Theramed

OCTOBER 2001
Issue # 4

Molecular Genetics of Thyroid Cancer

Shereen Ezzat, MD, FRCP(C), FACP1
and
Sylvia L. Asa, MD, Ph.D., FRCP(C), FCAP2

1 Associate Professor, Department of Medicine, University of Toronto;
Director, Freeman Centre For Endocrine Oncology, Mount Sinai Hospital
2 Professor, Department of Laboratory Medicine and Pathobiology, University of Toronto;
Pathologist-in-Chief, University Health Network;
Consultant Pathologist, Freeman Centre For Endocrine Oncology, Mount Sinai Hospital

Inherited Forms of Thyroid Carcinoma

The etiology of most endocrine tumors is not known. A small minority is due to inherited genetic defects. The genes responsible for the Multiple Endocrine Neoplasia (MEN) syndromes, MEN-1 and MEN-2, have been cloned and characterized, and the mutations have clarified our understanding of mechanisms of disease 1,2 3. MEN-2 is the best example of inheritance of a mutant proto-oncogene. The identification of an activating ret mutation in members of kindreds is now accepted as an indication for prophylactic thyroidectomy in early childhood, since these individuals will develop medullary thyroid carcinoma that can metastasize and is lethal in more than half of patients. Moreover, distinct ret mutations are associated with distinct clinical phenotypes. Mutations in exons 10 and 11 that encode the extracellular domain of the ret protein are implicated as the cause of familial medullary thyroid carcinoma alone. Specific mutations, usually in exon 11 involving codon 634, are associated with MEN-2A and specifically codon 634 mutations replacing cysteine with arginine are more often associated with parathyroid disease and pheochromocytoma that characterize this disease complex. Activating mutations in exon 16 that replace a codon 918 methionine with threonine alter the tyrosine kinase domain of ret and result in MEN-2B, a more aggressive variant of MEN-2 with mucosal neuromas and a marfanoid habitus in addition to tumors of thyroid C cells, parathyroids and adrenal medulla4.

The MEN-1 gene is expressed in thyroid, but it has not been implicated in the pathogenesis of thyroid carcinoma.
Medullary thyroid carcinoma, a tumor derived from the calcitonin-producing C cells of thyroid, is the most well recognized form of familial thyroid carcinoma, however, there is evidence that carcinomas of follicular epithelial derivation, usually papillary carcinomas, may also have familial predisposition 5. The genes implicated in most patients with a family history of papillary thyroid carcinoma are not yet clarified.

Two examples of familial papillary and follicular carcinomas are known to be associated with other diseases, and the genes implicated have been identified. Mutations of the PTEN tumor suppressor gene on chromosome 10q23 have been implicated in the pathogenesis of Cowden disease 6,7, an autosomal dominant inherited syndrome associated with a wide variety of malignancies including breast, skin, and thyroid (follicular subtype). Another syndrome of gastrointestinal neoplasia associated with thyroid carcinoma is familial adenomatous polyposis coli. The gene conferring predisposition to this disorder has been identified (APC) and mapped to chromosome 5q21 8,9. Although patients with this disorder exhibit a curious morphologic phenotype of papillary thyroid carcinoma 10, the role of the specific genetic mutation in the development of thyroid carcinoma remains unclear 11.

Molecular Genetics of Sporadic Thyroid Neoplasms
Although the genetic basis of the inherited endocrine tumors of MEN-1 and -2 is now understood, the genetic abnormalities underlying the far more common sporadic tumors are not clear 12, 13 .

Clonality assessment
The technique of clonality assessment using X chromosome inactivation patterns has evolved from the Lyon hypothesis which states that only one X chromosome is active in any female somatic cell; the inactivation occurs early in embryogenesis and persists throughout the lifespan of the cell and its progeny. A molecular approach to the determination of clonality takes advantage of X chromosome inactivation patterns; activated genes can generally be distinguished from their inactive counterparts because of differences in the degree of methylation of cytosine ( C ) residues which are typically hypomethylated in active genes. X chromosomes genes which have been utilized for these studies include hypoxanthine phosphoribosyltransferase (HPRT), phosphoglycerate kinase (PGK), the human androgen receptor gene (HUMARA) and M27ß. Molecular analyses have proven that most thyroid nodules, even those considered traditionally to be hyperplastic, are monoclonal neoplasms 14.

Oncogenes
Proto-oncogenes are normal cellular genes that play an essential role in the proliferation and differentiation of normal cells. They function at each step of signal transduction pathways as growth factors (eg. c-sis), membrane receptors (eg. C-erb-B, c-neu, c-fms), GTP binding proteins (eg. ras family) and nuclear proteins (eg. c-myc, c-fos). Proto-oncogenes may be activated by point mutations, translocations or by increased expression. Genetic alteration in these genes leads to sustained activation of the gene product in the absence of the normal control mechanisms. Activated oncogenes have been associated with a large number of human tumors.
Cell surface receptors. Activating mutations of receptors that regulate hormone synthesis and secretion have been anticipated as the molecular solution to the problem of endocrine tumorigenesis. In some cases of hyperfunctioning thyroid adenomas activating mutations of the thyrotropin (TSH) receptor have been identified and proven to be associated with disease 15-17.
G Proteins. One oncogene that plays an important role in endocrine tumorigenesis is the a-subunit of the Gs protein 18.
G-proteins are heterotrimeric membrane-anchored peptides that play a central role in transducing signals from the cell surface ligand-receptor complexes to the downstream effectors. The a-subunit dissociates from the ß– and gamma-subunits of Gs when GTP displaces its bound GDP, stimulates adenylyl cyclase to produce cyclic AMP from ATP. Cyclic AMP (cAMP) in turn activates c-AMP-dependent protein kinases, increases intracellular calcium transport, and may potentiate the effect of activated inositol phospholipid-dependent protein kinases. The weak intrinsic GTPase activity of Gsa and the action of GTPase activating peptides (GAP) dissociate GTP from Gsa and terminate the response. One of the earliest and most exciting molecular defects to be described in endocrine tumors involved point mutations in two critical domains of the Gsa subunit at codon 201 where Arg is switched to a Cys or codon 227 where Gln is replaced with Arg. Substitutions at these codons (the gsp mutations) activate adenylyl cyclase by inhibiting the hydrolysis of GTP and thereby maintaining Gsa in a constitutively activated state. Activating mutations of this protein are reported to occur in a large proportion of hyperfunctioning thyroid adenomas 19,20.
Ras Proteins. The three ras proteins (H- K- and N-) are involved in transducing signals from the cell surface to a number of ligand-receptor complexes. The commonest mutational sites alter the GTP-binding domain (codons 12/13) or more rarely the GTPase domain (codon 61). Ras mutations occur in thyroid tumors but they are controversial. They are considered rare but not indicative of biological behavior, since they are found in some adenomas, as well as carcinomas 21-26.
Others. Unique chromosomal rearrangements involving the ret proto-oncogene (ret/PTC gene rearrangements) are found in papillary carcinomas 27. These ret/PTC oncogenes are the result of DNA damage with rearrangements that transpose various cellular genes adjacent to the gene encoding the intracellular tyrosine kinase domain of the ret proto-oncogene. They are transforming and appear to be early events in the development of papillary carcinoma 28,29. The reported frequency of ret/PTC rearrangement varies widely among different series but is present in up to two thirds of sporadic papillary thyroid carcinomas 28,30 to 87% of papillary carcinomas attributed to the Chernobyl nuclear disaster 31. The discordant incidence data in multiple series reflect in part the different sensitivity of the techniques utilized, as well as the influence of environmental factors such as ionizing radiation exposure. The identification of ret/PTC gene rearrangements provides a novel diagnostic tool for papillary carcinoma 30,32,33 However, they do not appear to be potent oncogenes in promoting growth or dedifferentiation of thyroid carcinoma34.
The molecular basis for the development of follicular carcinomas is now thought to involve another novel gene rearrangement involving the thyroid transcription factor PAXX-8 and the peroxisome proliferator-activated receptor ?(PPAR?) gene 35. Normal thyroid follicular cells express Pax 8 at high levels; this transcription factor is essential for thyroid development, involved in regulating expression of the endogenous genes encoding thyroglobulin, thyroperoxidase, and sodium/iodide symporter. PPAR?, a transcription factor that is implicated in the inhibition of cell growth and promotion of cell differentiation, is also expressed by normal thyroid follicular epithelium. However, this in-frame rearrangement results in a fusion protein that likely interferes with the normal function of both differentiating factors, thereby explaining its potential role in thyroid tumorigenesis.

Tumor Suppressor Genes

Products of tumor suppressor genes (TSG) act as sequestering agents for transcription factors, thereby, modulating physiologic growth by arresting cell division in he G1 phase. This delay may allow for repair of genomic damage or may trigger apoptotic cell death. Deletion or reduced expression of TSGs appear to be a commonly shared mechanism in human tumorigenesis.
p53. The p53 protein plays a role in cell cycle regulation; point mutations, deletions, or rearrangements in the p53 gene which result in an altered protein are considered to be among the commonest genetic mutations in human neoplasms and have been implicated in tumor progression in several types of cancer. Progressive transformation to the malignant phenotype may be the result of mutational inactivation of the p53 TSG. In thyroid cancer, p53 mutations are late events that have been described only in anaplastic carcinomas 36-38 or in differentiated lesions that are likely to progress to more aggressive disease 39,40 .
APC. Although this gene has been implicated in predisposition to familial papillary thyroid carcinoma in patients with the familial polyposis coli syndrome, there is no evidence of APC mutations in sporadic thyroid neoplasms 41,42 . This is consistent with a lack of APC inactivation by loss of heterozygosity in patients with a mutated APC gene, suggesting that loss of APC is not the critical pathway for thyroid carcinogenesis in patients with the genetic disorder 11 PTEN. The PTEN tumor suppressor gene is implicated in the etiology of Cowden’s syndrome that includes thyroid follicular neoplasms. This Gene is only occasionally inactivated in sporadic follicular thyroid tumors 43.

Implications of Molecular Genetics in Thyroid Cancer

The value of novel molecular markers in the diagnosis and prognosis of thyroid cancer is beginning to be accepted. The use of ret/PTC as diagnostic tools, even on cytologic evaluation of thyroid aspirates, is emerging in clinical laboratories 30,32,33. Screening for endocrine tumors is now being applied systematically in cases of familial disease. Patients with activating mutations of the ret proto-oncogene will almost certainly develop medullary thyroid carcinoma, a disease that is lethal is not detected early or prevented, and therefore current guidelines recommend prophylactic thyroidectomy in childhood, usually by age 5 for those with MEN 2A or FMTC, and at or around age 1 year for those with the more aggressive mutation of MEN-2B. However, some families have unidentified mutations and hormonal screening remains the standard mechanism of tumor detection, with the addition of radiological and biochemical investigations where indicated.
The role of genetic influences in the etiology of most sporadic thyroid carcinomas remains incompletely understood, but there is accumulating evidence of a causal role for radiation. This is true of radiation therapy, for example in patients who have received external beam radiotherapy for malignancies of the head and neck as well as for cosmetic therapy for facial acne. It is also true in populations exposed to radioactive fallout from nuclear disasters, such as in Japan after the nuclear bomb disasters and in Ukraine and Belarus after the Chernobyl episode. The exposure to radioactivity has its highest impact in the young, and the disease is more often multifocal than in sporadic cases, however, the prognosis in patients who have been exposed to radiation does not appear to differ from those with no history of radiation 44. More recent evidence suggests that radiation maybe directly implicated in the genesis of the ret/PTC gene rearrangements 45. These findings bring together a conceptual framework linking an environmental influence with a molecular pathway responsible for thyroid cell transformation.

Shereen Ezzat, MD, FRCP(C), FACP1
and
Sylvia L. Asa, MD, Ph.D., FRCP(C), FCAP2

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EDITORIAL COMMENT

From ROBERT VOLPÉ, M.D., FRCP (C), MACP

The article of Ezzat and Asa details the nature of the genes involved in the pathogenesis of thyroid carcinoma, and have indicated the current view of the place these genes have in diagnosis and management of these disorders.
The most obvious value of this information has had to do with familial medullary thyroid carcinoma. In families with members manifesting this condition, children without thyroid nodules or other clinical evidence of thyroid disease, should be tested for the presence of the appropriate genes. If the genes for medullary thyroid carcinoma are present, the child should undergo prophylactic thyroidectomy which will prevent a subsequent development of medullary thyroid carcinoma.

If, on the other hand, the genetic tests are negative, patients and their families can be reassured that there is no danger from this familial scourge. In follicular and papillary carcinoma, while certain genetic markers are increased, these have not had a significant influence thus far in diagnosis or the outcome of treatment.

It is however, of interest that radiation induces thyroid carcinoma via genetic mutations. Further studies of the genetic makeup of thyroid carcinoma will undoubtedly elucidate the precise role of specific gene markers in these diseases.

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	Thyroid Canada Newsletter - Issue 4