1 MELODI 2012 workshop Helsinki 12 th - 14 th September, 2012 Molecular markers in radiation-induced thyroid cancer Horst Zitzelsberger Helmholtz Zentrum München Research Unit Radiation Cytogenetics
2 Radiation marker Radiation marker Integration of molecular biology and epidemiology; improved risk estimation (talk E. Pernot) Understanding molecular mechanisms of radiocarcinogenesis Targets for diagnosis and therapy Biodosimetry and emergency response
3 ? Why thyroid cancer?
4 Papillary thyroid carcinoma Chernobyl accident: ~ 6000 radiation-associated thyroid cancer cases in young patients in Belarus, Russia and Ukraine Epidemiology: relative risk of 3-6 per Gy absorbed dose during childhood related to iodine-131 exposure (Cardis et al., JNCI 2005; Abend et al., PLoS One 2012)
5 Papillary thyroid carcinoma Radiation-induced PTC as a model for mechanisms in radiocarcinogenesis Young age at exposure and age at diagnosis likelihood of radiation-related cancer Tumour tissue bank storing exposed cases and age-matched controls Individual dose estimates
6 ? Which radiation markers? Selected candidates or signatures from whole genome screening?
7 RET/PTC gene rearrangements RET (chromosome 10q11.2) codes for a receptor tyrosine kinase Intra- or interchromosomal rearrangements (inversion, translocation) Fusion of tyrosine kinase domain of RET with constitutively expressed promoter of partner gene: activation Radiation-induced generation of RET/PTC through microhomology-mediated end joining (model by Klugbauer et al. 2001) RET Pierotti M.A., Cancer Lett. 166:1-7, 2001 (modified) Klugbauer S. et al., Genomics 73: , 2001
8 RET/PTC gene rearrangements GENRISK-T Patients matched for age at diagnosis, residence and gender Group 1 Born before 26 th April 1986 Exposed to radioiodine fallout Group 2 Born after 1 st January 1987 Not exposed to radiation Estimated average thyroid dose: 150 mgy (Jacob et al., Radiat Res 2006) RET/PTC rearrangements (Imperial College London) Copy number alterations (Helmholtz Zentrum München) mrna gene expression (UAB Brussels, MSC Gliwice) mirna expression Imperial College London)
9 RET/PTC gene rearrangements GENRISK-T Is there qrt-pcr an association analysis of of Ret 86 expression GENRISK-T with cases radioiodine (exposed exposure? and non-exposed) NO association with radiation exposure!
10 RET/PTC gene rearrangements Conflicting evidence to which extent RET/PTC is radiation-induced PROS RET/PTC3 is frequent in post-chernobyl thyroid cancers Thomas et al. JCEM 1999; Rabes et al., Clin. Cancer Res. 2000; Ciampi and Nikiforov, Endocrinol RET/PTC prevalent in thyroid cancers from atomic bomb survivors Hamatani et al., Cancer Res RET/PTC prevalent in adults after external radiation? Bounacer et al., Oncogene 1997; Elisei et al., JCEM 2001 CONS RET/PTC activation is not associated with individual radiation dose Tuttle et al., Thyroid 2008 RET/PTC activation is common in sporadic thyroid cancer in children Nikiforov et al., Cancer Res. 1997; Fenton et al., JCEM 2000; Powell et al. J.Pathol 2005 RET/PTC3 in children is linked to age and morphology
11 Biomarkers identified in radiation-associated tumours Timeline Biomarker of exposure Types of biomarkers (from Pernot et al., 2012) Biomarker of susceptibility Biomarker of late effect Biomarker of persistent effect Genomic copy number alterations Alterations of mrna expression SNPs on 9q22.33 (FOXE1) Alterations of mrna expression Genomic copy number alterations Alterations of mrna expression Abend et al., 2012 Hess et al., 2011 Takahashi et al., 2010 Dom et al., 2012 Dom et al., 2012 Port et al., 2007 Boltze et al., 2009 Stein et al., 2009 Hess et al., 2011 Abend et al., 2012
12 mrna gene expression Comparison of radiation-induced PTC with PTC of unknown aetiology Detours et al., Br J Cancer, 2005 Detours et al., Br J Cancer, 2007 Port et al., Radiat Res, 2007 Stein et al., Thyroid, 2010 Ugolin et al., PLoS One, 2011 Dom et al., Br J Cancer, 2012 No radiation signature Signature of 118 genes (extension of 2005 study) Signature of seven genes Signature of 141 genes Signature of 227 genes Signature of 403 genes (in corresponding normal tissues of sporadic /radiation-induced tumours) Radiation-induced PTC with individual dose estimates Abend et al., PLoS One, genes with dose-dependent expression Limitations of studies No overlap of genes between different studies, small case numbers, no independent validation Different microarray platforms and biostatistics (methodological variability) Non-matched controls for Detours et al., Port et al., Stein et al., Ugolin et al. confounding factors: age, ethnicity, iodine supply, histopathology of tumours
13 mrna expression in normal tissues (Dom et al., 2012) Patients matched for age at diagnosis, residence and gender Group 1 Born before 26 th April 1986 Exposed to radioiodine fallout Group 2 Born after 1 st January 1987 Not exposed to radiation Estimated average thyroid dose: 150 mgy (Jacob et al., Radiat Res 2006) RET/PTC rearrangements (Imperial College London) Copy number alterations (Helmholtz Zentrum München) mrna gene expression (UAB Brussels, MSC Gliwice) mirna expression Imperial College London)
14 mrna expression in normal tissues (Dom et al., 2012) 45 PTC, 22 paired RNA samples from exposed and from 23 paired samples from non-exposed cases (tumour and corresponding normal tissue) Affymetrix full-genome mrna microarrays Exposed/non-exposed tumours and normal corresponding tissues have similar global expression profiles a large fraction of the transcriptome is dysregulated in the tumours Significance Analysis of Microarray (SAM) revealed no differences between exposed and non-exposed tumours a 403-gene signature (differentially expressed genes) between exposed and nonexposed corresponding normal tissues an accuracy of 67% of correctly classified cases Radiation susceptibility (altered proliferation pathways) or late effect of radiation Signature allows the identification of radiation-associated cases Independent validation is needed
15 Dose-dependent mrna expression (Abend et al., 2012) UkrAm cohort (exposed patients with individual dose estimates) Doses from to 8.6 Gy (mean 1.25 Gy) Three dose categories (means) of 0.11 (n=23), 0.57 (n=23) and 2.63 Gy (n=27) Dose-dependent expression of 11 genes Curvilinear relationship for dose-dependent gene expression Genes ACVR2A, AJAP1, CA12, CDK12, FAM38A, GALNT7, LMO3, MTA1, SLC19A1, SLC43A3, ZNF493 are involved in cell adhesion, energy metabolism, transcription, DNA methylation, growth Late or long-lasting effect of radiation (From Abend et al., 2012)
16 ? Why novel approaches?
17 Genomic radiation marker and data integration Hypothesis: primary radiation damage at DNA level that determines expression alterations Integration of genomic and expression data Two aspects of data integration: filtering and reduction of complexity of molecular data functional relationship between molecular levels (talk K. Unger)
18 Genomic copy number alterations GENRISK-T Patients matched for age at diagnosis, residence and gender Group 1 Born before 26 th April 1986 Exposed to radioiodine fallout Group 2 Born after 1 st January 1987 Not exposed to radiation Discovery set 52 cases Independent validation set 28 cases Estimated average thyroid dose: 150 mgy (Jacob et al., Radiat Res 2006) RET/PTC rearrangements (Imperial College London) Copy number alterations (Helmholtz Zentrum München) mrna gene expression (UAB Brussels, MSC Gliwice) mirna expression Imperial College London)
19 Array CGH Discovery set DNA gain on 7p14.1-q11.23 exposed group Region on chromosome 7 exclusively gained in PTC from exposed patients chr 7p14.1-q /33 cases of the exposed group (39%) showed a DNA gain on 7p14.1-q11.23 (p=0.0015, FDR=0.035) Hess et al., PNAS 108(23): , 2011.
20 Candidate genes on 7q Literature & Gene Ontology Analysis Literature: Genes were selected based on their functional role in conjunction with cancer Gene Ontology Analysis: To gain insights into the functional meaning of genes located in the altered region, significantly overrepresented GO terms were identified (p-values<0.05) Hess et al., PNAS 108(23): , 2011.
21 Candidate gene CLIP2 on chromosomal band 7q11 mrna expression (qrt-pcr) The candidate gene CLIP2 was specifically overexpressed in the exposed cases at mrna level Hess et al., PNAS 108(23): , 2011.
22 Candidate gene CLIP2 on chromosomal band 7q11 Protein expression
23 Candidate gene CLIP2 on chromosomal band 7q11 Protein expression Results of IHC staining of CLIP2 (34 CTB cases) 18 non-exposed 16 exposed cases An increased expression of CLIP2 in the exposed cases was also shown at the protein level by IHC
24 7q11 radiation marker Identification of CLIP2 Integrative data analysis identified radiation-specific alterations DNA gain 7q11 GENES? mrna non-exposed PROTEIN exposed What What do is we still know missing? so far? INTEGRATION Validation Functional role of CLIP2 Dose dependency Integration of the marker into epidemiology CLIP2 Smc domain chromosome segregation and cell division
25 Candidate gene CLIP2 on 7q11 Validation Association of gain on chromosome 7 with radiation exposure was confirmed in an independent validation cohort by array CGH Non-exposed cases Validation set: - 16 exposed cases - 12 non-exposed cases Exposed cases Further validation on an independent Ukrainian PTC cohort Validation set: - 20 exposed cases - 20 non-exposed cases
26 Summary & Conclusion In radiation-induced PTC potential radiation markers/signatures have been identified in the genome and transcriptome that need further validation in independent tumour cohorts No consistent radiation-specific gene expression signatures have been identified so far in different post-chernobyl PTC studies A dose-dependent expression signature of eleven genes have been reported in the UkrAm cohort A novel approach of integrating genomic copy number and gene expression data reduced data complexity and resulted in the identification of a radiation-specific copy number gain of 7q11 and a radiation-specific over-expression of CLIP2 An independent validation of 7q11 and CLIP2 was achieved within Genrisk-T at genomic, transcriptomic and proteomic level 7q11 and CLIP2 must be further investigated for - dose dependency - functional role - validation in other tumour cohorts - standardisation of biomarker detection for its use in epidemiologic studies ALL presented and future studies need access to a collection of quality assured tumour material (CTB) that also allows for integrative data analysis Post-Chernobyl PTC is a highly valuable model for the understanding of general mechanisms of radiation-associated carcinogenesis
27 Thank you Helmholtz Zentrum München Research Unit of Radiation Cytogenetics H. Braselmann J. Heß M. Selmansberger K. Unger V. Zangen All partners from Genrisk-T, EpiRadBio and INT-Thyr C. Maenhaut, G. Dom, B. Jarzab W. van Wieringen, M. van der Wiel E. Cardis, E. Pernot, J. Grellier, M. Lushchyk, Y. Demidchik Imperial College London G. Thomas U. Schötz A. Galpine K. Unger THANK YOU FOR YOUR ATTENTION! Institute of Endocrinology and Metabolism, Kiev T. Bogdanova Chernobyl Tissue Bank
Evidence Report/Technology Assessment Number 160 Impact of Gene Expression Profiling Tests on Breast Cancer Outcomes Prepared for: Agency for Healthcare Research and Quality U.S. Department of Health and
Gene expression profiling and expanded immunohistochemistry tests for guiding adjuvant chemotherapy decisions in early breast cancer management: MammaPrint, Oncotype DX, IHC4 and Mammostrat Issued: September
Health risk assessment from the nuclear accident after the 2011 Great East Japan Earthquake and Tsunami based on a preliminary dose estimation Health risk assessment from the nuclear accident after the
Technology Evaluation Center Special Report: acgh for the Genetic Evaluation of Patients with Developmental Delay/ Mental Retardation or Autism Spectrum Disorder Assessment Program Volume 23, No. 10 April
AFFYMETRIX MICROARRAY BULLETIN www.microarraybulletin.com Researchers Develop Improved Molecular Classification System for Rare Childhood Cancer Dr., of Childrens Hospital Los Angeles talks about using
Vol. 10, 1253 1258, December 2001 Cancer Epidemiology, Biomarkers & Prevention 1253 Lack of Interaction between Asbestos Exposure and Glutathione S-Transferase M1 and T1 Genotypes in Lung Carcinogenesis
CHALLENGES IN THE CLINICAL USE OF GENOME SEQUENCING Chapter in Ginsburg and Willard (eds) Genomic and Personalized Medicine, 2 nd Edition, 2012, in press. Robert C. Green, MD, MPH Heidi L. Rehm, PhD, FACMG
NIH Public Access Author Manuscript Published in final edited form as: Nat Rev Cancer. 2013 March ; 13(3): 153 159. BAP1 and Cancer Michele Carbone *, University of Hawaii Cancer Center, 701 Ilalo Street,
Open Access: Full open access to this and thousands of other papers at http://www.la-press.com. Clinical Medicine Insights: Oncology The Importance of Multidisciplinary Approach in Early Detection of BAP1
THYROID Volume 15, Number 11, 2005 О Mary Arm Uebert, inc. Iodine Excretion in Regions of Ukraine Affected by the Chornobyl Accident: Experience of the Ukrainian-American Cohort Study of Thyroid Cancer
STATE OF THE ART 3B-PL2 ISBT Science Series (2011) 6, 107 111 ª 2011 The Author(s). ISBT Science Series ª 2011 International Society of Blood Transfusion Human cord blood reprogrammed into embryonic-like
Principal Principal Investigator s Investigator Organization Project Title Alan Pollack University of Miami Integrated Biomarker Profiling for Individualized Prostate Cancer Therapy General Project Abstract
MedPage Tools Guide to Biostatistics Study Designs Here is a compilation of important epidemiologic and common biostatistical terms used in medical research. You can use it as a reference guide when reading
Annual Report Julius Center for Health Sciences and Primary Care Annual Report 2007 Julius Center for Health Sciences and Primary Care UMC Utrecht The Julius Center for Health Sciences and Primary Care
Methodology for Copy Number Variant Detection from High Throughput DNA Exome Sequencing and Application to the Genetic Mapping of Rare Genetic Disorders Case Presentation 3 Word Count - 4996 Michael Epstein
1 ontrac@petermac Victorian Adolescent & Young Adult Cancer Service A practice framework for working with 15 25 year-old cancer patients treated within the adult health sector Dr Susan Palmer & Associate
PERSPECTIVE doi:10.1038/nature09764 Charting a course for genomic medicine from base pairs to bedside Eric D. Green 1, Mark S. Guyer 1 & National Human Genome Research Institute* There has been much progress
Down Syndrome Directions The National Institutes of Health Research Plan on Down Syndrome Down Syndrome Directions National Institutes of Health Research Plan on Down Syndrome U.S. Department of Health
NCRI Lung CSG Mesothelioma workshop in association with Department of Health Workshop Report The Royal Society of Medicine, London 2nd May 2014 Sponsored by: Mesothelioma workshop Workshop Report Workshop
Novel Blood Biomarkers of Human Urinary Bladder Cancer Iman Osman, 1 Dean F. Bajorin, 2 Tung-Tien Sun, 1 Hong Zhong, 1 Diah Douglas, 1 Joseph Scattergood, 2 Run Zheng, 3 Mark Han, 3 K.Wayne Marshall, 3
Carcinogenesis vol.21 no.5 pp.1023 1029, 2000 mrna expression patterns in different stages of asbestos-induced carcinogenesis in rats H.Sandhu, W.Dehnen, M.Roller, J.Abel and induced. After a long period
Early Detection of Malignant Pleural Mesothelioma in Asbestos-Exposed Individuals with a Noninvasive Proteomics-Based Surveillance Tool Rachel M. Ostroff 1 *., Michael R. Mehan 1., Alex Stewart 1, Deborah
A REVIEW OF RESEARCH IN MESOTHELIOMA AND OTHER ASBESTOS-RELATED DISEASES NOVEMBER 2010 A Review of Research in Mesothelioma and other Asbestos- Related Diseases November 2010 PREFACE The catalyst for this
Epidemiology Article Critique 1 Running head: Epidemiology Article Critique An Article Critique - Helmet Use and Associated Spinal Fractures in Motorcycle Crash Victims Ashley Roberts University of Cincinnati
Are children more sensitive to radiation than adults? By Madan M. Rehani Director of Radiation Protection, European Society of Radiology There is a commonly held belief that children may be two to three