Molecular markers in radiation-induced thyroid cancer

Transcription

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