group was radiation-associated PTC carcinogenesis.15 also revealed using the visual scoring approach. Correlation analysis of the data from both evaluation approaches showed a strong correlation between digital analysis and visual scoring RESULTS − (Spearman’s correlation coefficients: 0.83, P-value: 1.63 × 10 6, CLIP2 protein expression is elevated in PTCs from patients Figure 4). In order to obtain an individual biomarker classification exposed to radioiodine fallout compared with a nonexposed for each case, cases with a visual score of 0 or 1 were considered control group as CLIP2 biomarker negative, whereas cases with visual score 3 In order to evaluate the expression of CLIP2 at the protein level were classified as CLIP2 biomarker positive. For a classification of in exposed and nonexposed cases of the Genrisk-T cohort, an intermediate CLIP2 staining (score 2), the genomic copy immunohistochemical staining with an antibody against CLIP2 number status (gained or not gained) of chromosomal band was performed in a highly standardized manner, followed by 7q11.23 (localization of CLIP2) was taken into account. Preferably, digital image analysis using the Definiens software (Definiens AG, data from array comparative genomic hybridization (array CGH) Munich, Germany). The obtained average marker staining were used. However, if array CGH data were not available, intensities within the analysed tumour regions (regions of interest) interphase fluorescence in situ hybridization analysis with a probe are listed in Supplementary Table 1. Statistical testing revealed specific for 7q11.23 was performed. Cases with a visual score of 2 significantly increased staining intensities (Mann–Whitney
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