Papillary Thyroid Cancer: Radiation induced versus Genetic Causes

Abstract

Carcinoma of the Thyroid is clinically acknowledged to be a rare malignancy and contributing for less than 1 per cent of all human malignant neoplasms although it is the most common amongst cancerous growths in the human endocrine system. Apart from the conventional form of Papillary Thyroid Carcinoma (PTC), a selection of some special variants are categorised in accordance with their manifestation and cell organisation in the tumour architecture, some variants being more aggressive than the conventional type. PTC originates in follicular cells of the thyroid tissue and is associated with a prior exposure to external ionising radiation or high intake of iodine. However, therapeutic treatment with radioactive iodine, unless excessive, has not been observed to increase the incidence of thyroid cancers. Persistent research aimed at an intuitive understanding of the causative factors behind papillary thyroid carcinoma has also led to a better appreciation of the pathogenesis involved in the disease. A significant increase in incidence of paediatric papillary thyroid cancer ensued the Chernobyl accident that has been attributed categorically to external radiation exposure.
Research has mainly involved the role of RET/PTC1, RET/PTC3 and BRAF oncogenes in tumour initiation and progression of PTC. RET/PTC rearrangements have been studied both in sporadic as well as in radiation-induced PTCs and it can be reasonably maintained that they seem to be more frequently encountered in tumours associated with ionising radiation. However, RET/PTCs though known to mark the onset of thyroid carcinogenesis, they are not appropriately known about their function in the progression of papillary thyroid cancer and their possible use as prognostic markers of the disease. In sporadic PTC, the RET/PTC1 rearrangement was the most common followed by RET/PTC3. In subjects with prior history of radiation however, RET/PTC3 was the more frequent rearrangement encountered. BRAF mutation has been found mostly in PTCs and some anaplastic thyroid cancers derived from the papillary variant and studies reveal that BRAF mutations are the most frequent genetic aberration found in sporadic PTCs with quite low frequency in radiation induced PTCs. The frequency of BRAF mutation in PTC in the region of Chernobyl was found to be very low in cases of childhood thyroid cancer while the frequencies are comparable in the case of adult papillary thyroid carcinomas when analogy is drawn with sporadic cases in other uncontaminated areas. However, there is still widespread disparity amongst the results obtained by the various studies, which have been performed and it can be argued that some studies were deficient in the appropriate use of controls. Comparatively little is known about the BRAF genes and its role in the development of PTC cannot be categorically stated at present. The discordance in the results exhibited by studies probing the prevalence of RET/PTC in sporadic papillary thyroid cancer in different geographical regions, sometimes even in the same population, may be attributed to method differences, sensitivity of the methods used and the question of whether the subjects studied were indeed a representative sample of the population concerned. The role of ethnic background in the results cannot be entirely disregarded.

Introduction

The thyroid gland, one of the major endocrine glands in the human body is anatomically positioned below the laryngeal prominence in the anterior neck, its primary function being secretion of the thyroid hormone that regulates the physiological functions in the body.
Carcinoma of the Thyroid is clinically acknowledged to be a rare malignancy and contributing for less than 1 per cent of all human malignant neoplasms although it is the most common amongst cancerous growths in the human endocrine system (Biersack & Grünwald, 2005).
After the Chernobyl nuclear power plant accident in April 1986, a large increase in the incidence of childhood thyroid cancer was reported in contaminated areas. Researchers have striven to establish the aetiological characteristics of Papillary Thyroid Cancer (PTC) and to seek the solutions to fundamental aspects like the apparently higher susceptibility of children and adolescents to PTC in the aftermath of the Chernobyl disaster in April 1986. A key aspect involved in recent research has been the factors relating to the frequency of the RET/PTC and BRAF mutations, the understanding of which are critical in the carcinogenesis of papillary thyroid carcinoma. Epidemiological features concerning the role of age, sex and geographic distribution in the prevalence of papillary thyroid carcinoma has also been as aspect of fervent research. Researchers have also looked into the susceptibility of particular ethnic groups to this disease. Key questions regarding the differences in the prevalence of papillary thyroid carcinoma with respect to age of the individuals, sex and their geographic location are being investigated. The ultimate question that needs answering however is whether papillary thyroid carcinoma is a direct consequence of exposure to external ionising radiation and the comparison between radiation induced papillary thyroid carcinoma and sporadically occurring PTCs.
This review aims to elucidate initial questions, which include an explanation of papillary thyroid carcinoma and the fundamental aspects surrounding its occurrence followed by an attempt at seeking possible answers to the more involved questions mentioned above.

Classification of thyroid carcinoma

Carcinoma of the thyroid gland, which was first described in the late eighteenth century, is medically accepted to be an uncommon cancer although it is the most common malignancy of the endocrine system and persistent research has resulted in a better understanding of the disease in the later half of the 20th century (Biersack & Grünwald, 2005). Malignant neoplasm in the thyroid is categorised under the ‘carcinoma’ form of cancer due to its epithelial origin and can be classified into four distinctive types based on differences in the cytological and morphological characteristics of the cancerous growth (Amdur & Mazzafferi, 2005).
Primarily, thyroid carcinomas are categorised on the basis of the cell from which they originate as depicted in Table 1.1. Apart from medullary carcinoma, which arises from parafollicular C-cells all the other types arise from the follicular epithelial cells. Moreover the papillary, follicular, insular and anaplastic forms of carcinoma arise from the epithelial cells of the thyroid follicle (Amdur & Mazzafferi, 2005) and are also, in essence, follicular growths. The extent of differentiation of thyroid cancers originating in the follicular cells forms the basis of further subdivision of this category of carcinoma (Table 1.1). Well-differentiated thyroid carcinomas include the papillary and follicular forms of thyroid carcinoma, which possess clearly demarcated structural and cytological features (Amdur & Mazzafferi, 2005).
Apart from the classification based on their cell architecture, the histological forms of thyroid carcinoma may also be classified according to their ability to assemble radioactive iodine, which is significant from the comparative prognosis point of view (Amdur & Mazzafferi, 2005). The conventional forms of papillary and follicular carcinoma generally concentrate radioactive iodine while the aggressive variants of papillary and follicular thyroid carcinoma are less efficient in concentrating quantifiable amounts of radioactive iodine. Anaplastic and medullary carcinoma do not concentrate radioactive iodine to an extent that is clinically pertinent.

Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC), which originates in follicular cells, is the more common between the two manifestations of differentiated thyroid cancer (papillary and follicular) and is associated with a prior exposure to radiation or high intake of iodine (Biersack & Grünwald, 2005). Interestingly, it is also the most curable amongst thyroid cancers (Schlumberger, 2004). This malignant growth appears in otherwise normal thyroid tissue as an uneven, solid or cystic multifocal mass and is identified by its distinctive malignant cytologic transformations which including enlargement, hypochromasia, intranuclear cytoplasmic inclusions, nuclear grooves and dissimilar nucleoli (Amdur & Mazzafferi, 2005). In general, PTC is clinically illustrated as being single, solid, symptomless thyroid nodules not easy to tell apart from benign nodules, which are a component of the thyroid anatomy (Schlumberger, 2004).
Apart from the conventional form of PTC, a selection of some special variants are categorised in accordance with their manifestation and cell organisation in the tumour architecture, some variants being more aggressive than the conventional type. Even though treatment of these variants are analogous to that of the conventional form of papillary thyroid cancer, (Biersack & Grünwald, 2005) more persevering treatment and attentive monitoring may be necessitated. The known variants of PTC have been briefly outlined below (Amdur & Mazzafferi, 2005).

Encapsulated Variant

This variant of papillary thyroid carcinoma is clearly demarcated from the neighbouring normal thyroid parenchyma by a well-defined fibrous capsule and it accounts for approximately 10 per cent of all PTCs reported. It may be mentioned that most PTCs do not involve such encapsulation and consequently invade the proximate thyroid tissue. The encapsulated variant has the characteristic formational transformations of PTC and has a superior prognosis than most other variants of PTC.

Follicular Variant

The follicular variant of papillary thyroid carcinoma is so called because its structural arrangement is that of follicle arrangement. Although this variant does not possess the normal papillary architecture, it is identified as a papillary carcinoma by means of the cytological transformations it entails that are identical to those in conventional papillary thyroid carcinoma. Prognosis for this variant is comparable to that for conventional PTC which can be attributed to the follicular variant having an affinity as good as conventional PTC for radioactive iodine (Biersack & Grünwald, 2005).

Diffuse Sclerosing Variant

Diffuse sclerosing papillary carcinoma is generally graded as being aggressive owing to the higher percentage of extrathyroidal extension (metastasis) occurring in patients with this variant in comparison with the conventional form of papillary thyroid carcinoma. This variant is uncommon and is typified by extensive sclerosis, lymphocytic infiltrate and psammoma bodies.

Tall Cell Variant

The tall cell variant retains the conventional papillary architecture as well as the cytological features of the usual type of papillary carcinoma although unlike the latter, its growth involves mitosis. In addition, each cell has a special structural feature wherein the length of each cell is at least twice its width. The requirement for any visible abnormal structural change in the thyroid tissue to be categorized as a tall cell variant is that it should be made up of at least 70 per cent tall cells.

Columnar Cell Variant

This variant is particularly infrequent and demonstrates a remarkably layered nuclear configuration in line with papillary architecture. Conversely, the customary nuclear features of papillary thyroid cancers are rarely observed.
An outline of the pathology of papillary thyroid carcinoma (Amdur & Mazzafferi, 2005)
The pathology of thyroid carcinoma consists of an unambiguous histopathological arrangement, which lays the foundation for clinical, diagnostic, and treatment modes as well as the development of application of new methods thereby increasing knowledge of basic mechanisms in molecular pathology.
Papillary carcinoma is derived from follicle cell of thyroid tissue and is the most common amongst the different forms of thyroid carcinoma, routinely containing rotund laminated calcifications called psammoma bodies. Often the papillary structures have a follicular configuration and have been termed “mixed carcinomas”. The distinctive nuclear characteristics of papillary thyroid carcinomas are the ground-glass nuclei, which are inflamed, sphere-shaped formations, with a pale karyoplasm condensing continuously to the nuclear membrane. These nuclei have been labelled as the main tactic used for diagnosis and their occurrence most certainly indicates the presence of PTC The ground-glass nuclei are compactly ordered and often overlie each other to form what is known as the “shingle roof pattern”. The nuclear criterion of ground-glass nuclei annuls any other structural form required in the differential diagnosis of follicle cell-derived tumours and “mixed” tumours. The papillary thyroid carcinomas also possess identifiable features, such as lymphocytic-type thyroiditis, which may further corroborate any diagnosis. The encasing stroma may also exhibit a dense fibrosis, which forms an active ingredient for research into clinical diagnosis and medical prognosis. Further, a collagenous capsule may possibly enclose papillary thyroid carcinoma. However, the carcinoma may not penetrate the capsule while infiltrating into the surrounding parenchyma, in which case there would not be any capsule formation. There is minimal chance that the PTC may break into the surrounding veins, a feature quite disparate from follicular carcinoma, where vascular infiltration is major condition of malignancy. Akin to follicular carcinoma, papillary thyroid carcinoma does exhibit variations of the cytoplasm of the tumour cells called the oncocytic cell type (Hürthle cell), which brings about diagnostic difficulty since it blurs the pattern of ground-glass nuclei. Since the ground-glass nuclei are hyperchromatic and often contain compacted chromatin structures diagnosis needs the additional criterion of papillary structure and/or infiltrating growth.

Aetiology

Persistent research aimed at an intuitive understanding of the causative factors behind papillary thyroid carcinoma has also led to a better appreciation of the pathogenesis involved in the disease. While an unambiguous aetiology of papillary thyroid carcinoma is yet to be illuminated upon, the medical research community has revealed links relating the malignancy to its cause (Biersack & Grünwald, 2005; Schlumberger, 2004). Thyroid tumours appear in people exposed to huge quantities of environmental radiation either from atomic weapon tests, nuclear reactor disasters or even the excessive thyroid irradiation during external radiation therapy.
Plentiful evidence exists indicating that external radiation is an important factor leading papillary thyroid carcinoma in particular when the patients are young at the time of exposure (Ron et al, 1995). The reason why children and adolescents are particularly susceptible is not yet known. A number of cases of thyroid carcinoma have been identified in epidemiological studies of individuals who had undertaken neck and chest radiation therapy for both benign and malignant disorders. Regardless of the dosage of radiation they were exposed to (Ron, 2003). It was observed that therapeutic doses for Hodgkin’s disease resulted in almost a 20-fold increase in thyroid cancer (Hancock et al, 1991). Of concern is the fact that in every report the amplified cancer was of the papillary form (Szanto, 1990).
Assessment of cases of thyroid cancers within the family (familial thyroid cancers) raises the question of the significance of the role of genetics in papillary thyroid cancer. Cancers occur due to an anomaly in the DNA, which develops in a clone of cells the ability to divide faster and infiltrate surrounding tissues, a property attributed to the oncogenes (mutations). Studies have shown that external radiation can alter the mechanism of oncogenes and rearrange them. The balance between familial and carcinogenic factors is a clinical dilemma. There have been reports where members of the same family have had significantly different responses to the cancer (Fisher, 1980). The contributory factors in such cases are important for the clinical community to comprehend. In the case of internal radiation, radionuclides of iodine used diagnostically or therapeutically are not connected with the risk of an increased possibility of thyroid cancer (Ron et al, 1998). This view was altered since the remarkable raise in thyroid cancer incidences in children who were exposed to radioactive iodine released from the Chernobyl nuclear power plant disaster (Williams, 2003). Another factor that seems to play a role in the variability of research studies carried out is ethnic background. Almost every study done needs has taken this aspect into account. The reason may lie in a high dietary intake of iodine in some geographic regions but an alternative explanation may well be plausible (Horn-Ross et al, 2001). It may be noted that prior benign thyroid conditions such as goitre are associated with an increased incidence of thyroid cancer (Burgess et al, 1997).
The issue of familial background has been looked into by a number of studies. (Kraimps et al, 1999) found that the familial incidence was 10.5 per cent when they examined the families of 105 individuals. 15 positive cases were found in 7 families. (Pal et al, 2001) made a comparative study of the incidence of thyroid cancer amongst the relatives of 339 individuals with thyroid cancer with that in families of 319 matched control samples and determined almost a 10 times increase. In yet another related study, an investigation of 1025 patients with thyroid cancer and 5457 relatives was conducted in Norway (Frich et al, 2001) and a 5.2 times increase in men and 4.9 times increase in women was found.
However, the chances of finding members of the same family having thyroid carcinoma are extremely slim (Malchoff et al, 1999).
As discussed later in the review, studies have unravelled reorganized structures of the RET oncogene, which have been identified as the vulnerable genes for progress of sporadic forms of papillary thyroid cancer. Located on chromosome 10q11.2 the RET gene encodes a trans-membrane receptor of the tyrosine kinase family. RET/PTC1, RET/PTC/2 and RET/PTC3 are chimeric oncogenes formed from the rearrangement of the RET oncogene. They have been identified as one genetic event leading to the development of sporadic cases of papillary thyroid cancer (Bongarzone et al, 1996). Rearrangement of the RET oncogene has also been identified in children who developed papillary cancer after exposure to radioactive iodine released during the Chernobyl reactor accident (Bongarzone et al, 1997). Papillary thyroid cancer is known to express c-MET located on the 7q31 chromosome. Moreover, it has been shown that papillary thyroid cancer has especially low rates of loss of allelic heterozygosity (Zhang et al, 1998). A genetic disorder that stands out is the Cowden’s syndrome, which is an autosomal dominant disorder distinguished by its associated increased risk of thyroid cancer grouped with other abnormalities such as development of hamartomas and an augmented risk of breast cancer (Yeh et al, 1999). It has been found that the deficiency of a tumour suppressor gene is responsible for susceptibility to this syndrome. A technique involving deletion mapping by examination of loss of heterozygosity of polymorphic markers was employed to ascertain the fine structure of a region of chromosome 10 (Yeh et al, 1999). Using this technique, the tumour suppressor gene whose removal is responsible for Cowden’s syndrome was identified as the PTEN tumour suppressor gene located on the chromosome10q23.3 (Dahia et al, 1997). The PTEN suppressor gene encodes for a phosphatase important in the phosphatidylinositol 3-kinase signal conduction pathway.
Yet another familial disorder that may be linked to papillary thyroid carcinoma is the familial adenomatous polyposis, which is an inherited autosomal dominant tumour syndrome categorized by colonic polyps with a final malignant transformation. The disorder is caused by germ-line mutations of the APC gene, which has been mapped to chromosome 5q21. (Malchoff et al, 1999) studied 18 family members with both familial polyposis as well as papillary cancer. The study revealed that the two conditions are not caused by the same genetic abnormality. The association between differentiated thyroid cancer and this familial tumour syndrome is well known. Researchers have used linkage analysis, utilising polymorphic markers located close to the APC gene, to establish whether familial papillary thyroid cancer is related to loss of the APC gene. It was concluded that familial papillary thyroid cancer is genetically distinct from familial adenomatous polyposis. A further genetic disorder called Gardner’s syndrome is analogous to familial adenomatous polyposis in its inheritance pattern, its connection with papillary thyroid cancer and the risk of carcinoma. In research studies assessing the genetic association of Gardner’s Syndrome with papillary thyroid cancer, patients with Gardner’s syndrome have been grouped together with patients with familial adenomatous polyposis from a comparative point of view (Hizawa et al, 1997).

Oncogenes

The mutations involved in papillary thyroid carcinoma are labelled as oncogenes. Mutations include instigation of receptor tyrosine kinases (RET/PTC, TRK, MET), through rearrangement or gene amplification specifically aimed at transforming the normal thyroid follicular cells into papillary thyroid carcinomas (Schlumberger, 2004). Such rearrangements generate chimeric proteins by means of tyrosine kinase activities that play a role in the progression of the malignant phenotype (Schlumberger, 2004). Nearly 40 per cent of adults with sporadic papillary carcinoma have RET gene rearrangement while about 15 per cent have NTRK1 rearrangement, the latter has been observed to be higher (60 per cent) in children (Biersack & Grünwald, 2005). Somatic point mutation in the BRAF gene may be the most common mutation among papillary thyroid cancers, as has been quite recently discovered. This gene encodes serine/threonine kinase acting on the RAS-RAF-MEK-MAPK signalling pathway. BRAF mutations seem to be much less common in childhood thyroid carcinomas.
Along with uncontrolled doses of radioactivity from the environment, such as the Chernobyl fallout, a couple of more reasons have been identified which lead to incidence of papillary thyroid carcinomas (Biersack & Grünwald, 2005).
Iodine excess: Papillary thyroid cancer has been successfully induced in animals with the administration of excess iodine. Its effects were observed in areas where goitres are widespread, where the addition of iodine in the diet had increased the fraction of papillary thyroid carcinomas relative to follicular thyroid cancer.
Radiation: External ionising radiation to the neck raises the chances of incidence of papillary thyroid carcinoma in later years. Irradiation during childhood has been associated with the greatest risk for acquiring papillary thyroid cancer. Studies have revealed that children irradiated for benign conditions such as tonsillar hypertrophy, thymic enlargement, and acne have later developed thyroid cancer over a latency period of roughly 20 years. Almost 7 per cent of the survivors of the atomic bomb explosion in Japan developed papillary thyroid cancers, which is a relatively higher percentage with respect to the normal population. Radiation exposure only increases the risk of developing thyroid cancer; it does not affect the prognosis or the aggressiveness of the tumour. Thyroid cancer was first expressed at the last decade of the eighteenth century. One and a half centuries of treatment saw surgery as the only effectual therapeutic option for this cancer, until in 1946 radioiodine therapy was performed for the first time. However, therapeutic treatment with radioactive iodine, unless excessive, had not been observed to increase the incidence of thyroid cancers until the Chernobyl nuclear disaster after which researchers looked more deeply into the effects of radiation therapy using radioactive iodine.
(Cardis et al, 2005) carried out a population-based case control study of thyroid cancer in Belarus and Russia in order to appraise the risk of thyroid cancer after exposure to radioactive iodine (I131) in childhood and to inspect the factors, environmental or otherwise that may alter this risk. The study involved 276 case subjects with thyroid carcinomas and 1300 matched control subjects, aged 15 years or under at the time of the disaster. Their study revealed that exposure to I131 in childhood increases the risk of thyroid cancer incidence, which is altered both by iodine deficiency as well as iodine supplementation.

The Chernobyl Incident and Papillary Thyroid Cancer

April 26, 2007 saw the 21st anniversary of the Chernobyl Nuclear Plant Accident. The Chernobyl nuclear reactor accident had resulted in the release of huge quantities of radioactive compounds, including radioactive iodine, into the atmosphere with extensive contamination of the regions surrounding the plant. Following the explosion of the reactor, prevailing winds carried the radioactive fallout from the northern parts of Ukraine to Belarus, territories of Western Russia, the Scandinavian Peninsula and regions of Western Europe. Belarus has by far been the most heavily contaminated, afflicted by almost 70% of the released nuclear activity (International Atomic Energy Agency, 1996).
Whether two decades of clinical surveillance is adequate to comprehensively appraise the radiological effects of this accident still remains to be seen. Nevertheless, 20 years of worthy research did reveal vital information concerning the clinical and molecular epidemiological ramifications of sporadic and radiation induced papillary thyroid carcinomas.
A significant increase in incidence of paediatric papillary thyroid cancer ensued the Chernobyl accident that has been attributed categorically to radiation exposure (Fagin, 2005). It has drawn exclusive attention of researchers towards identification of risk factors, such as iodine deficiency (Ashizawa et al, 1997) in radiation induced thyroid cancer. Attempts at evaluating the implications of exposing the thyroid gland to radiation in childhood thyroid cancer have been made since the accident (Kazakov et al, 1992). In Poland, suitable prophylactic methods like administration of iodine just after the accident were identified (Nauman & Wolff, 1993), which could contribute in alleviating the intensification of childhood thyroid cancer as well as reduce the future risk of thyroid cancer occurrence. Childhood thyroid cancer is infrequent under normal circumstances and supported with a good prognosis. It was observed that incidence of thyroid cancer in children and adolescents increased radically in the neighbourhood of Chernobyl with over 5000 cases being diagnosed from 1990 to 2005 albeit the mortality rate being considerably lower at 0.4 per cent (Hatch et al, 2005).
The thyroid gland along with the bone marrow is believed to be the most sensitive cancer site to radiation in the human body (Ron et al, 1995).
The extent of cytological damage resulting from exposure to ionising radiation depends on the amount of dosage (Biersack & Grünwald, 2005). Exposure to high dosage brings about bulk damage to cells ultimately resulting in cell death whereas lower dosage effectuates genetic consequences including rupture of the DNA double helix, introduction of point mutations and the triggering of chromosomal instability.
Microbiological divergence between sporadic and radiation induced papillary thyroid cancer can be appreciated by the use of specific approaches (Yamashita, 2006)

• Scrutinising the alteration of chromosomes in radiation-induced and sporadic thyroid cancers
• Evaluating gene expression in radiation-induced and sporadic thyroid cancers
• Molecular epidemiological analyses of individuals who contracted radiation-associated thyroid cancers

As observed by certain studies (Demidchik et al, 1999) the incidence of thyroid cancer in children from Belarus, Ukraine and the western parts of Russia has been increasing since 1990. However, data relating to the transmission and control of PTC has been particularly useful from Belarus (Demidchik et al, 1999). The relative incidence of PTC in the region increased alarmingly with Gomel being the worst affected by the radioactive fallout. Incidence of PTC in adults too showed a remarkable increase. A total of 673 cases of childhood thyroid cancer were detected and operated on by the between 1986 and 1997 (Demidchik et al, 1999), 52 per cent of who lived in the Gomel region. 94 per cent of the cancerous growths were categorised as papillary thyroid cancer.
Therapeutic treatment for the papillary thyroid cancers was carried out primarily using radioactive iodine (I131) along with nuclear medicine staging procedures.
The role of REarranged during Transfection/Papillary Thyroid Carcinoma (RET/PTC) oncogenes in tumour initiation of PTC – a discussion
The RET/PTC oncogenes are significant amongst the genetic activities that have been identified to execute a contributory role in the pathogenesis relating to papillary thyroid carcinomas with particular emphasis to radiation exposure and the RET oncogene activation in the papillary thyroid carcinoma has been described in diverse populations with dissimilar frequencies.
The RET (REarranged during transfection) gene was recognized in 1985 as a unique oncogene which was activated by DNA rearrangement. The resulting chimeric oncogene encoded a fusion protein, having an amino-terminal half with a presumed zinc finger motif, and a carboxyl-terminal half with a tyrosine kinase domain. This fusion resulted from recombination that occurred between two unlinked human DNA segments, which took place during the transfection process (Kodama et al, 2005). The name RET (RET proto-oncogene) has been retained to designate the gene coding for the tyrosine kinase (Kodama et al, 2005).
Genetic studies investigating radiation induced papillary thyroid cancer resulted in the discovery of the combination between RET gene which is located on the 10q11.2 chromosomal band with other genes that are specifically found in tissue affected by papillary thyroid cancer, the resulting chimeric genes being jointly labelled as RET/PTC rearrangements. The RET/PTC1 and RET/PTC3 genes are by far the most common amongst the 10 different types of RET/PTCs known and account for nearly 90 per cent of all chimeric genes (Kodama et al, 2005). A paracentric inversion of the long arm of chromosome 10 results in fusion of RET with a gene named H4/D10S170 thus forming the RET/PTC1 oncogene. Likewise, the RET/PTC3 oncogene is the end result of a similar intrachromosomal rearrangement and is formed by fusion of RET with the RFG/ELE1 gene (Fagin, 2004). The fusion proteins produced by the chimeric genes dimerise in a ligand-independent mode and trigger the tyrosine kinase function of RET (Fagin, 2005).
While several observations support the concept that a specific carcinogenic factor (radiation) can be directly related to a specific type of cancer (PTC) through a specific molecular event (rupture of DNA double helix) and specific gene fusions (RET intrachromosomal rearrangement), there is still scope for substantiation of the hypothesis. (Rabes et al, 2000) pointed out that the occurrence of RET/PTC rearrangements varies from 11 per cent to 43 per cent in sporadic papillary thyroid cancers and while the figure for individuals with a prior exposure to radiation ranges from 50 to 80 per cent. The group based its results on the analysis of 191 PTCs that developed between April 1993 and January 1998 in individuals between the age of 0 and 18.3 years exposed at a young age to radiation exposure after the Chernobyl nuclear reactor accident. The study observed a high ratio (~ 56 per cent) of malignant growths in children under the age of 4 years, which confirmed an earlier hypothesis (Ron et al, 1995) that at a young age the thyroid gland is most sensitive to ionising radiation. The largest fraction of all tumours observed in the study was found to have occurred in the most highly contaminated areas of Belarus. On the basis that the epidemiological data was found to be similar to that of post Chernobyl PTCs (Pacini et al, 1997) the group concluded that the 191 PTCs availed were a representative cohort of post Chernobyl cancers for the five year interval considered in the study. In the representative cohort it was found that in papillary thyroid malignancies that developed less than a decade after the accident, nearly two thirds of the intrachromosomal rearrangements were of the RET/PTC3 while PTCs, which occurred after a longer latency period were largely the RET/PTC1 rearrangement type. Earlier studies (Elisei et al, 2001; Mizuno et al, 2000) showed that adults exposed to external radiation mainly induced the RET/PTC1 rearrangements whereas in children exposed to external radiation the major rearrangements were of the RET/PTC1 and those exposed to Chernobyl radiation the RET/PTC3 rearrangements were the major type (Nikiforov et al, 1997; Santoro et al, 2000).
However, the RET/PTC oncogenes are not exclusive to radiation-induced malignancies, since they are also found in sporadic paediatric papillary thyroid cancers (Fagin, 2005). (Rossella et al, 2001) evaluated patterns of RET/PTC activation in thyroid tumours in different groups of individuals including both children and adults with benign or malignant tumours exposed as well as not exposed to radiation. The study involved the variables of age, radiation exposure and histological tumour variant and included 65 subjects with benign nodules and 89 with papillary thyroid cancer, the latter consisted of 25 Belarus children exposed to the post-Chernobyl radioactive fallout, 17 Italian adults exposed to external radiotherapy for benign diseases, and 47 Italians (25 children and 22 adults) with no history of radiation exposure. Their attributed earlier conflicting results in similar studies to the use of different experimental methods being done by different groups and designed their study in the single laboratory using same methodologies.
The average frequency of RET/PTC rearrangements in papillary thyroid cancer was found to be 55 per cent, the highest (76 per cent) being found in children exposed to the post-Chernobyl fallout which was appreciably higher than that in Italian children not exposed to radiation.
The group found no difference between RET/PTC rearrangements in the samples taken from adult subjects both irradiated and not as well as between children and adults with naturally occurring thyroid cancer. In conclusion, (Rossella et al, 2001) asserted that the occurrence of RET/PTC rearrangements in thyroid cancers is not limited to the malignant phenotype, which is consistent with earlier findings. A conflicting result however, was that neither exposure to radioactive iodine/external radiation nor young age increases the presence of RET/PTC rearrangements in radiation-induced tumours in comparison with naturally occurring thyroid cancer indicating that it is not affected by the mode of irradiation. These results are in disagreement with an earlier opinion that no RET/PTC rearrangements appear to be associated with benign thyroid lesions (Santoro et al, 1992; Thomas et al, 1999) and that RET/PTC rearrangements are only related to radiation exposure (Nikiforov et al, 1997) and young age (Bongarzone et al, 1996).
Although RET/PTCs are known to mark the onset of thyroid carcinogenesis, comparatively little is well known about their function in the progression of papillary thyroid cancer and their possible use as prognostic markers of the disease. In a clinical study, (Puxeddu et al, 2003) examined the frequency of RET rearrangements in adult PTCs, and investigated the possibility of RET/PTCs influencing the biological behaviour and clinical features of the cancers. Tissue samples of a cohort of 48 PTC cases in Italian adults between the age of 22 to 80 years were studied and the effects of RET/PTC positivity, preferential RET tyrosine kinase domain (RET-TK) expression and RET/PTC plus RET-TK positivity, on age, sex, tumour size, staging, number of neoplastic foci, and histological subtype were genetically evaluated using the RT-PCR-Southern blot technique. The genetic study established the positivity of RET/PTC1 and RET/PTC3 in 27 per cent of the cases and revealed that RET/PTC may not be a convincing prognostic marker for papillary thyroid carcinomas.
An interesting comparative study was performed by (Christofaro et al, 2005) in which the occurrence of RET/PTC1 and RET/PTC3 in thyroid tumours from 21 liquidators was analysed against the sporadic thyroid cancer cases in 31 adult Ukrainian subjects from uncontaminated regions of Ukraine and 34 adult French subjects. Liquidators are people who performed the post explosion cleaning in the most contaminated parts of Chernobyl. The group observed the occurrence of RET rearrangements in 83.3% of the liquidators, 64.7% of Ukrainian subjects, and 42.9% of French subjects with a considerably high prevalence of RET/PTC3 in the liquidators compared to the sporadic French subjects. The prevalence study of RET/PTC1 and RET/PTC3 was carried out using reverse transcriptase PCR (RT-PCR) and hybridisation with a radiolabeled probe. The sporadic Ukrainian subjects showed intermediate prevalence of RET/PTC3. RET/PTC1 prevalence in all three groups was found to be statistically comparable. The results are in agreement with (Rosella et al, 2001) in the hypothesis that the outcome of irradiation is independent of age. In addition the higher prevalence of RET/PTC3 in sporadic adult Ukrainian subjects in comparison with sporadic adult French subjects calls attention to the likelihood of genetic susceptibility or low-level exposure to radiation in the former. In an earlier study concerning Chernobyl liquidators, (Ivanov et al, 1997) regarded the liquidator group in their research as being a representative sample, and that the excess relative risk of thyroid cancer is equivalent for all liquidators, regardless of ethnic background. More recently, (Cherenko et al, 2004) reported that thyroid cancer in liquidators was significantly more aggressive than in sporadic adults’ cases of thyroid cancer. The substantial increase in the occurrence of RET rearrangements in papillary thyroid carcinoma from liquidators agrees with earlier results that have revealed the relative higher prevalence of RET rearrangements in thyroid cancer from adults exposed to radioactive fallout in Chernobyl with respect to sporadic subjects with no history of exposure to radiation (Rabes et al, 2000; Elisei et al. 2001).
(Sadetzki et al, 2004) explored the involvement of RET/PTC initiation in radiation induced thyroid cancers using RT-PCR analysis and Southern blotting techniques in individuals exposed to low doses of external ionising radiation and matched the results with non irradiated individuals of same genetic origin in the distinctive populace of the Israeli Tinea Capitis cohort, who were treated between 1948 and 1960 with ionising radiation to the head area for Tinea Capitis (a fungal illness of the scalp). Although the possibility of a selection bias cannot entirely be condoned in this study, its dissimilarity with related studies performed earlier is that the subjects considered were exposed to low-dose external beam irradiation during childhood and had an extended latency period before the development of thyroid carcinoma. RET/PTC1 was the predominant rearrangement observed. However, the RET/PTC activation rate was found to be lower (~ 35 per cent) than that observed earlier (~ 50 – 80 per cent) in individuals exposed to radiation after the Chernobyl accident, which may be attributed to contrasting aspects among the two subject populations such as dosage, manner of irradiation, age at diagnosis and latency time.
Studies pertaining to the prevalence of RET expression in sporadic papillary thyroid cancer demonstrate considerable disparity between geographic regions.
(Zou et al, 1991) analysed the frequency of RET/PTC oncogene in 40 sporadic cases of papillary thyroid cancers in Saudi Arabia using the RT-PCR technique and observed only one case (2.5 per cent) that showed RET/PTC rearrangement. Apart from showing an uncertainty about the prevalence of RET/PTC rearrangements in PTC cases in the Saudi population, the study asserts that genetic background and environmental factors may play a role in the activation of the RET/PTC oncogene.
In a separate study much later (Fenton et al, 2000) examined 33 cases of sporadic papillary thyroid cancer that developed during childhood or adolescence in North American subjects, using RT-PCR amplification. The overall frequency of RET/PTC mutations was found to be 45.5 per cent. The RET/PTC1 rearrangement was the most common followed by RET/PTC3, which was consistent with previous studies.
(Bongazone et al, 1996) looked at 9 cases of sporadic PTC from Italian children between 4 and 19 years using RT-PCR and Southern Blotting techniques and found that 67 per cent contained RET/PTC mutations, the majority of them being RET/PTC 1 rearrangements. (Motomura et al, 1998) examined sporadic PTC cases in 8 Japanese children between 9 and 14 years and found that 37.5 per cent contained RET/PTC mutations, the prevailing rearrangements being RET/PTC1 type. (Nikiforov et al, 1997) studied 17 children between 5 and 18 years from the North America and observed that 71 per cent contained RET/PTC rearrangements, again with RET/PTC1 being the dominant rearrangement type.
(Chua et al, 2000) probed into the prevalence and distribution of RET/PTC1, 2, and 3 in 28 sporadic papillary thyroid carcinoma cases in New Caledonia, which has the highest incidence of thyroid cancer in the world (Blot et al, 1997) and compared the observed pattern with 20 Australian cases using RT-PCR and Southern Blotting methods. Possible unwanted effects in the results due to ethnic differences within each study populace was avoided by restricting the New Caledonian cases to Melanesians and the Australian cases to Caucasian Australians with no significant age and gender disparity between the two populations groups. RET/PTC was found to be present in 70 per cent of the New Caledonian and in 85 per cent of the Australian samples, which is amongst the highest prevalence rates reported. Interestingly, the study found the occurrence of multiple RET/PTC rearrangements in a reasonably high percentage of tumours from both populations. In an earlier Australian study however, (Learoyd et al, 1998) used RT-PCR and direct sequencing methods and showed an RET/PTC incidence of 4.2 per cent, which is significantly lower.
In the United Kingdom, (Williams et al, 1996) reported that 10 out of the 21 cases (47 per cent) of sporadic papillary thyroid cancer studied in children had RET/PTC mutations. More recently, (Finn et al, 2003) assessed the prevalence of the two common RET chimeric transcripts (RET/PTC1 and RET/PTC3) in an Irish cohort of sporadic papillary thyroid cancer. Of the 28 samples studied RET/PTC prevalence was found to be 60 per cent with RET/PTC1 significantly higher (43 per cent) than RET/PTC3 (18 per cent), which is higher amongst the prevalence of RET/PTC reported.
The discordance in the results exhibited by studies probing the prevalence of RET/PTC in sporadic papillary thyroid cancer in different geographical regions, sometimes even in the same population, may be attributed to method differences, sensitivity of the methods used and the question of whether the subjects studied were indeed a representative sample of the population concerned. The role of ethnic background in the results cannot be entirely disregarded.
In view of the above RET/PTC rearrangements have been documented both in sporadic as well as in radiation-induced papillary thyroid cancers, it can be reasonably maintained that they seem to be more frequently encountered in tumours associated with ionising radiation.
Within the last 5 years, substantial research has been undertaken in looking mostly at the oncogenic T1799A transversion mutation, formerly termed as T1796A, of BRAF (the gene for the B-type Raf kinase) and its role in sporadic thyroid cancer (Xing, 2005). BRAF fits into the RAF family of kinases, which comprises of two other isoforms: ARAF and CRAF. BRAF mutation has been found mostly in papillary thyroid cancers and some anaplastic thyroid cancers derived from the papillary variant but no evidence of its occurrence in follicular or medullary thyroid cancer has been reported (Xing, 2005). (Xing, 2005) describes the T1799A BRAF mutation in that it occurs entirely in PTC and PTC-derived anaplastic thyroid cancer and can be an unambiguous diagnostic marker for this cancer when identified in cytological and histological samples. Moreover, it can also act as a unique and objective molecular prognostic marker in the risk assessment of thyroid cancer.
Several studies were conducted that have consistently upheld a high prevalence of BRAF mutation in thyroid cancer, ranging from 29 to 83% (Namba et al. 2003, Kim et al. 2004), even as the last 5 years of determined research grew increasingly important.
(Xu et al, 2003) reported that BRAF mutation (at codon 600) occurred in 21 of the 56 papillary thyroid carcinomas (38%) studied in the early research aimed at unearthing the role of BRAF mutation in thyroid cancer. With respect to sporadic papillary thyroid cancer, the study also revealed a significantly higher frequency of BRAF mutation in male subjects than female. In addition, it was found that BRAF mutation occurred in 45.4 per cent of subjects over the age of 40 and only 26.1 per cent in subjects younger than 40 years. In search for the question of whether BRAF mutation concurs with RET/PTC mutations in sporadic PTCs, the group found that out of 21 BRAF-mutated samples, 8 samples (38%) showed positive RET/PTC rearrangements, a result indicative of an interconnection between BRAF mutation and RET/PTC rearrangements in sporadic PTC. This result is in disagreement with an earlier study reporting that BRAF mutation at the codon 599 does not overlap with RET/PTC rearrangements in sporadic papillary thyroid cancer (Kimura et al, 2003). It may also be mentioned that earlier studies (Soares et al, 2003; Kimura et al, 2003) showed that RET/PTC rearrangements did not coexist with BRAF V600E mutation no matter what.
In an interesting study, (Lima et al, 2004) set out to find if an equivalent relationship between BRAF mutation and RET/PTC rearrangements existed in PTCs induced from radiation background. A cohort of 34 cases of PTC were analysed which occurred in contaminated regions of Ukraine. They showed that the frequency of BRAF mutations is considerably lower (12 per cent) in post-Chernobyl PTC than in adult sporadic PTC (46 per cent), whereas there is no marked difference relating to post-Chernobyl and sporadic childhood PTCs. Thus, findings of this study indicate that BRAF mutations are unusual in PTC in children and adolescents, both sporadic and radiation associated and moreover, highlight the significance of considering the age factor when evaluating mutation frequency. In a contrasting study, (Xing et al, 2004) explicit to radiation induced PTC that occurred as a result of the Chernobyl fallout, it was found that the frequencies of BRAF mutations in the radiation induced PTCs and sporadic adult PTCs was comparable. However, the same study revealed a much lower BRAF frequency when analysing only radiation induced PTCs in children.
Related research carried out by (Collins et al, 2006) investigated a cohort of individuals exposed to external beam radiation treatment as children for benign conditions, who later developed papillary thyroid cancer as adults. Out of the 23 cases studied only one showed positive BRAF mutation. This study adds to earlier results that BRAF mutation occurs at low frequencies in radiation associated papillary thyroid carcinomas.
(Puxeddu et al, 2004) analysed 60 PTC cases and reported the results of a BRAF mutation and RET/PTC rearrangement screening in an Italian thyroid cancer cohort. The study confirmed the frequent occurrence of BRAF mutations in sporadic adult PTCs and its exclusive association with this thyroid cancer histotype. Furthermore, a low rate of RET/PTC rearrangements was found in the cohort, which was attributed to its unique environmental background although none of the subjects had any prior history of radiation exposure. The study further speculated that the simultaneous discovery of a high prevalence of BRAF mutations with a low rate of RET/PTC rearrangements in adult sporadic PTC may reflect ionising radiation as the primary factor in the former case and a possible spontaneous mutations of the BRAF gene in the latter. (Xing, 2005) lists the pooled data on sporadic adult thyroid cancer patients from 29 studies, which displays an overall prevalence of BRAF mutation of 44% in PTC.
An investigation towards the clarifying the disparity between the features of adult and childhood PTCs was carried out by (Kumagai et al, 2004). 31 childhood Japanese cases and 48 post Chernobyl Ukrainian thyroid carcinomas, all of who were less than 17 years at the time of the Chernobyl accident, were studied. Thus two distinct childhood carcinoma groups were selected with an aim to reduce the effect of genetic background on the results. In assessing whether radiation exposure effectuates BRAF mutation in thyroid carcinoma, the study found no BRAF mutations in the Ukrainian childhood PTCs, whereas it was a very low frequency (3.2 per cent) of this mutation was found in the Japanese childhood PTCs. The study also did not come across any connection between the behaviour of PTC in childhood and the occurrence of BRAF mutation. Therefore the study has revealed that unlike RET/PTC, exposure to radiation does not seem to induce BRAF mutations. Further, it was suggested that BRAF mutation might perhaps be one of the factors that contribute to the conflicting biological behaviours observed between childhood and adult PTCs.
An independent study to investigate the frequency of BRAF mutation in a Korean population was performed by (Kim et al, 2004). 70 cases of sporadic papillary thyroid carcinoma were evaluated and the BRAF mutation at the V600E (earlier termed as V599E) codon was found in 58 of the 70 PTCs (83 per cent), a figure higher compared to earlier studies. The results indicated that BRAF mutation plays a major role in the carcinogenesis of PTC, especially with respect to the Korean population. Although the study is confined within a narrow histological boundary, it further asserts that BRAF mutation offers a genetic, prognostic and diagnostic marker for use in cancer therapeutics.
It can be seen that BRAF mutations are the most frequent genetic aberration found in sporadic PTCs.

Results

In papillary thyroid carcinoma the RET oncogene activation has been intensively studied. There is substantial evidence that indicates the involvement of RET/ PTC rearrangements as the initiating step in thyroid cancer pathogenesis.
External irradiation of human the thyroid or paediatric human thyroid cells induces RET/PTC1 rearrangements in a dose-dependent manner. A number of observations have demonstrated that radiation can directly induce RET/PTC rearrangements resulting in an atypical manifestation of the chimeric oncogene. Furthermore, analysis of the DNA from papillary thyroid carcinomas from children exposed to radiation after the Chernobyl disaster offers sound confirmation that RET/PTC3 is the most common type of fusion gene found in this patient population. These experimental results indicate that ionising radiation is a key risk factor for development of PTC.
In children afflicted by the Chernobyl radiation associated papillary thyroid carcinomas, it has been shown that the RET/PTC1 rearrangement is prevalent.
The prevalence of RET/PTC rearrangements ranges has been found to be considerably lower in sporadic adult papillary thyroid cancers than in individuals with a history of radiation exposure. It has also been shown that in children affected by the Chernobyl accident, RET/PTC3 was the most common type in tumours developed within a latency period of less than 10 years, whereas papillary thyroid cancers which occurred after a longer latency, had predominantly RET/PTC1. Nonetheless, it must be kept in mind that the high prevalence of the RET/PTC rearrangements is an observed trait of papillary cancer in young patients, which is not restricted to PTC caused by irradiation. The BRAF mutation is the other kind of gene rearrangement recently claimed to have a profound effect in the carcinogenesis of papillary thyroid cancers. The studies revealed no appreciable variation in the BRAF mutational frequencies between radiation-induced and sporadic thyroid cancers when similar age groups of subjects were compared. Also, the frequency of BRAF mutation in papillary thyroid carcinomas in the region of Chernobyl is very low in cases of childhood thyroid cancer while the frequencies are comparable in the case of adult papillary thyroid carcinomas when analogy is drawn with sporadic cases in other uncontaminated areas.
However, at this stage the role of BRAF mutations in the development of PTC cannot be categorically stated. There is still widespread disparity amongst the results obtained by the various studies, which have been performed. It can be argued that some studies were deficient in the presence of apposite controls, in other words, an adequate number of sporadic papillary thyroid carcinomas both in children as well as adults, if at all possible from the same geographical areas and coming up from the same genetic background, with no noticeable history of exposure to external ionising radiation. Understandably, the trouble in obtaining adult specimens and the insufficiency of sporadic children thyroid carcinomas greatly affected the possibility of such evaluation. Nevertheless, discrepancies have been observed among the results taken from different populations and sometimes even within the same population. An added aspect may be that many of the cohorts studied are numerically small and may not be representative of the population being studied, while some studies scrutinized only a small number of adult cases and made no reference to childhood carcinomas.

Conclusion

Measures to dovetail the interpretations of the results from the numerous studies taking into consideration the demographic and genetic factors are not easy to think through. Additional detail is essential to fully elucidate the patterns of papillary thyroid carcinogenesis. On the whole, a scrutiny of the studies relating to mutation in the case of the Chernobyl thyroid malignancies reveals that a possible marker for the aetiology has not yet been established to categorically distinguish the patterns of gene expression in radiation-induced papillary thyroid cancers and sporadic papillary thyroid cancers.