Linking Molecular Biology and Radiation Epidemiology J.C. Kaiser Helmholtz Zentrum München, Institut für Strahlenschutz, Munich, Germany

Nuclear and Radiation Studies Board Planning towards the BEIRVIII report Washington DC, 17 November 2014

Content

• Radiation biomarkers for use in epidemiological studies Summary of a multi-disciplinary European study

Two examples:

• 1. Thyroid cancer: Increased radiation risk in children is associated with overexpression of the CLIP2 gene presented material is partly unpublished

• 2. Colon cancer: Radiation risk in the two main molecular pathways Kaiser et al. 2014, PLoS ONE 9(10):e111024

• Concluding remarks

Timing of radiation-induced disease processes and relation to biomarker types

Pernot et al. Mutat. Res. 2012

Biomarkers of - Exposure suitable for dose estimation - Susceptibility predict increased risk of radiation effects notably before exposure - Late effects assessment of health effects long after exposure but before clinical detection - Persistent effect assessment of radiation effects long time after exposure

Overview of radiation biomarkers • cytogenetic biomarkers • biomarkers related to

nucleotide pool damage and DNA damage

• biomarkers related to germline inherited mutations and variants

• biomarkers related to induced mutations

• biomarkers related to

transcriptional and translational changes

• biomarkers related to

epigenomic modifications • other biomarkers, including

biophysical markers of exposure

Pernot et al. Mutat. Res. 2012

Discussion on the potential use of radiation biomarkers in epidemiological studies

Only a limited number of studies have stored biological samples (Mayak workers, Chernobyl children, LSS)

Clear potential to identify suitable biomarkers of susceptibility by GWAS

Can biomarkers provide inside into the shape of the risk-response curve? Yes, by interfacing molecular analysis with epidemiological studies

This talk: two examples of thyroid cancer and colon cancer partly funded by the European Commission in FP7-Project

Pernot et al. Mutat. Res. 2012

Papillary thyroid cancer (PTC): Increased radiation risk in children is associated with overexpression of the CLIP2 gene Incidence data of the Ukrainian-American (UkrAm) cohort presented material partly unpublished

Radiation-associated DNA gain in chromosomal band 7q11: Overexpression of the CLIP2 gene in PTC

DNA mRNA

protein

Selmansberger et al. 2014

Genrisk-T cohort: Comparison of exposed vs. unexposed cases UkrAm cohort: Dose estimates available CLIP2 biomarker is a binary variable

Hess et al. PNAS 2011

1st neighbours of CLIP2 interactome suggest that CLIP2 is involved in chromosomal/genomic instability, a hallmark of cancer development

Hanahan and Weinberg, Cell 2011

CLIP2 as surrogate for genomic instability

CLIP2 overexpression in PTC tissue represents a surrogate marker of genomic/chromosomal instability

Unpublished material deleted

Dose response of probability for positive CLIP2 marker at age at exposure (AaE) < 5 yr and ≥ 5 yr

Mechanistic risk model

Radio-epidemio logical cohort

PTC risk after exposure to IR

Molecular biology measurements

Mechanistic risk models link epidemiology and molecular biology

Unpublished material deleted

Radiation path model for radiation-induced PTC

Unpublished material deleted

Link to molecular biology: Comparison of probabilities for positive CLIP2 biomarker

Unpublished material deleted

Link to molecular biology: Comparison of probabilities for positive CLIP2 biomarker

Colon cancer: Radiation risk in the two main molecular pathways Incidence data of Japanese A-bomb survivors 1958-1998 Kaiser et al. 2014, PLoS ONE 9(10):e111024

Mutation frequencies in colorectal cancer

Exome sequencing of tumor and normal tissue pairs from 224 patients

Clear separation of hypermutated and non-hypermutated samples

p

The Cancer Genome Atlas Network Nature 2012

Analysis of genomic changes in 195 colorectal cancers reveals chromosomal instability (CIN) in non-hypermutated tumors

The Cancer Genome Atlas Network Nature 2012

Copy number changes in 22 autosomes red: gain blue: loss

Two molecular pathways to colorectal cancer

• mostly a disease of the right colon • often starts with silencing of mismatch repair gene MLH1 • hyper-mutated tumors (> 10 mutations per 106 bases) • slow growth of (non-polypoid or flat) adenoma • BRAF gene often mutated • hereditary form: HNPCC • 15-20% of sporadic cases in Western countries Front view

• tumors appear in right, left, transverse colon and rectum • often starts with mutations in both alleles of the APC gene • heterogeneous chromosomal copy number and structure (aneuploidy) • faster growth of adenoma (polyps) compared to MSI • hereditary form: FAP • 80-85% of sporadic cases in Western countries

Micro-satellite instability (MSI)

Chromosomal instability (CIN)

Two path model of colon cancer

Kaiser et al. 2014, PLoS ONE 9(10):e111024

Life

styl

e

Radiation target

Radiation target (only men)

Radiation target

Quality of fit: Comparison with descriptive models

Model Reference Parameters Δ AIC

Mechanistic two molecular

pathways Kaiser et al. 2014 11 0

Descriptive ERR Preston et al. 2007 20 29

Descriptive EAR Preston et al. 2007 20 33

Link to molecular biology: Predicted share of cases in the MSI pathway

Men Women Both sexes

Follow-up period

Total cases

MSI share [%]

Total cases

MSI share [%]

Total cases

MSI share [%]

1958-1998 688 18 820 31 1508 25 1958-1980 161 41 206 63 367 53 1981-1998 527 11 614 21 1141 17

Observed (e.g. TCGAN

2012): 15-20%

• Imbalance between number of cases in MSI and CIN emerged in 1980s • Biological explanation: Rate of CIN events λCIN increased exponentially for subsequent birth cohorts • Case shares possibly determined by westernization of the Japanese diet

Link to molecular biology

Biological process Estimated rate per stem cell and year

Independent estimate from biological data

Value Reference APC mutation (single hit) 1.2 x 10-5 10-5 Hornsby et al. 2008

MLH1 methylation (single hit) 1.2 x 10-5 2 x 10-5 Herrero-Jiminez et al.

2000 Crypt fission (Monocryptal

adenoma) 0.057 0.06 – 0.03 Humphries et al.

2008

CIN event (leading to aneuploidy)

0.02 - 1 0.2 Lengauer et al. 1997 (in vitro)

Comparison of rates for biological processes which are accessible to independent investigation

Link to epidemiology: Subtype-specific incidence in dependence of age

Most of early incidence is MSI

Most of late incidence is CIN

females Total incidence males

Link to epidemiology: Radiation risk coefficients

ERR EAR per 104 PY Model Men Women Men Women

Descriptivea

0.77 (0.29; 1.4)

0.33 (0.066; 0.70)

14 (6.3; 24)

2.8 (-0.23; 6.4)

Two path

0.54 (0.32; 0.95)

0.13 (0.073; 0.35)

10 (5.3; 16)

1.3 (0.67; 3.2)

MSI path

0.39 (0.21; 0.68)

0.94 (0.42; 1.7)

CIN path

0.56 (0.31; 1.1)

0.047 (0.0095; 0.27)

9.1 (4.4; 15)

0.34 (0.057; 2.2)

aPreston et al. 2007

Maximum likelihood estimates (95% confidence intervals in brackets) for persons of attained age 70, exposed to 1 Gy at age 30

• Risk estimates from Two Path model slightly lower than from descriptive model • 64 (of 1508) cases induced by radiation (54 in CIN, 10 in MSI)

Concluding remarks

• „Good“ biomarkers should be indicators of key pathogenic processes: genomic/chromosomal instability, deregulated signalling pathways, growth of pre-neoplastic lesions and tumors

• Molecular data needed for long time periods between radiation exposure and tumor growth (analysis of healthy tissue)

• Mechanistic modelling can be helpful in linking molecular biology and radiation epidemiology

• Close collaboration of epidemiologists, dosimetrists, biologists and modellers is essential

Acknowledgments

Helmholtz Zentrum München Institut für Strahlenschutz & Abteilung für Strahlenzytogenetik Peter Jacob Horst Zitzelsberger Jan Christian Kaiser Kristian Unger Markus Eidemüller Julia Hess Reinhard Meckbach Martin Selmansberger Denise Güthlin

Johannes-Gutenberg Universität Mainz Institut für Medizinische Biometrie, Epidemiologie und Informatik Maria Blettner

Process-oriented integration approach

• Focus on key events in molecular biology such as: gene overexpression, chromosomal instability, growth of pre-neoplastic lesions, tumor growth • Design blueprints for mechanistic models which describe carcinogenesis as a progression of key events • Integrate molecular data into mechanistic models • Identify molecular pathways in cancer incidence data • Derive radiation risk estimates from mechanistic models validated with both molecular data and goodness-of-fit criteria

CLIP2 gene regulatory network

• Interaction with RGS4/ BAG2/NEURL1 leads to deregulation of MAPK signalling

• KIF3C: link to

“genomic/chromosomal instability“

• Validation (qRT-PCR) of all CLIP2 interactions except for GOLM1

Reconstructed CLIP2 network: global mRNA expression data of UkrAm cases (Abend et al. PONE 2012) – 1st neighbours

MAPK signaling

Protein-folding Apoptosis

Golgi-apparatus

Kinesin-family

Microtubule associated complex

RNA splicing protein-folding

Notch signaling Apoptosis

GTPase activator MAPK

inactivation Golgi-

apparatus

Selmansberger et al. 2014

Life Span Study of Japanese a-bomb survivors: Colon cancer incidence 1958 - 1998

Item Males Females Both sexes

Cohort members 42,762 62,384 105,146

Mean colon dose (mGy) 85 81 83

Mean dose of cases (mGy) 142 92 115

Colon cancer cases 688 820 1508

Mean age at diagnosis 67 71 69

RERF data set lssinc07.csv Preston et al. Radiation Research 2007

Key elements of a mechanistic model for radiation-induced carcinogenesis in the colon

Radiation epidemiology with covariables • Attained age • Birth cohort / Age at exposure • Gender • Radiation dose

Key elements must leave imprints in the incidence data

Molecular biology • Two molecular pathways of CIN and MSI • Early silencing of tumor supressor genes (TSGs: APC or MLH1) • Crypt fission (renewal of healthy of colonic tissue) spreads TSG -/- • Subtype-specific impact of lifestyle trends • Subtype-specific adenoma growth • Subtype-specific radiation action

Secular trend of colon cancer incidence in Japan

Increase of colon cancer incidence after 1950 reaching US level in mid-1990s Possibly due to a westernization of the Japanese diet Yiu et al. Int J Cancer 2004

Red meat intake inversely associated with high-level MSI tumors, and positively associated with low-level MSI or CIN tumors in European populations Diergaarde et al. Cancer Epidemiol Biomarkers Prev 2003

Excess Relative Risk

black: descriptive red: Two Path model green: CIN path blue: MSI path

Estimates with 95% confidence intervals for persons exposed to 1 Gy at age 30

Sojourn time

Time scale of adenoma developement in the colon

LSS SEERa

Male 52 yr 51 yr Female 58 yr 49 yr

Sojourn time from the birth of a small adenoma (containing CIN properties and surviving extinction) to the birth of the first malignant cell)

aSEER cohort from NCI Luebeck et al. 2013

Availability of experimental data

Healthy organ irradiation tumor

Wealth of data from radiation experiments

Growing pool of molecular data (TCGAN)

Lack of data in during pathogenesis

(deregulated signalling pathways, genomic instability, growth advantage of pre-neoplastic cells)