Approved projects
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
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: sarah.forbes_robertson@hotmail.com
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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