Vladimir Saenko

Department of Health Risk ControlAtomic Bomb Disease Institute Nagasaki University Graduate School of Biomedical Sciences

10th AOTA CongressOctober 21-24, Bali, Indonesia

Radiation and thyroid carcinogenesis

Content

Radiation epidemiology of thyroid

cancer

Pathology (Chernobyl)

Clinical prognosis (Chernobyl)

Molecular characteristics

(Chernobyl)

I. Radiation epidemiologyRadiation exposure of the thyroid at young age is the most clearly defined environmental factor associated with thyroid cancer

E.Ron 2002

Rela

tive

risk

Chernobyl (0-17 y.o.)

Thyroid dose (Gy)

ERR/Gy~7.7 [1.1 – 32]

External exposure

OR at 1 Gy~5.5 – 8.4 [ERR/Gy 1.9 – 19]

Internal radiation exposure  Therapeutic radioiodine Hanford (fall-out) Chernobyl

External radiation exposure  A-bomb hibakusha Marshall Islanders (fall-

out) Children exposed to EBT

V.Ivanov 2010

26 April 1986the accident at the Chernobyl nuclear power plant

More than 200 000 sq kmof Europe were contaminatedwith > 37 kBq/sq m of 137Cs

Radioactive substance release -13 EBq:131 I - 1.8 Ebq 137Cs - 0.085 Ebq90Sr - 0.01 EbqPu isotopes - 0.003 EBq

Over 70 % of this area was in the three most affected countries,Belarus, Russia and Ukraine.

The Chernobyl Forum: 2003–2005, IAEA

Estimated average thyroid doses to children and adolescents around Chernobyl

UNSCEAR 2008 Report Annex D

Incidence of thyroid cancer in the residents of radiocontaminated

territories

'90 '95 '00 '05

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Incidence of thyroid cancerdiagnosed in 1986-2002

Belarus Russian Ukraine TotalFederation

No. of casesAge atexposure(yr)

0-14 1,711 349 1,762 3,822

15-17 299 134 582 1,015

Total 2,010 483 2,344 4,837

Health Effects of the Chernobyl Accident and Special Health Care ProgrammesReport of the UN Chernobyl Forum, 2006

UNSCEAR 2008 Report, Annex D:Over 6,000 thyroid cancers by 2006

Dose-dependent excess thyroid cancer incidence in children/adolescents 0-4 and 5-18 years old at the time of accident in 6 most contaminated regions of Ukraine

1990-2000

Thyroid dose, GyThyroid dose, Gy

Addi

tiona

l inc

iden

ce p

er 1

0*5 P

Y

Tronko 2008

Latency Incidence by age at exposure

Effect of iodine deficiency and of stable iodine consumption

Cardis 2005

Major radiation epidemiology conclusions

Dose-response relationship (up to 2 Gy)

Young age at exposure is a risk factor (0-5 y.o.)

Latency may be short (4-5 years)

Iodine deficiency increases risk (~3-fold at 1 Gy)

No significant risk for childhood thyroid cancer for radiation doses below 100 mSv

No increase in cancer incidence in the population with accumulated doses <100 mSv during 25 years

No increase in cancer incidence in emergency workers with doses <150 mSv

Rela

tive

risk

Chernobyl (0-17 y.o.)

Thyroid dose (Gy)

OR at 1 Gy~5.5 – 8.4 [ERR/Gy 1.9 – 19]

V.Ivanov 2010

PTC FTC MTC0

10

20

30

40

50

60

70

80

90

100

4.7% 5.1% 0.4% 1.5%

94.9% 93.2%

Ukraine, based on 2658 cases

II. Pathology

Major histotypes of thyroid cancer in Ukraine and Belarus after Chernobyl

Belarus, based on 740 cases

PTC is the only type of thyroid carcinoma showing a significant increase after Chernobyl

Prev

alen

ce, %

PTC FTC MTC

Chernobyl PTC subtypes 2478 cases from Ukraine

Pap30%

Fol18%

Sol8%

Mix42%

DSV1%Other

1%

PapFolSolMixDSVOther

Courtesy T.Bogdanova

P

F SMix

Age-related pathological features

pT N0 N1 Total M1 (% sub-total)

T1 915 255 1170 (47.2%) 5 (0.4%)

T2 301 111 412 (16.6%) 5 (1.2%)

T3 286 610 896 (36.2%) 170 (19.0%)

Total (%) 1502 (60.6%) 976 (39.4%) 2478 (100%) 180 (7.3%)

2478 PTCs from Ukraine, age 0 – 18 years old at 26 April, 1986, diagnosed in 1990-2010

Courtesy T.Bogdanova

Children 67.6%Adolescents 52.4%Adults 34.0%

Children 27.9%Adolescents 17.3%Adults 3.3%

Is radiation-induced PTC pathologically more aggressive than sporadic?

Number of cases RAD SPOR

Mean age F:M

Children 114 111 11.6 12.0 1.5:1 3.0:1

Adolescents 66 97 16.8 16.8 2.0:1 2.9:1

Adults 59 56 21.2 20.8 2.9:1 4.6:1

Age-matched groups of radiogenic and sporadic PTC from Ukraine

Courtesy T.Bogdanova

Parameters associated with radiogenic PTC (born before Chernobyl) vs sporadic PTC (born after Chernobyl),

logistic regression analysis in age-matched groups, final models

Variables P-value* OR CI1 Sex (M vs F) 0.041 1.88 1.02-3.472 Tumor capsule (full vs absent+partial) 0.025 0.30 0.10-0.863 Subtype (S+SF vs other) 0.025 1.95 1.08-3.514 pN (N1 vs N0) 0.042 2.05 1.02-4.125 M (M1 vs 0) 0.046 2.30 1.01-5.236 Intrathyroidal extension (Y vs No) 0.008 2.74 1.28-5.83

Children <15 years old at surgery (N= 114 vs 111)

Variables P-value* OR CI1 M (M1 vs 0) 0.017 4.61 1.31-16.21

Adolescents 15-<18 years old at surgery (N= 66 vs 97)

Variables P-value* OR CI1 Age at operation (years) 0.017 1.50 1.07-2.112 pN (N1 vs N0) 0.049 2.80 1.00-7.853 Extrathyroidal extension (Y vs No) 0.021 3.63 1.21-10.94

Adults 18-23 years old at surgery (N= 59 vs 56)

* Based on likelihood ratio test CI, confidence interval

Courtesy T.Bogdanova

Major pathological conclusions

Time-related trendsPrevalence of less differentiated structures (solid component) decreases

Pathological aggressiveness (extrathyroidal extension, vascular invasion and nodal disease) declines

Proportion of encapsulated and small tumors increases

Age-related trendPathologically, the aggressiveness declines in the rowchildren > adolescents > adults

Radiation-induced PTC may be pathologically more aggressive than sporadic PTC in age-matched groups

III. Clinical prognosis

Mortality

Country No. N1 M1 Death Recurrence

Belarus 133 60% 5% 0.75% -

Belarus 472 65% 8% - -

Ukraine 232 62% 17% - -

Ukraine 330 57% 15% 1.2% 7%

Belarus 22 55% 0%

Russia 48 60% 8% -

Belarus 740 69% 10% 0.8% 28%

Russia 172 55% 4% 0% 15%

Totals 2149 60% 8% 0.69% 17%

Tuttle 2011

% S

urv

iva

l

98.8%

Years follow-up

Demidchik 2006

Belarus, 740 PTC

Disease-specific mortality rate is low ~ 1% (15-20 years)

Risk factors for disease-free survival in radiation-induced and sporadic PTC

from Russia

Prognosis: recurrence

Does radiation-induced PTC recur more readily than sporadic?

Cohort:172 Chernobyl PTC with individual thyroid doses > 50 mGy325 PTC with individual thyroid doses < 5 mGy

Matched by:• Sex• Age• Calendar time of treatment

Proportional hazard model of disease-free survival in radiation-induced and sporadic PTC

Variables Comparison P-value HR Wald’s CI

Radiation exposure yes vs no 0.104 0.54 0.26–1.13

Tumor size >10 mm yes vs no 0.472 1.47 0.51–4.20

pN Na+Nb vs N0 0.0053 5.21 1.63–16.7

Tumor capsule yes vs no 0.0003 0.17 0.06–0.45

Treatment according to the Guidelines

yes vs no 0.0002 0.16 0.06–0.420 50 100 150 200

0

25

50

75

100RI PTC

Sporadic PTC

Follow-up, months

Per

cent

sur

viva

l

RI-PTC

SP-PTC

HR=0.702 [0.465-1.090], P=0.118

(Logrank test)

None of the 12 variables tested in the model interacted with radiation exposure attesting to the absence of risk factors for recurrence specific to radiation

Rumyantsev 2011

Effects of tumor capsule, nodal disease and treatment adequacy

Risk factors 0 1 2 3

Major clinical conclusions

Disease-specific 15-20 years mortality is low (~1%)

Chance of recurrence is comparable to that in sporadic thyroid cancer

No etiology-specific risk factors for recurrence

Radiation-induced thyroid cancer is suggested to be treated and followed in the same way as sporadic thyroid cancer

IV. Molecular characteristics

From A.Chiloeches, R.MaraisClin.Cancer Res., 2006

MAP kinase pathway activation in PTC

(Table 12

RET/PTC3 RET/PTC1 BRAF, RASPr

eval

ence

Latency

Morphology Sol, Sol-Fol Classic Classic, Encaps

Clinical course Aggressive↑ Typical Aggressive↓

Latency, years 4 - 10 7 - 17 15 - …

Evolution of mutational events in timeChernobyl Japanese Hibakusha

Nakachi 2006

Alteration Chernobyl PTC Sporadic PTC

RET/PTC 50-86% 13-43%

NTRK1 3% 5-13%

AKAP9/BRAF 11% 1%

BRAFT1799A 0-16% 29-69%

RAS family 0-10% 0-21%

Major oncogenic events in PTC

Williams 2008

Gene expression patterns in radiation-related and sporadic PTCs are similar on a global scale.

Comparative gene expression studiesComparative gene expression studies

13 Adenomas12 Chernobyl and 8 Sporadic PTC

2400 genes

Detours 2005

Gene expression in 12 Chernobyl and 14 sporadic PTCs

Gene expression in 12 Chernobyl and 14 sporadic PTCs

Detours 2007

Based on 8,000 genes:

classifiers included from one to several

thousand genes (median 256)

overall error rates in discrimination

12-27%

Sporadic Chernobyl

Gene expression in 11 Chernobyl and 41 sporadic PTCs

Gene expression in 11 Chernobyl and 41 sporadic PTCs

Port 2007

Among 29,000 genes: 646 were upregulated and 677 were downregulated (>5-fold difference)

a 7-gene classifier

Gene expression signature distinguishes normal tissues of sporadic and radiation-induced PTC

unsupervised

N-SporN-Rad

T-SporT-Rad

Dom et al, Sep 2012

DNA copy number variation (CNV)DNA copy number variation (CNV)

Richter 2004Kimmel 2006Unger 2008Stein 2010

• 30% PTCs display CNV• RET/PTC-positive and –negative cases could be distinguished• Besides carcinogenesis-related changes, CNV also depends on radiation exposure• Prevalence of DNA gains is 2-4 times higher in Chernobyl PTCs• Recurrent gains are ~10 times more frequent in Chernobyl PTCs

Gain of chromosome band 7q11 in Chernobyl PTC Hess 2011

Learning set:33 Chernobyl (13/33=39.4%)19 Sporadic (0/19=0%)

Validation set:16 Chernobyl (6/16=37.5%)12 Sporadic (0/12=0%)

Mutational studies: NONE of the oncogenes or tumor suppressors implicated in of PTC has proved a distinctive molecular signature of radiation-induced thyroid cancer

Gene expression and DNA copy alteration analyses are suggestive that molecular classifiers of radiation-induced PTC may exist

Validation in the larger independent series (matched for ethnicity/ age/ sex) of radiation-induced and sporadic cancers is urgently needed to understand if the establishment of a reliable radiation signature is achievable

Are there molecular signaturesof radiation-induced PTC?

Molecular epidemiology studies

Molecular epidemiology is an approach to the identification of genetic factors that influence health and disease

Studies are conducted using advanced technologies to rapidly and cost-effectively analyze genetic differences between people with specific illnesses compared to healthy individuals

The purpose is to explore the connection between specific genes, i.e. genotype, and phenotype, to facilitate the determinationof genetic risk factors for the development of disease

Phenotype

Gene 1Linkage

disequilibriumMarker 1

Linkageassociation

Gene 2

Gene 2Individualenvironment

Commonenvironment

Polygenicbackground

Adapted from:Weiss and Terwilliger , 2000

(complex)

Mode ofinheritance

Multi-stage design of an association study

Genotype full set of SNPsin relatively small sample setat liberal P-value (GWAS)

Genotype full or narrower set of SNPsin relatively small sample setto achieve more stringent P-value

Validation:genotype narrow set of SNPsin extended sample setto increase stringent P-value

* Reduces study cost but also decreases power to detect modest associations on meta

rs# Chr Gene OR published Source

rs965513 9q22.33 FOXE1 upstream 1.75 / 1.69 Gumundsson et al, 2009Matsuse et al, 2011

rs1867277 9q22.33 FOXE1 5'UTR 1.49 Landa 2010

rs944289 14q13.3 NKX2-1 or MBIP 1.37 Gumundsson et al, 2012

rs116909374 14q13.3 MBIP 2.09 Gumundsson et al, 2012

rs2439302 8p12 NRG1 1.36 Gumundsson et al, 2012

rs966423 2q35 DIRC3 1.34 Gumundsson et al, 2012

Results of genetic association studiesin sporadic thyroid cancer

Areas of sampling of Chernobyl PTCs and controls

Map from UNSCEAR 2008 Report

Annex D

POLAND

448 controls from

the previous study

620 controls from

the previous study

Cases, approx 30 persons (total, 1098)

Controls, approx 30 persons (total, 2309)

rs10759944rs965513

rs7850258

rs925489rs7024345rs907580

Result: pooled analysis

rs10759944rs965513

rs7850258

rs925489rs7024345rs907580

FOXE1NKX2-1 or MBIP : NO association

SNPs associating with radiation-induced PTC also associate with sporadic PTC

DIRC3 : NO association

FOXE1 locus at 9q22.33 is confirmed as the strongest

NO association with 2q35 (DIRC3) and 14q13.3 (NKX2-1 or MBIP)Weak association with NRG1 at 8p12

Chromosomes1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122

NRG1 :Weak association

Genome-wide significance

Final analysis

Cases: 953(Bel) + 145(Ukr) = 1098 (1057 after QC)Controls: 1084(Bel) + 157(Ukr) + 448(Rus) + 620(Pol) = 2309 (2287 after QC)

rs# Chr GeneGWAS

837 Cs + 1,242 CtrValidation

220 Cs + 1,045 Ctr MetaOR (95% CI)

OR published Etiology

rs965513 9q22.33 FOXE1 upstream 1.13E-16 3.62E-04 5.80E-19 1.69 (1.51-1.90) 1.75 / 1.69 Sp & Rad

rs1867277 9q22.33 FOXE1 5'UTR 7.50E-03 3.75E-04 1.38E-05 1.52 (1.26-1.83) 1.49 Sp & Rad

rs944289 14q13.3 NKX2-1 or MBIP 0.0208 0.093 4.50E-03 1.17 (1.05-1.30) 1.37 Sp

rs116909374 14q13.3 MBIP 0.0438 0.0756 0.0169 2.19 (1.15-4.16) 2.09 Sp

rs2439302 8p12 NRG1 8.85E-04 0.0182 9.11E-05 1.35 (1.16-1.57) 1.36 Sp & Rad

rs966423 2q35 DIRC3 0.235 0.316 0.125 1.9 (0.98-1.21) 1.34 Sp

Candidates in Chernobyl PTC

rs6920544 6q21 LOC442245 4.71E-07 0.645 6.03E-06

rs4697477 4p15.2 ATP5LP3 1.19E-05 0.417 5.03E-05

rs10455038 5q23.2 PPIC 2.57E-06 0.0703 1.55E-03

rs7666030 4p15.3-p15.1 SOD3 1.13E-04 0.618 7.59E-04

rs3014966 13q14.13 COG3 5.33E-06 0.190 8.67E-04

rs11197463 10q26 ATRNL1 1.60E-04 0.371 4.97E-03

rs7199669 16p13.12 ERCC4 4.42E-05 0.505 1.31E-04

rs7861296 9p21.2 LRRN6C 7.40E-07 0.716 1.01E-05

rs7241128 18q11.2 LOC390843 2.15E-05 0.944 1.90E-04

rs2691546 7q21 MAGI2 4.59E-05 0.624 1.21E-03

rs2691542 7q21 MAGI2 1.08E-05 0.710 9.41E-05

Risk factors for radiation-induced PTC

• Radiation dose for the thyroid• Age at exposure• Iodine deficiency• Sex(?)

FOXE1 NRG1

Sporadic Radiation-induced

DIRC3NKX2-1or MBIP

NOstrong

markers

• Genetic predisposition

Predisposition to thyroid cancer

Etiology-specific modifiers (?)

weak (?)

In conclusion

• Displays dose-response relationship; the highest risk is in the youngest children; no risk at doses below 100 mSv

• Morphological aggressiveness declines with time and in the row children > adolescents > adults

• May be morphologically more aggressive than sporadic

• Is recommended to be treated and followed in the same way as sporadic thyroid cancers

• May have molecular signature that combines gene expression pattern and DNA CNV

• Has genetic risk markers common with sporadic

Radiation-induced thyroid cancer…

Acknowledgements

Nagasaki University Tatiana RogounovitchNorisato MitsutakeNoboru TakamuraShunichi Yamashita (福島県立医科大学 )

Kyoto UniversityMeiko TakahashiTakahisa Kawaguchi Ryo YamadaFumihiko Matsuda

MRRC RAMSPavel RumyantsevAnatoly TsybAlexander AbrosimovValery Stepanenko

BelMAPGE Valentina DrozdLarisa DanilovaMaxim LushchikNatallia AkulevichYuri Demidchik

CNGSimon HeathMark Lathrop

IEMTatiana BogdanovaMykola TronkoVladimir PushkarevVictor Shpak

CNG

Thank you for your attention!