Supporting Information

Heß et al. 10.1073/pnas.1017137108 SI Materials and Methods housekeeping gene, GADPH (4); all cases showed good quality Patient Data and Tumor Tissues. For this study a frequency-matched (amplification products of 400 bp). For array CGH, 450 ng tu- case-control design (1) was used, and sequential cases of PTC mor DNA and 450 ng male or female reference DNA (Promega) who were born after January 1, 1987 were identified from the were labeled with Cy3-dCTP and Cy5-dCTP (Perkin-Elmer), Chernobyl Tissue Bank (CTB; www.chernobyltissuebank.com) respectively, using the BioPrime-Labeling Kit (Invitrogen) in an database. For inclusion in the Genrisk-T cohort, these cases must overnight incubation at 37 °C. Hybridizations were carried out have frozen tissue available for extraction of RNA and DNA “sex-mismatched” (i.e., female cases were hybridized against from both normal and tumor tissue and be resident in one of the male reference DNA, and vice versa). After labeling, the purified following areas of Ukraine (receiving an average thyroid dose of samples were coprecipitated with 135 μg cot-1 DNA (Roche), 40–120 mGy): Zhytomir, Cherkasse, Chernigov, Kiev, Rovno, or dissolved in hybridization buffer [50% formamide, 7% dextran Sumy. Cases were matched as closely as possible on residency, sulfate, 0.1% Tween20, 2× SSC, 10 mM Tris (pH 7.4), and 25 age at operation and sex to cases of PTC born before April 26, mM EDTA (pH 8.0)], and hybridized onto the array for 40 h in 1986. To allow for the possibility that cases that were operated an automated hybridization station (HS400; Tecan) including more than 5 y before the start of this study may have relatively prehybridization, washing, and drying steps. Fluorescence in- more degraded DNA and could potentially fail the quality as- tensity ratios were measured using a microarray scanner (Gen- surance (QA) step before array, we included more cases in the ePix Personal 4100A; Axon Instruments, Molecular Devices). exposed group. However, we did not find this to be the case. We Fluorescence intensities were extracted using GenePix Pro-6.0 therefore included all cases in the primary analysis, whether software (Axon Instruments, Molecular Devices) and were im- matched to unexposed cases or not. There are many fewer cases ported into the R statistical platform (5). Median replicate in- of PTC diagnosed at a young age in this population in the cohort tensities were only accepted if the replicate SE was <10% and born after January 1, 1987 and therefore not exposed to radio- the foreground to background ratio (both channels) >3. Cases iodine in fallout from the accident. The exploratory set of cases with an overall rate of surviving clones <80% were excluded included those that were available from cases reviewed by the from the study. Data normalization was performed using an al- CTB Pathology Panel before March 2006, when this project was gorithm for spatial normalization of array CGH data [R package initiated. The validation set of cases born after January 1, 1987 MANOR (6)]. The normalized data were segmented using cir- was identified from sequential cases of PTC that were operated cular binary segmentation [DNAcopy (7)] and the CGHcall between March 2006 and November 2008, fitting the same cri- package (8) for calling of gains and losses. To reduce data teria as the exploratory set. The validation set of exposed cases complexity, copy number calls were transformed into regions was selected as described for the exploratory set. A description of using the R package CGHregions (9). the clinical features of the cases included in the study is given in Table 1 (main text). The tumor samples were from 80 patients High-Resolution Oligo Array CGH. For high-resolution copy number aged <25 y (median age 16.9 y, range 7.7–24.6 y) presenting with typing, 180-k SurePrint arrays (Agilent) comprising ≈180,000 PTC developed after the Chernobyl reactor accident and were probes were used. Different from the standard 180-k array design, obtained from the CTB. All tumors were diagnosed as papillary we used a design (kindly provided by Bauke Ylstra, VU University carcinomas according to the TNM classification of malignant Medical Center Amsterdam, The Netherlands) that provides a tumors (2) by the International Pathology Panel of the CTB and higher probe density at regions of cancer genes. Tumor DNA further subdivided according to their dominant histological ar- (500 ng) and sex-mismatched reference DNA (500 ng) (Promega) chitecture (papillary, follicular, and solid) (by T.B.). We analyzed were labeled with Cy3 and Cy5, respectively, using the CGH la- a cohort of 52 cases (median age 16.6 y) and an independent beling kit for oligo arrays (Enzo), and unincorporated nucleotides validation cohort of 28 cases (median age 18.5 y) by array com- were removed using Microcon YM-30 columns (Millipore). La- parative genomic hybridization (CGH). In addition, the PTCs beled DNA was hybridized to the 180-k CGH arrays (Agilent) and were characterized for the presence of RET/PTC rearrangements subsequently washed and scanned according to the manufacturer’s and for the most frequent BRAF mutation, V600E, via DNA di- protocol. The fluorescence intensities were extracted using Fea- rect sequencing (details in ref. 3). Expression of the RET gene was ture Extraction software (Agilent) and the resulting text files im- classified as balanced (BAL) when expression of the extracellular ported into the R statistical platform. The data were filtered for domain (EC) and tyrosine kinase domain (TK) were the same, as quality outliers using the QA measurements generated by the unbalanced TK when the expression of the TK was significantly Feature Extraction software and subsequently normalized, seg- higher than expression of the EC domain, as unbalanced EC when mented, and called for gains and losses using the same methods as the EC domain was more highly expressed compared with ex- described above for the 1-Mb BAC arrays. pression of the TK domain, and as nonRET expressor (nre) when no expression, or very low expression, of both domains was de- Array CGH: Secondary Data Analysis. Unsupervised hierarchical tectable. The patients’ demographic and clinical data are sum- cluster analysis (correlation distance and Ward linkage, R Package marized in Tables S1 and S4. Tumor DNA and RNA isolated amap) based on smoothed log2 ratios (median log2 ratios of seg- from fresh-frozen tissue were provided by the CTB. ments) was used to identify groups of cases according to similar aberration patterns. Associations of the resulting groups (main Array CGH: Primary Data Analysis. To detect copy number aberra- clusters) with tumor phenotypes were statistically tested using tions (CNAs), array CGH analysis on 52 PTC cases (main data Fisher’s exact test (95% confidence interval). Associations of CNAs set, Table S1) and on additional 28 PTC cases (validation set, (regions) with tumor phenotypes and clinical data were tested by Table S4) using 1-MB bacterial artificial chromosome (BAC) multiple comparisons using the χ2 test. Testing was performed using arrays (≈3,400 BAC clones; Centre for Microarray Resources, the CGHtest method [http://www.few.vu.nl/∼mavdwiel/CGHtest. Cambridge, UK) was performed. Isolated tumor DNA was as- html (10)], including permutation procedures with 10,000 permu- sessed for the required quality by multiplex size PCR of a tations. P values were corrected for multiple-testing error using

Heß et al. www.pnas.org/cgi/content/short/1017137108 1of7 a false discovery rate (FDR) controlling procedure (11), whereas were selected on the basis of their functional role in conjunction FDR values <0.05 were considered statistically significant. with cancer, particularly thyroid cancer. Further, to gain insights into the functional meaning of genes located in the associated Fluorescence in Situ Hybridization. We applied Interphase-FISH regions, GO term enrichment analysis was performed using the analysis to validate the DNA gain on chromosome 7q11.22–11.23 online analysis tool DAVID [http://david.abcc.ncifcrf.gov (13)] detected by array CGH. BAC clones mapping the candidate genes with default parameters. RFC2 (RP11-331N10) and CLIP2 (RP11-422O01) were selected from the 32-k Re-Array BAC Library (BACPAC Resources Quantitative RT-PCR (qRT-PCR) of Selected Candidate Genes. To Center, Children’s Hospital Oakland Research Institute, Oakland, validatetheeffectofthecopynumbergainonchromosome7q11.22– CA) and hybridized to formalin-fixed, paraffin-embedded (FFPE) 11.23 at the transcriptional level, nine genes (CLDN3, CLDN4, tissue sections of the same PTC cases that were analyzed by array CLIP2, LIMK1, PMS2L2, PMS2L3, PMS2L11, RFC2, and CGH. To correct for polyploidy a reference clone (RP11-136K15) STAG3L3) were selected for analysis by qRT-PCR. Total RNA mapping to a region near the centromere of chromosome 2 that from the same cases analyzed using BAC array CGH was pro- did not show any copy number changes in array CGH was cohy- vided by the CTB. We used Taqman probes (Applied Biosystems) bridized. Chromosome 7 was not suited for reference hybridiza- Hs00185593_m1 for analyzing the gene CLIP2, Hs00265816_s1 for tion because it showed various copy number changes including the CLDN3, Hs00533616_s1 for CLDN4, Hs00242731_m1 for LIMK1, centromere. BAC DNA was extracted according to standard Hs03989376_mH for PMS2L2, Hs00389106_m1 for PMS2L3, procedures and was labeled with digoxigenin-11-dUTP or biotin- Hs03653709_mH for PMS2L11, Hs00267983_m1 for RFC2, and 16-dUTP by nick translation. Hybridization of BAC clones, slide Hs00219216_m1 for the gene STAG3L3. Reactions (20 μL) were washing, and detection of fluorescent signals were performed as carried out in duplicate according to the manufacturer’s protocol in described previously by Unger et al. (12). Differentially, biotin- an ABI7300 real-time PCR thermocycler (Applied Biosystems) in labeled DNA probes were detected with fluorescein (DTAF)- conjugated streptavidin (Jackson ImmunoResearch Laborato- combination with the analysis software SDS (Applied Biosystems). μ Relative expression levels were determined using the ΔΔCt method ries). Tissues were analyzed by taking 3D images (0.5- m steps) β with the AxioImager system using the ApoTome (Zeiss). Signals (14), whereas a Taqman probe for -actin was used for endogenous fi from 100 cell nuclei per tumor tissue were scored, whereas only normalization, and the total RNAs from ve normal tissues were cells with at least two reference BAC clone signals were counted. used as calibrators. The relative expression levels of the genes were tested for differential expression in the exposed cases that showed Candidate Genes and Gene Ontology (GO) Analysis. The genomic againon7q11.22–11.23 and cases with normal copy number using positions of copy number regions associated with phenotypes or the Mann-Whitney test. Additionally, the relative expression levels clinical data were used to extract candidate genes from the latest of the genes were tested for differential expression in the groups build of the Ensembl database using the R package biomaRt. To exposed and not exposed to irradiation. The test result was con- define tumor-related candidate genes, the genes from the gene lists sidered statistical significant if the P value was <0.05.

1. Katz MH (2006) Study Design and Statistical Analysis: A practical Guide for Clinicians 8. van de Wiel MA, et al. (2007) CGHcall: Calling aberrations for array CGH tumor (Cambridge Univ Press, Cambridge, UK). profiles. Bioinformatics 23:892–894. 2. Sobin LH, Wittekind C; International Union Against Cancer (2002) TNM: Classification 9. van de Wiel MA, Wieringen WN (2007) CGHregions: Dimension reduction for array of Malignant Tumours (Wiley-Liss, New York), 6th Ed. CGH data with minimal information loss. Cancer Inform 3:55–63. 3. Powell N, et al. (2005) Frequency of BRAF T1796A mutation in papillary thyroid 10. van de Wiel MA, Smeets SJ, Brakenhoff RH, Ylstra B (2005) CGHMultiArray: exact carcinoma relates to age of patient at diagnosis and not to radiation exposure. J P-values for multi-array comparative genomic hybridization data. Bioinformatics 21: Pathol 205:558–564. 3193–3194. 4. van Beers EH, et al. (2006) A multiplex PCR predictor for aCGH success of FFPE samples. 11. Manduchi E, et al. (2000) Generation of patterns from gene expression data by Br J Cancer 94:333–337. assigning confidence to differentially expressed genes. Bioinformatics 16:685–698. 5. R Development Core Team R: A Language and Environment for Statistical Computing 12. Unger K, et al. (2004) Heterogeneity in the distribution of RET/PTC rearrangements (R Foundation for Statistical Computing, Vienna). Available at http://www.R-project. within individual post-Chernobyl papillary thyroid carcinomas. J Clin Endocrinol org. Metab 89:4272–4279. 6. Neuvial P, et al. (2006) Spatial normalization of array-CGH data. BMC Bioinformatics 13. Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of 7:264. large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57. 7. Olshen AB, Venkatraman ES, Lucito R, Wigler M (2004) Circular binary segmentation 14. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real- for the analysis of array-based DNA copy number data. Biostatistics 5:557–572. time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408.

Heß et al. www.pnas.org/cgi/content/short/1017137108 2of7 Fig. S1. Cluster analysis. Unsupervised hierarchical clustering (Ward distance) separated array CGH profiles into two main groups (cluster 1 and 2); cluster 2 was further separated into two subclusters (cluster 2-1 and 2-2). Losses are represented by red, gains by green, and unaltered regions by gray vertical bars. The copy number profiles are ordered according to the physical position of copy number clones in the genome (from left to right). On the right-hand side colored boxes represent, from left to right, the RET/PTC status, tumor size (TNM), and lymph node status (TNM) of each case. Color codes: RET/PTC status: light-blue, RET expression negative; dark-blue, RET expression positive. Tumor size: light purple, T1; purple, T2; violet, T3. Lymph node status: gray, lymph node negative; black, lymph node positive. White boxes indicate nonavailable values. The cluster hierarchy and distances are shown by the dendrogram on the left-hand side.

Fig. S2. FISH validation. Interphase-FISH on an FFPE tissue section of the PTC case UA0135 captured using optical sectioning microscopy. Red signals (indicated by yellow arrows) represent the BAC clone RP11-422O01, which maps the middle part of 7q11.22–11.23 that we found to be specifically gained in a subset of PTCs from patients exposed to the radioiodine fallout. Green signals (indicated by white arrows) represent the reference BAC clone RP11-136K15, which binds close to the centromere of chromosome 2. Cells with two green and more than two red signals represent the copy number gain of 7q11.22–11.23, whereas cells with two red and two green signals represent a normal copy number. The FISH probe (≈150 kb) binds along the chromosome axis. When the axis of the chromosome is in line with the viewing angle the signal looks smaller, and it looks largest when the angle between chromosome axis and viewing axis is 90°, whereas the copy number of the region the probe binds to is the same in both situations.

Heß et al. www.pnas.org/cgi/content/short/1017137108 3of7 Fig. S3. Expression of candidate genes in exposed cases showing and not showing gain of 7q11.22–11.23. Expression of the seven candidate genes located in the irradiation-associated region on 7q11 in exposed cases not showing the gain (gray boxplots) and those showing the gain (green boxplots). The P value was calculated using Mann-Whitney test, and expression fold changes (FC) were calculated according to the median expression of the genes in the two groups.

CLIP2 Relative Expression Relative 0.5 1.0 1.5 2.0

non−exposed (n=30) exposed (n=17)

p−value: 0.039 FC: 1.5

Fig. S4. CLIP2 expression in the exposed and in the unexposed group. Shown is the relative expression of the CLIP2 gene after qRT-PCR (Taqman) in non- exposed cases (red boxplot) and exposed cases (blue boxplot). The P value was calculated using Mann-Whitney test, and expression fold changes (FC) were calculated according to the median expression of the genes in the two groups.

Heß et al. www.pnas.org/cgi/content/short/1017137108 4of7 Table S1. Patient data of main data set Case Sex County Age (y)* Type TNM (1) Subtype RET RET/PTC BRAF Exposed† Pair Type

UA0144 Female Chernigov 14.7 PTC T1N1aM0 F TK RET/PTC1 Neg Yes 1 Case UA0307 Female Zhytomyr 13.1 PTC T1N0M0 F TK — Neg No 1 Control UA0484 Female Rovno 16.4 PTC T2N1aM0 F (P areas) nre — Neg Yes 2 Case UA1053 Female Sumy 16.1 PTC T1N0M0 F (P,S areas) TK — NA No 2 Control UA0601 Male Kiev 16.9 PTC T3N1abM0 F (S areas) NA — Pos Yes 3 Case UA0368 Male Sumy 10.3 PTC T3N1abM1 F TK — NA No 3 Control UA0400 Female Zhytomyr 16.9 PTC T1N0M0 FP nre — NA Yes 4 Case UA0796 Female Chercassy 17.1 PTC T3N0M0 FP TK — NA No 4 Control UA0103 Female Zhytomyr 15.0 PTC T3N1abM1 FS TK RET/PTC3 Neg Yes 5 Case UA0615 Female Sumy 15.4 PTC T3N1abM0 FS TK RET/PTC3 Neg No 5 Control UA0436 Female Zhytomyr 17.1 PTC T3N0M0 PF nre — NA Yes 6 Case UA0939 Female Sumy 17.7 PTC T1N0M0 PF nre — Pos No 6 Control UA0502 Female Zhytomyr 16.9 PTC T3N1abM1 PS TK RET/PTC3 Neg Yes 7 Case UA0692 Female Zhytomyr 16.4 PTC T3N1aM0 P (S areas) TK — Neg No 7 Control UA0208 Male Chernigov 15.1 PTC T3N1abM1 F (S areas) TK RET/PTC3 Neg Yes 8 Case UA0312 Male Chernigov 11.4 PTC T3N1abM0 PS nre — Neg No 8 Control UA0180 Female Kiev 16.3 PTC T1N0M0 P TK RET/PTC1 Neg Yes 9 Case UA0691 Female Chercassy 7.7 PTC T3N0M0 P (F areas) nre — Neg No 9 Control UA0147 Female Zhytomyr 16.5 PTC T2N0M0 P nre — Pos Yes 10 Case UA0162 Female Zhytomyr 8.8 PTC T3N1abM1 P nre — Pos No 10 Control UA0515 Female Chernigov 17.7 PTC T1N0M0 P (F areas) NA — Pos Yes 11 Case UA0710 Female Chernigov 16.7 PTC T1N1aM0 P (F areas) nre — Neg No 11 Control UA0242 Female Kiev 17.7 PTC T3N1aM0 P TK — Neg Yes 12 Case UA1091 Female Kiev 18.5 PTC T2N0M0 P EC — NA No 12 Control UA0648 Female Chernigov 17.4 PTC T1N1aM0 S (P, F areas) TK RET/PTC3 Neg Yes 13 Case UA1005 Female Chernigov 18.7 PTC T1N1aM0 SP TK RET/PTC1 NA No 13 Control UA0135 Male Zhytomyr 15.6 PTC T1N1aM0 SP BAL — Neg Yes 14 Case UA1030 Male Sumy 12.8 PTC T3N1aM0 S TK RET/PTC3 NA No 14 Control UA0616 Male Kiev 18.0 PTC T3N0M0 S nre — Neg Yes 15 Case UA0421 Male Rovno 13.8 PTC T3N1aM0 SP TK — NA No 15 Control UA0138 Male Zhytomyr 14.0 PTC T3N1abM0 S (P areas) TK RET/PTC3 NA Yes 16 Case UA0465 Male Chernigov 13.9 PTC T3N0M0 S TK RET/PTC3 Neg No 16 Control UA0456 Female Chercassy 17.7 PTC T1N0M0 P (S areas) nre — Pos Yes 17 Case UA0574 Female Kiev 13.6 PTC T2N1aM0 P (S areas) BAL — Neg No 17 Control UA0249 Female Rovno 16.8 PTC T2N1aM0 F nre — Neg Yes 18 Case UA0991 Female Kiev 18.4 PTC T1N0M0 SP nre — Neg No 18 Control UA0580 Female Rovno 16.9 PTC T3N1abM1 S TK RET/PTC3 Neg Yes 19 Case UA0496 Female Chercassy 14.1 PTC T3N1abM0 P (F areas) TK RET/PTC3 NA No 19 Control UA0139 Female Kiev 14.1 PTC T3N1abM0 S TK RET/PTC1/3 Neg Yes um Case UA0145 Male Zhytomyr 15.8 PTC T1N1aM0 F (P areas) BAL — Neg Yes um Case UA0165 Female Chernigov 16.7 PTC T1N0M0 F nre — Neg Yes um Case UA0192 Female Sumy 15.0 PTC T1N1aM1 PF NA — Neg Yes um Case UA0286 Female Zhytomyr 18.9 PTC T1N1aM0 F nre — NA Yes um Case UA0343 Female Kiev 18.5 PTC T2N1aM0 PFS TK — Neg Yes um Case UA0363 Male Chernigov 15.9 PTC T1N1abM0 PF nre — Neg Yes um Case UA0366 Male Sumy 15.6 PTC T1N0M0 P nre — NA Yes um Case UA0463 Female Chernigov 18.6 PTC T3N0M0 PF nre — Pos Yes um Case UA0469 Male Rovno 17.0 PTC T3N1abM1 PF NA — Neg Yes um Case UA0503 Female Kiev 18.6 PTC T1N0M0 F (S areas) nre — Neg Yes um Case UA0583 Female Kiev 19.0 PTC T2N0M0 FS TK RET/PTC3 Neg Yes um Case UA0597 Female Rovno 17.1 PTC T3N0M0 FS TK RET/PTC3 Neg Yes um Case UA0686 Male Chernigov 18.1 PTC T3N1aM0 P NA — Neg Yes um Case

F, follicular; P, papillary; S, solid; TK, unbalanced RET expression in favor of RET-TK domain; nre, nonRET-expressor; EC, unbalanced RET expression in favor of RET-EC domain; BAL: balanced expression of RET-TK and RET-EC domain; um: unmatched. *Age at operation. †Exposed to radioiodine fallout.

1. Sobin LH, Wittekind C; International Union Against Cancer (2002) TNM: Classification of Malignant Tumours (Wiley-Liss, New York), 6th Ed.

Heß et al. www.pnas.org/cgi/content/short/1017137108 5of7 Table S2. Regions of copy number alteration associated with tumor size (TNM), patient sex, and exposure Chromosome Location Start (BAC) End (BAC) Start (bp)* End (bp)* Size (Mb) Gain/loss Association P value FDR

1 q21.1–23.3 RP11-315I20 RP11-15G16 145440576 161929198 16.5 Gain TNM T1 0.008 0.028 5 q23.3–31.3 RP1-247F3 CTD-2332G20 130546647 142017094 11.5 Loss Females 0.002 0.046 † 7 p14.1-q11.23 RP5-1032B10 RP11-107L23 43183638 75314803 32.1 Gain Exp 0.002 0.035 ‡ 7 q11.22-q11.23 RP11-409J21 RP11-107L23 71050338 75314803 4.3 Gain Exp 0.024 NA§ 7 q22.1 RP4-550A13 RP11-333G13 98867726 101602962 2.7 Gain TNM T1 0.020 0.040 9 p24.3 GS1-41L13 GS1-77L23 11190 345203 0.3 Gain TNM T1 0.008 0.028 10 p15.3–15.1 CTC-306F7 RP11-336A10 270607 5803775 5.5 Gain TNM T1 0.006 0.028 10 q26.13–26.3 RP13-238F13 CTB-137E24 126018441 135396841 9.4 Gain TNM T1 0.008 0.028 11 p11.12-cen RP11-227P3 RP11-100N3 50057493 56640040 6.6 Gain TNM T1 0.009 0.029 12 q24.11–24.23 RP11-256L11 RP11-322N7 109695428 119752625 10.1 Gain TNM T3 0.007 0.028 16 q22.1–23.3 RP11-63M22 RP11-2L4 66596260 82721053 16.1 Gain TNM T3 0.017 0.036

*Clone positions according to February 2009 Homo sapiens high-coverage assembly from the Genome Reference Consortium (GRCh37). †Exposed to irradation from radioiodine fallout. ‡Exposed to irradation from radioiodine fallout (validation set). §The P value was generated from one test only verifying the validity of the hypothesis that gain of 7q11 is associated with radiation exposure in the validation set. Therefore, no FDR correction was required.

Table S3. Cancer-related candidate genes of regions associated with patient sex and tumor size (TNM) Type of Chromosomal location Candidate genes aberration Association

1q21.1–23.3 PVRL4, F11R, VANGL2, COPA, TAGLN2, CRP, NTRK1, CCT3, RAB25, CKS1B, SHC1, RAB13, Gain TNM T1 JTB, S100A1, S100A13, S100A4, S100A6, S100A7, S100A10, S100A11, PRUNE, CTSK, MCL1, BCL9, CHD1L, PIAS3, EFNA4, PLEKHO1, PMF1 5q23.3–31.3 SPRY4, CTNNA1, EGR1, JMJD1B, TGFBI, CXCL14, PITX1, PPP2CA, GDF9, IRF1, PDLIM4 Loss Females 7q22.1 ARPC1A, MCM7, EPHB4, TRIP6, SERPINE1, CUX1 Gain TNM T1 9p24.3 KANK1 Gain TNM T1 10p15.3–15.1 AKR1C1, AKR1C2, AKR1C3, NET1 Gain TNM T1 10q26.13–26.3 MMP21, BCCIP, DHX32, ADAM12, MKI67 Gain TNM T1 11p11.12-cen No tumor related candidate gene Gain TNM T1 12q24.11–24.23 TBX3, PTPN11 Gain TNM T3 16q22.1-q23.3 NOL3, CTCF, NFATC3, CDH3, HAS3, TERF2, NQO1, KIAA0174, BCAR1, MAF Gain TNM T3

Heß et al. www.pnas.org/cgi/content/short/1017137108 6of7 Table S4. Patient data of validation data set Case Sex County Age (y)* Type TNM (1) Subtype RET RET/PTC BRAF Exposed† Pair Type

UA0672 Female Chernigov 17.7 PTC T3N0M0 F (S areas) TK RET/PTC1 Neg Yes 20 Case UA1240 Female Chernigov 11 PTC T2N1aM0 PF nre — Pos No 20 Control UA0954 Female Kiev 19.8 PTC T3N1aM0 F (S areas) NA — Neg Yes 21 Case UA1208 Female Kiev 15.2 PTC T2N1aM0 P NA — Neg No 21 Control UA0756 Female Zhytomyr 20.9 PTC T3N1aM0 P nre — Pos Yes 22 Case UA1247 Female Zhytomyr 15.6 PTC T1N0M0 P nre — Pos No 22 Control UA0417 Female Kiev 20 PTC T3N0M0 PF nre — Neg Yes 23 Case UA1190 Female Kiev 16.6 PTC T1N0M0 PS NA — Neg No 23 Control UA0374 Male Kiev 18.3 PTC T3N1a,bM0 S (P areas) TK RET/PTC3 na Yes 24 Case UA1175 Male Kiev 16.6 PTC T1N0M0 F nre — Neg No 24 Control UA0679 Female Chernigov 18.7 PTC T3N0M0 F BAL — Pos Yes 25 Case UA1224 Female Chernigov 17 PTC T3N0M0 PF NA — na No 25 Control UA0905 Female Sumy 21.1 PTC T1N0M0 PF nre — Pos Yes 26 Case UA1328 Female Sumy 18 PTC T3N1a,bM0 SP nre — Neg No 26 Control UA0886 Female Chercassy 24.5 PTC T3N0M0 SP NA — Neg Yes 27 Case UA1486 Female Chercassy 20.7 PTC T1N1aM0 F nre — Pos No 27 Control UA0173 Female Kiev 19.8 PTC T3N1a,bM0 FP TK RET/PTC1 Neg Yes 28 Case UA1423 Female Kiev 19.6 PTC T3N1a,bM0 F NA — na No 28 Control UA0482 Female Kiev 17 PTC T1N1aM0 P nre — Pos Yes 29 Case UA1367 Female Kiev 17.7 PTC T1N0M0 F (S areas) nre — Neg No 29 Control UA0053 Female Kiev 16.4 PTC T1N0M0 P nre — Neg Yes 30 Case UA1319 Female Zhytomyr 12.4 PTC T3N1a,bM0 PS TK RET/PTC1 Neg No 30 Control UA0693 Female Kiev 19.6 PTC T2N0M0 PS nre — Neg Yes 31 Case UA1243 Female Chernigov 18.2 PTC T3N1a,bM0 S (P areas) nre — Neg No 31 Control UA0216 Male Kiev 19.2 PTC T3N1a,bM0 F (S areas) NA — Neg Yes um Case UA0329 Male Sumy 23.4 PTC T1N1aM0 PF nre — Pos Yes um Case UA0771 Female Kiev 20 PTC T1N0M0 FP EC — Neg Yes um Case UA1126 Female Sumy 21.4 PTC T1N0M0 F NA — Neg Yes um Case

F, follicular; P, papillary; S, solid; TK, unbalanced RET expression in favor of RET-TK domain; nre, nonRET-expressor; BAL: balanced expression of RET-TK and RET-EC domain; EC, unbalanced RET expression in favor of RET-EC domain; um, unmatched. *Age at operation. † Exposed to radioiodine fallout.

1. Sobin LH, Wittekind C; International Union Against Cancer (2002) TNM: Classification of Malignant Tumours (Wiley-Liss, New York), 6th Ed.

Table S5. Common CNAs of tumors included in present study and childhood tumors from a previous study (1) Chromosomal location Aberration Start (BAC) End (BAC) Start (bp) End (bp) Size (Mb)

1p31.3–13.3 Loss RP4-700A9 RP5-1077K16 65599782 107676036 42.1 1q23.3–32.1 Loss RP11-430G6 RP11-152M20 162947338 199646074 36.7 1q32.3–41 Loss RP11-286E7 RP11-308L13 213239930 221893823 8.7 6p21.1-q25.2 Loss RP3-441A12 RP11-495B4 44880799 154180965 109.3 9q33.3–34.3 Gain RP11-101K10 GS1-135I17 127089109 140997194 13.9 10q26.13–26.3 Gain RP13-238F13 CTB-137E24 126018441 135396841 9.4 11p14.1–12 Loss RP11-541L7 RP11-108L12 27267990 43063365 15.8 11q14.2–21 Loss RP11-320L11 RP11-338H14 85935688 95384834 9.4 13q13.1–33.3 Loss RP11-95N14 RP11-513N16 32170305 108683924 76.5 16p13.3 Gain RP11-344L6 RP11-35P16 72088 4657877 4.6 16q23.3–24.3 Gain RP11-483P21 CTB-121I4 83805115 90025221 6.2 19p13.3-q13.43 Gain CTD-3113P16 GS1-1129C9 233116 59078905 58.8 20q13.33 Gain RP13-152O15 CTB-81F12 62639242 62922571 0.3 21q22.13–22.3 Gain RP11-102E10 CTB-63H24 37844789 48100495 10.3

1. Unger K, et al. (2008) Array CGH demonstrates characteristic aberration signatures in human papillary thyroid carcinomas governed by RET/PTC. Oncogene 27:4592–4602.

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