Approved projects
DNA methylation patterns and radiation induced papillary carcinoma
Validation of the gene signature differentiating exposed from non-exposed PTCs, obtained in the Genrisk-T project (no.: 036495) with an independent QPCR method
EpiRadBio - integrative analysis of molecular data
A Sequence-based Approach to Identify Genetic Determinants of Tumorigenesis in Radiation-Induced Pediatric Papillary Thyroid Carcinomas
EpiRadBio - Validation of radiation-associated gain of chromosome band 7q11
Molecular specificities of radiation-induced thyroid tumors
EpiRadBio
Expression profiling of tyrosine kinase genes
in malignant tumours of the
thyroid gland.
Chernobyl, an Integrated Pan-European Study,
morphology, oncogenes, DNA repair and outcome in radiation
carcinogenesis.
RET proto-oncogene rearrangements and
tyrosine kinase gene expression in radiation induced papillary
thyroid carcinomas developed after the Chernobyl accident.
Mitochondrial DNA deletions and mutations
in post Chernobyl thyroid tumours and in the respective normal
thyroid parenchyma.
Expression of the RET proto-oncogene in post-Chernobyl
thyroid tumors and unirradiated controls: analysis of RET/PTC
rearrangements, RET wild type and RET-TK domain, with quantitative
assessment of mRNA and protein expression.
Analysis of RET/PTC transforming
ability in Thyroid cells using Oligonucleotide DNA Micro Array
A Comprehensive Molecular Profile
of ChildhoodRadiation Induced Papillary Thyroid Tumors Compared
to Adult Sporadic Papillary Tumors
A comprehensive analysis to find
out molecular biomarker(s) of radiation exposure and grade
of malignancy in human post-Chernobyl PTC
Molecular Change and Thyroid Cancer
Risk after Chernobyl
Analysis of Genetic and Epigenetic
Abnormalities in Radiation-induced Thyroid Cancers
Investigation of molecular genetic abnormalities
associated with progression of human thyroid follicular neoplasms
Molecular Definition of Gene Expression
in Chernobyl Thyroid Cancers
Genomic Analysis of Gene Copy Number in Thyroid
Cancer
The influence of genetic variation in DNA
repair pathways on cancer risk following exposure to ionising
radiation
Array CGH analysis of RET/PTC-positive and
RET/PTC-negative post-Chernobyl thyroid tumours
Expression profiling of childhood thyroid
cancer: a comparison of those exposed to radioiodine and those
exposed to low level radiocaesium
Gene expression in normal and cancerous tissue
in relation to I-131 exposure
Genetic predisposition to radiation-induced
carcinogenesis and to specific genetic alterations in post-Chernobyl
thyroid cancer.
Genrisk-T – defining the risk of low
dose radiation for thyroid cancer – the role of germline
SNPs.
Expression profiling of childhood follicular
tumours: a comparison of those exposed and not exposed to
radiation. Defining the genetic component of thyroid cancer
risk at low doses.
Array CGH analysis of follicular post-Chernobyl
thyroid tumours
Defining the genetic component of thyroid
cancer risk at low doses – request for RNA aliquots
for QPCR validation and for the exon arrays.
Identification of somatically acquired rearrangements in post-Chernobyl
pediatric thyroid cancers using genome-wide massively parallel
paired-end sequencing
miRNA profiles in childhood thyroid cancer
Project reference number 001/2001
Principal Investigator:Dr Heinz-Ulrich Weier, LBNL, University
of California, Berkeley Email: ugweier@lbl.gov
Expression profiling of tyrosine kinase genes in malignant
tumours of the thyroid gland.
Summary of project:
Abnormal expression of tyrosine kinase (tk) genes is a common
phenomenon in papillary thyroid carcinomas (PTCs), where it
is believed to alter cell growth and response to external
signals such as growth factors, hormones etc. While the pathogenesis
of radiation-induced PTC remains unclear, there is evidence
that tk genes such as the receptor tyrosine kinases ret and
NTRK-1 are abnormally expressed, and that the overexpression
of some tk genes due to gene amplification or changes in gene
regulation in the absence of structural alterations may lead
to oncogenic transformation of cells. Using a DNA microarray
based technique, we have identified several tk genes with
abnormal expression in human tumour cell lines. We now propose
to apply the technique to measure the relative expression
levels of more than 50 tk genes in the PTC's that arose after
the Chernobyl nuclear accident and to compare these expression
profiles with the gene expression pattern found in sporadic
PTC cases and tumors that arose following low level therapeutic
irradiation of the thyroid. Results from this study may allow
the identification of molecular markers that can be used to
facilitate tumor diagnosis and staging, and, eventually, provide
targets for therapeutic intervention.
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Project reference number 002/2001
Principal Investigator:
Dr GA Thomas, South West Wales Cancer Institute, University of Wales, Swansea, UK.
Email: gerry@mynydd-p.u-net.com
Chernobyl, an Integrated Pan-European Study, morphology, oncogenes, DNA repair and outcome in radiation carcinogenesis.
Summary of project
The objective of this study is to investigate the link between exposure of children to radiation, the subsequent development of tumours and how their morphology, molecular and cell biology influence clinical outcome. The project is an integrated approach involving 5 leading European centers. Samples of the same tumours will be studied by the 5 different centers to determibe tumour morphology and type; the degree of variation within the tumour, including the variation of the proportion of cells in cycle using antibodies to novel DNA replication associated peptides; the gene involved in the carcinogenic process, using DNA chip technology; specific studies of the pathways associated with one oncogene (ret) known to be linked to the tumour type involved; and studies of novel gene rearrangements using FISH technology. By using the same tumour/normal pairs in these studies, integrating the results from the different centers and studies and correlating these with detailed morphological analysis and patient details including evidence on tumour aggressiveness and recurrence, we will increase our understanding of the link between radiation exposure and cancer development, and provide evidence which will inform decisions on radiation protection and on clinical management of patients with radiation
associated cancers.
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Project reference number 004/2001
Principal Investigator: Professor Horst Zitzelsberger, GSF, Munich, Germany
Email: zitzelsberger@gsf.de
RET proto-oncogene rearrangements and tyrosine kinase gene expression in radiation induced papillary thyroid carcinomas developed after the Chernobyl accident.
Summary of project
The papillary thyroid carcinoma oncogene (RET/PTC) is a rearranged version of the tyrosine kinase RET. It is know that the incidence of RET/PTC activation is increased in radiation-induced papillary thyroid carcinomas compared to papillary thyroid carcinomas without a radiation history. The prognostic value of RET/PTC rearrangements and its importance as a radiation-specific marker is still unclear. It is proposed to screen 70 childhood cases from Belarus and Ukraine with interphase FISH in conjunction with RT-PCR to confirm the presence and type of the chimeric transcripts. Cases with indications for atypical RET/PTC rearrangements will be further investigated using 5'RACE for the presence of novel types of alteration. For this part of the proposal RNA samples as well as a limited number of paraffin-embedded sections would be needed. The RNA samples will be further investigated for expression profiles of other tyrosine kinase genes to identify other gene rearrangements which may occur as a sole abnormality or in addition to RET/PTC rearrangements.
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Project reference number 005/2001
Principal Investigator: Professor Manuel Sobrinho-Simoes, IPATIMUP, Porto, Portugal
Email: sobrinho.simoes@ipatimup.pt
Mitochondrial DNA deletions and mutations in post Chernobyl thyroid tumours and in the respective normal thyroid parenchyma.
Summary of project
Mittochondrial DNA (mtDNA) is a likely hotspot for mutation in cancer as it is preferentially modified by many carcinogens. We have previously shown that there is a specific association between sequence variants of Complex I genes and ATPase6, one of the two mitochondrial genes of Complex V, and the occurrence of malignancy and of oxyphil features in thyroid tumours. We have also found a significant association between mitochondrial sequence variants and the occurrence (and degree) of the so-called mitochondrial common deletion. In an attempt to elucidate the role of post-Chernobyl irradiation in mtDNA alterations and to find out whether or not such alterations are involved in the etiopathogenesis of thyroid tumours we will search for the mtDNA common deletion and for somatic mutations and sequence variants in the D-loop region, in the 13 coding genes and in the 22 tRNAs genes of cases from which there are RNA and DNA samples extracted from blood (set “without radiation”), normal thyroid (set “irradiation”) and tumours (set “irradiation and tumourigenesis”). In a first step, we will study exhaustively 20 cases. The results obtained in this first part will be used together with the data we and others have previously obtained to decide the most appropriate targets for the second part of the study. If possible we would like to correlate the results of our study on mtDNA deletions and mutations with those on ret oncogene. In case there are also clinico-pathyological data available, we would like to collaborate with the pathologists who have studied the cases in order to clarify the putative clinical significance of the mtDNA alterations.
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Project reference number 007/2001
Principal Investigator: Professor Aldo Pinchera, University of Pisa, Italy
Email: a.pinchera@endoc.med.unipi.it
Expression of the RET proto-oncogene in post-Chernobyl thyroid tumors and unirradiated controls: analysis of RET/PTC rearrangements, RET wild type and RET-TK domain, with quantitative assessment of mRNA and protein expression.
Summary of project
Genetic alterations of the ret proto-oncogene play a critical role in the pathogenesis of papillary thyroid carcinomas both naturally occurring and radiation induced. We have recently found that classical RET/PTC rearrangements are present also in benign thyroid nodular disease. Furthermore, in several cases the independent expression of tyrosine kinase (TK) and extracellular (EC) domains of RET was found. This finding may be interpreted as RET wild type gene expression. In other cases, especially radiation exposed, the TK domain in the absence of EC was found and was interpreted as unknown RET/PTC rearrangements (PTCX). Aim of this project is to clarify the modality of expression of RET protooncogene in nodular thyroid diseases. In cases with EC and TK expression, we will search for RET wild type by an extralong PCR encompassing the TK and EC domains, followed by sequencing of the PCR product. In cases of TK positive expression only (but not classical PTC1, PTC2 and PTC3) we will identify the 5' domain rearranged with the TK domain. As for other unknown RET/PTC rearrangements will use the 5'race approach.
All samples positive for TK expression will be submitted to quantitative PCR (ABI Prism 7700 sequence detector, Perkin Elmer) for TK mRNA. The TK mRNA expression will be correlated with the histological characteristics of the analysed tissues, and the immunohistochemical pattern of RET protein expression, using an antibody to recognize the TK domain.
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Project Reference Number 001/2002
Principal Investigator: Dr Giuliana Salvatore, University of Naples, Italy
Email: gsalvato@unina.it
Analysis of RET/PTC transforming ability in Thyroid cells using Oligonucleotide
DNA Micro Array
Summary of the project
The expression of RET/PTC oncogenes in thyroid PC Cl 3 cells induces a complex phenotype with a block of the differentiation program, hormone-independent proliferation and increased apoptotic rate. Oligonucleotide GeneChips were used to analyse gene expression profiles of PC Cl 3 cells expressing either RET/PTC1 or RET/PTC3 oncogenes in comparison to parental cells. About 2,000 genes showing at least two-fold increase and 2,000 genes showing at least two-fold decrease were identified in RET/PTC-expressing thyrocytes. Virtually all the genes up-regulated by more than 5-folds (about 100) were confirmed by RT-PCR and some of them by immunoblot. Genes upregulated by RET/PTC could be functionally divided in genes involved in proliferation (such as D-type cyclins), apoptosis, proteolysis, inflammation, and metabolism. We plan to extend these studies on the identified genes up and on the downregulated by analyzing their expression in human thyroid tumors of different histotypes. We propose to study their expression by semiquantitative reverse transcriptase –PCR and for selected genes by real time quantitative PCR. Immunohistochemical analysis will be use to verify protein expression.
The findings of these studies can reveal clues to the molecular pathways involved in papillary thyroid carcinoma and may provide biomarkers for clinical use.
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Project Reference Number 002/2002
Principal Investigator: Dr Lesleyann Hawthorn, Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, USA
Email: lesleyann-hawthorn@roswellpark.org
A Comprehensive Molecular Profile of ChildhoodRadiation Induced Papillary Thyroid Tumors Compared to Adult Sporadic Papillary Tumors
Summary of project
Papillary thyroid carcinoma (PTC) accounts for 80% of all thyroid malignancies. It has a variable disease course and to date no pathways or specific genes have been implicated as causative in this tumor. RET activation, through translocations involving several genes, have been noted in a high incidence of PTCs. However, the activation of this oncogene is found at all stages from benign through well-differentiated to undifferentiated carcinoma. This suggests that it represents an early event and that this defect is not in itself sufficient for carcinogenesis. It may also suggest that the classification of PTC covers more than one tumor subtype. The relationship between radiation exposure and PTC is well established. We plan to perform a genome wide scan using microarray analysis for alterations in tumors from children exposed to radiation and compare them to sporadic adult tumors to identify which genes are commonly altered and which genes are display differential alteration expression patterns. We plan to extend this study using high-resolution BAC-CGH to define a molecular pattern for these tumors and evaluate this approach for diagnostic applications. The study of molecular alterations which cause thyroid carcinoma is of importance since the identification of causative factors could lead to new approaches for treatment.
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Project Reference Number 001/2003
Principal Investigator: Dr Hiroyuki Namba, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan
Email: namba@net.nagasaki-u.ac.jp
A comprehensive analysis to find out molecular biomarker(s) of radiation exposure and grade of malignancy in human post-Chernobyl PTC
Summary of project
Development of papillary thyroid cancer (PTC), similarly to that of most of other human malignancies, is likely to comprise a multistep and multihit process. It is quite probable that mutational events initiating, promoting and/or driving the tumor progression are quite similar in the sporadic and radiation-induced PTC. Along with this, one may expect there may be unidentified to date molecular distinctive features peculiar to thyroid cancers of different etiology. Thus, a comparative study of various molecular characteristics in the two groups of PTC may provide additional information for the determination of the ”molecular signature” of radiation-induced thyroid cancerogenesis.
In the proposed project we intend to study the following molecular characteristics of the DNA extractedfrom normal and tumor tissue of radiation-induced PTCs: i) relative content of mtDNA and number of large-scale deletions in mtDNA; ii) prevalence of gene mutations of MAPK signal molecules, including the Ras,BRAF, Raf-1 and MEK genes; and iii) distribution of the codon 72 allelic variants of the TP53 .
After the data are obtained, we will perform a comprehensive univariate and multivariate statistical analysis against already available results of examination of sporadic PTC in order to identify molecular parameter(s) specific to
radiation-induced PTC.
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Project Reference number 002/2003
Principal Investigator: Professor Scott Davis, Fred Hutchinson Cancer Centre,
Seattle, USA
Email: sdavis@fhcrc.org
Molecular Change and Thyroid Cancer Risk after Chernobyl
Project Summary
This study investigates the occurrence and molecular characteristics of thyroid cancer in residents of the Bryansk Oblast of the Russian Federation, who were 0-50 years of age at the time of exposure to radiation from the Chernobyl Power Station accident (ATA) on April 26, 1986. The study has three primary purposes: 1) to characterize cases of thyroid cancer according to specific molecular markers of genetic change, and investigate whether the presence of such markers is associated with individual thyroid radiation dose from the Chernobyl accident; 2) to investigate whether age-at-exposure dependent radiation dose response for thyroid cancer differs between cancers that are positive versus negative for the molecular markers investigated; and 3) to investigate whether the presence of these same molecular markers is associated with clinical outcomes. Included will be thyroid cancer cases diagnosed between April 1, 2001 and March 31, 2006 and confirmed by a panel of expert thyroid pathologists. An equal number of controls will be individually matched to cases by sex, age, type of settlement and raion of residence on April 26, 1986. Data collected will include in-person interviews for all participants, and for cases only, paraffin embedded tissue or fresh frozen tissue, clinical history and outcome information.
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Project Reference number 001/2004
Principal Investigator: Dr Michael M Xing, John Hopkins University School of Medicine, Baltimore USA
Email: mxing1@jhmi.edu
Analysis of Genetic and Epigenetic Abnormalities in Radiation-induced Thyroid Cancers
Project Summary Thyroid cancers are the most common endocrine malignancies and the vast majority of them are papillary thyroid cancers (PTC). Several genetic abnormalities, including Ras mutations and RET/PTC rearrangements have been well characterized in these cancers. Recently, we and several other groups have reported the BRAF mutation in PTC with a high prevalence. We have also characterized aberrant DNA methylation in several genes in thyroid cancers, including novel tumour suppressor genes and some thyroid-specific genes. Except for the RET/PCT rearrangements, these genetic and epigenetic abnormalities have been studied mainly in sporadic thyroid cancers, and their role is unknown in the special group of thyroid cancers induced by radiation, the most common and well-established environmental risk factor for thyroid cancer. Chernobyl nuclear accident has been associated with a significant increase in the incidence of PTC, which represent an ideal thyroid tumour model for the study of radiation-induced thyroid tumorigenesis. In the present project, we propose to use such special thyroid cancer samples to study novel genetic and epigenetic abnormalities, their relationship, and their effects on the expression of key thyroid genes. Well-established experimental protocols and techniques, including RT-PCR, methylation-specific PCR, real-time quantitative PCR, and a recently established colorimetric mutation detection method will be used. The study is expected to result in important insights into radiation-induced thyroid tumorigenesis and provide novel clinical implications for this special group
of thyroid cancers.
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Project Reference number 002/2004
Principal Investigator: Professor D Wynford-Thomas, University of Wales College of Medicine, Cardiff UK
Email: kingTD@cardiff.ac.uk
Investigation of molecular genetic abnormalities associated with progression of human thyroid follicular neoplasms
Project Summary
Thyroid follicular carcinomas frequently exhibit RAS mutation, and closely resemble benign follicular adenomas with respect to morphology and differentiation. Cell culture studies suggest that at least one requirement for progression from an adenoma to a carcinoma is failure of an intrinsic mechanism that normally limits the proliferative lifespan of RAS-induced cell clones. One current candidate for over-riding the mechanism is the tumour suppressor gene p16 INK4a .
There is great clinical interest in this area, as currently there is no marker to distinguish between thyroid follicular adenoma and carcinoma when evaluating fine needle aspirates and biopsies of thyroid glands. This means that many people have unnecessary operations on the basis of presumed malignancy. We now therefore wish to carry out a comprehensive comparison between thyroid carcinoma and adenoma cells to identify differences that may confer an extended proliferative lifespan on carcinoma cells.
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Project Reference Number 003/2004
Principal Investigator: Professor Carine Maenhaut, ULB, Brussels
Email: cmaenhau@ulb.ac.be
Molecular Definition of Gene Expression in Chernobyl Thyroid Cancers
Project Summary
Chernobyl thyroid cancers represent a unique resource in oncology and radiation biology. They appear in a cohort of patients irradiated at the same time and in which there is no doubt that the cancer originated from radiation exposure. The precise timing of the course allows to follow precisely the kinetics of appearance of the cancers. On the other hand study of the gene expression pattern of cancers by microarrays allows a precise molecular definition of each cancer. Using this methodology we were able to show that the clustering of gene patterns of Chernobyl cancers of the first wave and European and US sporadic cases could not separate them. This work should now be extended to a larger series of cases.
1) to try to distinguish subtypes of Chernobyl and sporadic papillary carcinomas and their signature.
2) to relate patterns of gene expression with clinical variables such as the duration of the incubation period and with the genetic diagnosis.
3) to validate data at the RNA level by PCR and at the protein level by Western and to define potential diagnostic markers and therapeutic targets.
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Project reference number 001/2005
Principal Investigator:Dr PE Neiman, FHCRC, Seattle, USA
Email: pneiman@fhcrc.org
Genomic Analysis of Gene Copy Number in Thyroid Cancer.
Summary of project:
High copy-number gene amplification is known to take place at a few genomic loci in numerous human cancers, but widespread low-level copy-number changes in genomic DNA have not been described. cDNA microarray-based comparative genome hybridization yields high-resolution copy-number profiles that enable the detection of low-level amplification events at individual gene loci. We have shown in a small pilot study that pediatric thyroid carcinoma in residents of a region contaminated by 131I from the Chernobyl accident (the Bryansk Oblast) exhibits gene amplification at a higher frequency than that seen in pediatric thyroid carcinoma in US children with no history of radiation exposure. This result suggests that exposure to ionizing radiation from the environment may be associated with an increased rate of gene amplification in a human cancer. The consistent amplification of many genes among cases of post-Chernobyl thyroid carcinoma from Bryansk suggests the existence of a target pool of radiation-sensitive genomic loci that respond to exposure by initiating local amplification events. The pattern of gene amplification may represent a radiation signature that could be used to map amplicons likely to harbor participating oncogenes.
Based upon these differences observed in the pilot study in apparent gene amplification between post-Chernobyl and spontaneous pediatric papillary thyroid carcinoma (PTC), we hypothesize that radiation exposure leaves a measurable genomic signature in the form of stable changes in gene amplification. Some chromosomal regions identified by this method are likely to harbor participating oncogenes, but it is unreasonable to expect that so many genes be directly involved in oncogenesis. We hypothesize the presence of genomic “hot spots” in human DNA that are susceptible to radiation-induced amplification. These genomic targets are unlikely to be saturated by the doses of radiation delivered to these patients. The number of targets hit in each case should therefore be directly proportional to radiation dose.
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Project reference
number 002/2005
Principal Investigator:Dr S Forbes-Robertson, Swansea Medical
School, Swansea UK
Email: Gerry.thomas@imperial.ac.uk
The influence of genetic variation in DNA repair pathways
on cancer risk following exposure to ionising radiation.
Summary of project:
Genetic damage following radiation exposure is subject to
correction by the ‘caretaker’ systems of DNA repair. Our interest
is in the role that these systems may play in the molecular
pathogenesis of cancer. We propose a pilot project to assess
the influence of genetic variation on cancer risk following
exposure to ionising radiation. This will be achieved by investigation
of variation in the genes involved in the DNA repair pathways,
in DNA derived from blood samples or normal tissue samples
from patients with thyroid tumours of radiation-associated
and non-radiation-associated aetiology. There are two main
justifications for such a study, first to identify SNPs which
indicate possession of an ‘at risk’ genotype, and secondly,
to identify the genes in which genetic variation is a significant
modulator of cancer risk. This has particular and wider relevance
to the involvement of DNA repair-associated factors in the
pathology of other cancers as well, such as breast cancer.
Genotyping of genes involved in double-strand break repair
will be performed using a mass spectrometry-based SNPing platform
which has been developed by our group at the University of
Wales, Swansea. Data will be provided to the Chernobyl Tissue
Bank for correlation with pathology and expression of oncogenes
such as ret and BRAF.
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Project
reference number 001/2007
Principal Investigator: Professor H Zitzelsberger, Helmholtz
Zentrum München, Neuherberg, Germany
Email: zitzelsberger@helmholtz-muenchen.de
Array CGH analysis of RET/PTC-positive and RET/PTC-negative
post-Chernobyl thyroid tumours.
Summary of project: It is proposed to study chromosomal imbalances
in post-Chernobyl papillary thyroid carcinomas (PTC) by means
of array CGH using 1Mb BAC arrays. As derived from interphase
FISH experiments RET/PTC rearrangements are heterogeneously
distributed within tumour tissues leading to the assumption
that additional gene alterations may play an important role
in these tumours. To address this question post-Chernobyl
PTC, with and without RET/PTC rearrangements, will be analysed
by array CGH. Altered candidate genes will be derived from
recurrent regions of amplifications and deletions and will
be confirmed by interphase FISH on paraffin sections and further
studies by PCR-based approaches to investigate expression
of these genes. In a first pilot study it is intended to compare
10 RET/PTC-positive (RET/PTC3) and 10 RET/PTC-negative cases
with similar histological features, a comparable age range
of patients at time of exposure and a similar latency after
exposure is a first study. The pilot study will use cases
from the age-matched series with known RET/PTC status. If
successful this study will be extended to a larger series
of cases linked to the GENRISK-T project.
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Project
reference number 002/2007
Principal Investigator: Professor Carine Maenhaut, University
of Brussels School of Medicine, Brussels, Belgium
Email: cmaenhau@ulb.ac.be
Expression profiling of childhood thyroid cancer: a comparison
of those exposed to radioiodine and those exposed to low level
radiocaesium
Summary of project: The aim of this study is to investigate
whether different transcriptomic profiles can be related to
exposure to radioiodine in fallout from the Chernobyl accident
and to lower level radiocaesium exposure present in the contaminated
environment. We will use Affymetrix microarray technology
to define transcriptomic profiles in two cohorts of children,
matched on age, oblast and pathological type of tumour. The
research will be carried out in two separate laboratories
and cross-validated. This project is one of a series of projects
that will study the transcriptomic and genetic profile of
two well-defined cohorts to investigate the relative effects
of radioiodine and radiocaesium exposure on the development
of thyroid cancer.
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Project reference number 003/2007
Principal Investigator: Dr M Abend, Bundeswehr Institute of
Radiobiology, Munich, Germany
Email: michaelabend@bundeswehr.org
Gene expression in normal and cancerous tissue in relation
to I-131 exposure
Summary of project: Gene expression has received less attention
than the role of germline polymorphisms or somatic mutations
in studies of radiation and thyroid cancer. The increase in
papillary thyroid cancers (PTC) in exposed children following
the Chernobyl nuclear accident presents an opportunity to
pursue the role of gene expression further. Recently, we reported
on expression of seven genes, each of which was able to distinguish
post-Chernobyl PTCs from sporadic PTCs (Port et al 2007).
Our approach involved (i) a whole genome microarray used for
screening purposes; and (ii) quantitative examination of the
92 target genes with a high throughput RTQ-PCR technique (LDA).
The study had some limitations such as the origin of sporadic
PTCs (Eastern Germany), their different age at diagnosis and
a small number of cases (n=11). Subsequently another group
has reported on expression of thirteen genes involved in homologous
recombination suggesting a distinct radiation pattern of post-Chernobyl
PTCs (Detours et al 2007). Using the already established 2-stage
design, the purpose of the present application is to overcome
limitations in the previous study and extend the findings
(Port et al 1007) by evaluating a dose-dependent gene expression
pattern in 74 post-Chernobyl PTCs with individual I-131 dose
estimates.
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Project reference number 004/2007
Principal Investigator: Dr Y Nikiforov, University of Pittsburgh
School of Medicine, Pittsburgh, USA
Email: nikiforovye@upmc.edu
Genetic predisposition to radiation-induced carcinogenesis
and to specific genetic alterations in post-Chernobyl thyroid
cancer.
Summary of project:
Radiation exposure is a well established risk factor for thyroid
cancer. Ionising radiation is known to cause extensive DNA
damage including double strand breaks, which may lead to the
generation of somatic mutations in thyroid cells and cancer
initiation. However, environmental triggers cannot fully explain
the inter-patient heterogeneity in the individual response
to exposure to radiation, which points to the existence of
genetic variations that define the individual susceptibility
to radiation-related cancer. We propose to analyse several
candidate DNA repair genes and perform a genome-wide analysis
of single nucleotide polymorphisms (SNPs) in Ukrainian patients
who developed thyroid cancer after Chernobyl and in control
cancer-free individuals to identify mutations and SNPs that
are involved in genetic predisposition and to identify the
genes that are affected. We will then perform functional analysis
to find whether these genetic variations alter gene function.
We will also study the link between specific SNPs and known
or new mutations found in these tumours. The overall goal
of this study is to identify genes involved in genetic predisposition
to radiation-associated thyroid carcinogenesis and a pattern
of SNPs that can detect it.
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Project reference number: 001/2008
Principal Investigator: Professor GA Thomas, Imperial College
London, Hammersmith Hospital, London
Email: Gerry.thomas@imperial.ac.uk
Genrisk-T - defining the risk of low dose radiation for thyroid
cancer - the role of germline SNPs.
Summary of project:
Cancer of the non-medullary (follicular epithelium) component
of the thyroid is induced by external irradiation and by radionuclides
deposited within the thyroid tissue. Estimates of the radiological
risk of developing thyroid cancer are derived from epidemiological
studies performed in. Populations receiving high doses where,
according to Ron et al, the threshold in 100 mSv. Extrapolation
of this risk to exposures at much lower doses is compromised
by the lack of an accurate model of the dose response curve
for thyroid cancer at low doses. Moreover, such population
based estimates fail to take into account the contribution
of individual genetic variability to the risk estimate. Individuals
with an increased genetic predisposition to develop thyroid
cancer are not identified, and it is precisely these individuals
who will be at greatest risk at low doses. The GENRISK-T consortium
is composed of thyroid cancer experts with experience in the
fields of radiation biology, animal models of radiation-induced
cancer, tumour banking, cancer biology, molecular genetics,
histopathology, cyto genetics and risk modelling. We will
use this interdisciplinary knowledge to define the genetic
component influencing the risk of radiation-induced thyroid
cancer. This will be achieved through a combination of studies
using animal models and in human radiation-induced thyroid
tumours. This new understanding of the genetic risk modifiers
will be used to develop an animal model of thyroid cancer
that is responsive to low dose radiation in the cGy range,
thereby providing an experimental solution to resolving the
uncertainties of the low dose-response curve. This EC collaborative
project (PI Professor M Atkinson, Helmholtz Zentrum, Munich)
combines the use of animal models and human studies. This
particular application is to support the investigation of
human germline SNPs that may predispose to radioiodine induced
thyroid cancer in those exposed as children and adolescents.
Project
reference number 002/2008
Principal Investigator: Professor B Jarzab, Maria Sklodowska-Curie
Memorial Cancer Centre and Institute of Oncology, Gliwice,
Poland
E-mail: bjarzab@io.gliwice.pl
Expression profiling of childhood follicular tumours: a comparison
of those exposed and not exposed to radiation. Defining the
genetic component of thyroid cancer risk at low doses.
Summary of project:
The aim of the study is to investigate transcriptomic profiles
of follicular thyroid tumours (malignant and benign) that
arose after expose to radioiodine fallout from Chernobyl power
station and to low level radiocesium exposure present in the
contaminated environment. Affimetrix microarray technology
will be employed to define transcriptomic profiles in two
cohorts of children matched on age, oblast and pathological
type of tumour. This project is the continuation of a series
of projects that are carry on to investigate the transcriptomic
and genetic profile of radiation induced thyroid cancer.
Project
reference number 003/2008
Principal Investigator: Professor H Zitzelsberger, Helmholtz
Zentrum München, Neuherberg Germany
E-mail: zitzelsberger@helmholtz-muenchen.de
Array CGH analysis of follicular post-Chernobyl thyroid tumours
Summary of project:
Follicular thyroid cancers are less frequent than papillary
thyroid carcinomas (PTC), however, they are associated with
a poorer survival outcome than PTC. Although several genetic
changes have been identified so far, the molecular genetic
mechanisms of tumour development in follicular thyroid neoplasms
are still unclear.
To investigate novel gene alterations and potential radiation
signatures in follicular thyroid adenomas (FA) and follicular
thyroid carcinomas (FTC) it is proposed to investigate genome-wide
copy number changes of 100 thyroid tissue samples by array
CGH using 1 Mb BAC arrays. For this purpose we want to compare
genomic profiles of tumours (FA and FTC) developed pre- and
post-fallout of the Chernobyl accident. The proposed study
aims to identify gene alterations in follicular thyroid neoplasms
from altered genomic regions and to determine aberrations
patterns that correlate with the radiation history of patients
as well as with any of the clinical phenotypes of the tumours.
Project
reference number 001/2009
Principal Investigator: Professor B Jarzab, Maria Sklodowska-Curie
Memorial Cancer Centre and Institute of Oncology, Gliwice,
Poland
E-mail: bjarzab@io.gliwice.pl
Defining the genetic component of thyroid cancer risk at low
doses – request for RNA aliquots for QPCR validation
and for the exon arrays.
This project is an expansion of project 002/2008
Summary of project:
The aim of the study is to seek for differences in transcriptomic
profiles of childhood papillary thyroid cancers that arose
after radiation exposure from Chernobyl power station fallout
and sporadic cancers. As the first step Affimetrix microarray
technology was employed to define transcriptomic profiles
in two cohorts of children matched on age, oblast and pathological
type of tumour. The second step of the study is to validate
microarray results with Q-PCR analysis. We also plan to extent
the analysis to exon microarray study which allow to detect
transcript isoforms, chromosomal deletions and amplifications.
Project reference
number 002/2009
Principal investigator: Professor JA Fagin, Memorial Sloan-Kettering
Cancer Center, New York, USA
E-mail: faginj@mskcc.org
Identification of somatically acquired rearrangements in post-Chernobyl
paediatric thyroid cancers using genome-wide massively parallel
paired-end sequencing
Summary of project:
Post-Chernobyl pediatric thyroid cancers are associated with
a high frequency of recombination events leading to the generation
of fusion oncogenes, resulting in aberrant expression and
activation of RET, and less frequently of NTRK and BRAF. Altogether,
~60% of PTC arising in this patient population harbor one
of these abnormalities. The discovery of novel somatic rearrangements
using conventional methods has low sensitivity and/or resolution.
We propose to use genome-wide parallel paired-end sequencing
to identify somatic rearrangements in childhood thyroid cancers
induced by radiation, as compared to age-matched thyroid cancers
without radiation exposure. Altogether we will select 6 samples
of each population: 2 with a known rearrangement in RET, NTRK
or BRAF (as positive controls), and 4 without. We will construct
3kb insert Illumina libraries from each tumor DNA sample,
and ~35bp of sequence from both ends of each fragment will
be obtained. Each end will be mapped back to the reference
genome. Fragments for which the ends do not map back within
3kb of each other and/or are in inappropriate orientation
will be further studied as they may represent rearrangements
present in the thyroid cancer genome. We will acquire ~1 fold
genome coverage (~3Gb) from each sample. This will approximate
to 30-fold physical coverage, allowing detection of essentially
all rearrangements present in the dominant clone of the cancer.
Rearrangements will be processed using a suite of informatics
tools to predict which may generate an in-frame fusion gene.
RNA from each of the samples will then be tested by exon-exon
PCR to determine whether the fusion is expressed, and based
on its predicted function, whether it may correspond to a
driver mutation. The number of rearrangements in each cancer
and their architecture will then be compared between the two
classes. Sequences at the rearrangement junctions will also
be compared particularly to examine the complexity of the
rearrangement, sequence contexts of breaks, presence of repeats
and overlapping microhomology of the rearrangement. Each of
these indices may provide clues to the way large radiation
doses induce DNA double strand breaks and how they are repaired.
Project reference
number: 003/2009
Principal Investigator: Professor GA Thomas, Imperial College
London, Hammersmith Hospital, London
Email: Gerry.thomas@imperial.ac.uk
miRNA profiles in childhood thyroid cancer
Summary of project:
MicroRNAs (miRNAs) are 21-23 nucleotide long non-coding RNA
molecules that have been shown to regulate the stability or
translational efficiency of target messenger RNAs. Dysregulation
of miRNAs has been implicated in a variety of cancers, and
in the thyroid germline SNPs in miRNA binding sites and in
the coding sequence for miRNAs themselves, have been implicated
in Papillary Thyroid carcinogenesis. The Human Cancer Studies
Group is part of an EC sponsored consortium (Genrisk-T) that
has intensively studied papillary carcinomas from a series
of 100 patients, and is currently extending this approach
to follicular tumours. Half of this group were exposed to
radiation and half were born after 1/1/87 and not exposed
to radioiodine in fallout. The cohort is carefully age and
sex matched. The Genrisk-T project has provided data on RNA
expression using Affymetrix technology, bac array CGH data
on copy number variation in the tumours and SNP array data
from normal tissue from these patients. We now seek to add
miRNA data from this cohort and to correlate data on SNPs
and copy number variation in the tumour, with changes in miRNA
level, and miRNA expression levels with changes in RNA expression
levels. The combination of this data will give us a thorough
understanding of the regulation of a number of different growth
control pathways involved in carcinogenesis of the thyroid
follicular cell and their relationship to radiation exposure.
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Project reference number:
001/2011
Principal Investigator: Dr K Unger, Helmholtz Centre, Munich,
Germany
Email: unger@helmholtz-muenchen.de
EpiRadiBio
Summary of project:
The EU funded project EpiRadBio project seeks to model the
cancer of the lung, breast and the thyroid after exposure
to radiation in the low-dose range (cumulative dose < 100
mGy). The formalin-fixed paraffin-embedded (FFPE) papillary
thyroid cancer tissue sections we apply for will be used in
a work package of EpiRadBio that focuses on the cancer risk
of papillary thyroid cancer. In another, recently finished
EU funded project on young onset childhood thyroid carcinomas
that also used CTB material we found that a gain of the chromosome
band 7q11 was associated with exposure to low-dose ionising
radiation (Heß et al., PNAS, in press). The FFPE tumour
sections from patients, which are part of the UkrAm cohort
and which come with estimates on radiation dose the patients
have received will be used for validation and further characterisation
of this marker. The FFPE sections will be used for in situ
hybridisation (FISH) using 7q11 specific probes. DNA and total
RNA will also be extracted from the FFPE sections. The DNA
will be used for high-resolution array CGH and the total RNA
for qRT-PCR mRNA expression analysis of candidate genes from
the gained region. The copy number data and qRT-PCR data will
be integrated with the dose estimates in order to identify
a potential relationship between the radiation-associated
biomarker and radiation dose. The resulting data will be provided
to the modellers of the project who will use this information
to build and refine their models on radiation risk of papillary
thyroid cancer after exposure to low-dose ionising radiation.
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Project reference number:
002/2011
Principal Investigator: Dr C Ory, Commissariat à l’Energie
Atomique, Département Sciences du vivant, Institut
de radiobiologie cellulaire et moléculaire, Laboratoire
de Cancérologie Expérimentale (CEA), France
Email: catherine.ory@cea.fr
Molecular specificities of radiation-induced thyroid tumors
Summary of project:
The constituted network to realise this program involved two
teams well known in diagnosis and treatment of thyroid tumors
and one team which were already implicated in the search of
radiation-induced signatures in the thyroid and in the identification
of the molecular mechanisms of radiation-induced tumorigenesis.
To date, we focused our approach at the transcriptomic level.
We wish now to analyse miRNA and mRNA deregulations in a series
of post-Chernobyl thyroid tumors by microarray analysis: 1)
to identify a radiation-induced miRNA and mRNA signatures.
We will assess the robustness of these signatures for a potential
use as a diagnostic tool alone or in combination. 2) to obtain
an integrated miRNA/mRNA overview of radiation-induced tumorigenesis
by taking advantage of the deregulated pathway identified
by the transcriptomic approach. To date, no such integrated
analysis has been performed as most of the studies focused
on either transcriptome analysis or miRNA analysis separately.
Ultimately, we will cross the obtained data with others results
from analysis of post-radiotherapy tumors (Ory et al. 2011;
Ugolin et al. PLosONE under revision).
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Project reference number:
003/2011
Principal Investigator: Dr K Unger, Helmholtz Centre, Munich,
Germany
Email: unger@helmholtz-muenchen.de
EpiRadBio - Validation of radiation-associated gain of chromosome
band 7q11
Summary of project:
The project using the applied biomaterial is an extension
of the EU-funded GENRISK-T project and aims to validate a
gain on chromosome band 7q11 that was found to be associated
with papillary thyroid carcinomas from young patients (<
19 years) that were exposed to radiation from the Chernobyl
fallout at very young age (median: 2 years). The validation
set containing exposed and unexposed cases will be matched
for age, morphology and residence - these criteria were already
used for case selection of the GENRISK-T set. A high-definition
array CGH platform will be used to validate the gain and to
type for additional radiation-markers that are smaller in
size and were therefore not detectable by 1Mb BAC array CGH
that was used in GENRISK-T. Further, we will use RNA samples
and paraffin sections for characterisation of expression candidate
genes and proteins by qRT-PCR and immunohistochemistry. Fresh-frozen
tissue from selected samples expressing the candidate gene
CLIP2 from the gained region on 7q11 and those that do not
express the gene will be analysed using a whole proteomics
approach (liquid chromatography-tandem mass spectrometry,
LC-MS/MS). The results will be used to identify the dysregulation
networks in which CLIP2 is specifically involved. The projects
aims to validate the radiation marker on chromosome band 7q11,
to identify the networks that are dysregulated in tumours
harbouring the marker and to find new markers that are associated
with exposure to radiation.
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Project reference number:
004/2011
Principal Investigator: Dr L Hawthorn, Georgia Health Sciences
University, USA
Email: lhawthorn@georgiahealth.edu
A Sequence-based Approach to Identify Genetic Determinants
of Tumorigenesis in Radiation-Induced Pediatric Papillary
Thyroid Carcinomas
Summary of project:
Cancer is a genetic disease, a concept which has been consistently
observed for all tumors. Our approach is to survey the entire
genome of these tumors to look for mutations and other perturbations
that are involved in radiation-induced papillary thyroid carcinoma
(RI-PTC) and create a genomic profile of this tumor. A new
technology, termed Next-generation sequencing allows sequencing
of the entire human genome in 8 days. We will begin by sequencing
the DNA of 50 individual RI-PTC tumors from Chernobyl pediatric
patients to look for mutations in genes. We will correlate
this data with RNA sequencing from the same patient
samples. This data will provide information about events that
are taking place at the level of gene expression, providing
information about over and under expressed genes and the expression
of aberrant genes, and gene fusion products. This data will
be compared to RIP-PTC in age matched patients who were not
exposed to radiation to develop a radiation-specific profile.
We have bioinformatic specialists in the group who will integrate
these various kinds of data. This study will provide a comprehensive
profile of RIP-PTC.
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Project reference number:
001/2012
Principal Investigator: Dr W van Wieringen, VU University
Medical Centre, Amsterdam, The Netherlands
Email: w.vanwieringen@vumc.nl
EpiRadBio – integrative analysis of molecular data
Summary of project:
Within the scope of the EpiRadBio-project, Mark van de Wiel,
Carel Peeters and Wessel van Wieringen from the department
of Epidemiology & Biostatistics of the VU University Medical
Center are responsible for integrative analysis of the molecular
data from (radiation) exposed and non-exposed thyroid cancer
samples.
Integrative analysis combines heterogeneous biological data,
be it experimental (e.g., copy number, gene expression, microRNA
expression) or from the biological literature (e.g., gene
annotation, pathway information). Integration of data from
multiple sources is imperative for a mechanistic understanding
of cancer. By putting together partial views of a complex
process like tumorgenesis, we may obtain a more accurate and
complete picture of the molecular mechanisms underlying it.
No off-the-shelf methodology for the statistical analysis
of the data is available. We therefore aim to develop statistical
models from the experimental data of these biological processes.
Such models will enhance our ability to identify biomarkers
and therapeutic targets more unambiguously and to interpret
genotype information. This should enhance the understanding
of what distinguishes the exposed from the non-exposed thyroid
cancers. In addition, the biological insight these models
provide is likely to result in more targeted follow-up experiments
and efficient use of available resources.
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Project
reference number: 002/2012
Principal Investigator: Professor B Jarzab, Maria Sklodowska-Curie
Memorial Cancer Centre and Institute of Oncology, Gliwice,
Poland
Email: bjarzab@io.gliwice.pl
Validation of the gene signature differentiating exposed from
non-exposed PTCs, obtained in the Genrisk-T project (no.:
036495) with an independent QPCR method
Summary of project:
The aim of the study is to investigate transcriptomic profiles
of papillary thyroid carcinomas that arose after expose to
radioiodine fallout from Chernobyl power station and to low
level radiocesium exposure present in the contaminated environment.
This project is the continuation of a series of projects that
are carry on to investigate the transcriptomic and genetic
profile of radiation induced thyroid cancer.
As a first step of the study Affimetrix microarray technology
was employed to define transcriptomic profiles in two cohorts
of children matched on age, oblast and pathological type of
tumour. At present we want to validate the results with qPCR.
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Project
reference number: 004/2012
Principal Investigator: Professor GA Thomas, Imperial College
London, Hammersmith Hospital, London
Email: Geraldine.thomas@imperial.ac.uk
DNA methylation patterns and radiation induced papillary carcinoma
Summary of project:
Cancer is a complex disease that develops when normal cells
are "re-wired". This can be the result of changes
in the structure of DNA by addition, deletion or rearrangement
of parts of a chromosome, or by changes in the way the DNA
is switched on or off. One of the mechanisms for switching
parts of the chromosome on or off involves changing the pattern
of methylation of the DNA bases. Changes in DNA methylation
pattern are common in cancer, and have been shown
to occur in cells that have been exposed to radiation. This
project will investigate whether the exposure to radiation
from the Chernobyl accident altered global methylation patterns
resulting in the development of a specific type of thyroid
cancer, papillary carcinoma.
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