RET Genetic Analysis

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RET mutation analysis represents one of the most useful genetic screening tests in clinical practice. The mutation is inherited as an autosomal dominant trait: since the penetrance of RET mutations is near 100%, all gene heterozygous carriers will develop medullary thyroid

Ret Thyroid Cancer

Figure 21.11 Medullary thyroid carcinoma (MTC) and normal adjacent thyroid tissue (NT). A Immunohistochemistry for calcitonin. B Immunohistochemistry for chromogranin. C Immunohistochemistry for thyroglobulin. Both calcitonin and chromogranin, but not thyroglobulin,are positive in MTC.A positive immunostaining for thyroglobulin is present in NT (x100,Ventana Medical System antibodies). (Kindly provided by Professor F. Basolo, Department of Pathology, University of Pisa, Italy.)

Figure 21.11 Medullary thyroid carcinoma (MTC) and normal adjacent thyroid tissue (NT). A Immunohistochemistry for calcitonin. B Immunohistochemistry for chromogranin. C Immunohistochemistry for thyroglobulin. Both calcitonin and chromogranin, but not thyroglobulin,are positive in MTC.A positive immunostaining for thyroglobulin is present in NT (x100,Ventana Medical System antibodies). (Kindly provided by Professor F. Basolo, Department of Pathology, University of Pisa, Italy.)

carcinoma, which is lethal in almost 50% of cases if not adequately treated. The genotype-phenotype correlation has been well demonstrated by the analysis of 477 MEN2 families studied by the International RET Consortium: no evidence of false-positive RET mutation was described and all patients who underwent thy-roidectomy on the basis of the genetic screening were found to have medullary thyroid cancer [27]. Recently, a new mutation at codon 883 in exon 15 has been reported to result in the development of medullary thyroid carcinoma only in the homozygous condition [125].

Screening for RET gene mutations allows the early discovery of gene carriers, who can be treated with precocious and even prophylactic thyroidectomy, which may provide a definitive cure of this potentially lethal thyroid disease [126].

RET Gene

The RET proto-oncogene is a 21 exon gene that lies on chromosome 10q11-2 and encodes for a tyrosine kinase transmembrane receptor. The receptor is composed of an extracellular domain (EC), with a distal cadherin-like region and a juxta-membrane cystine-rich region, a transmembrane domain and an intracellular domain with tryosine kinase activity (TK) (Figure 21.12). RET is expressed in a variety of neuronal cell lineages including thyroid C cells and adrenal medulla. Recently data indicate that

PI3Kinase Others?

Pathway

RAS-RAF P ath wa y

Figure 21.12 Schematic representation of RET tyrosine kinase receptor. The interaction with the ligand and corresponding co-receptor induces the dimerization and phosphorylation of the receptor, resulting in the activation of the intracellular signaling pathway.

RET gene expression may also occur in follicu-lar thyroid cells [122]. In physiological conditions, the activation of RET protein is secondary to its dimerization due to the interaction with one of its ligands. Four different ligands have so far been recognized: GDNF, neurturin (NTN), persepin (PNS), and artemin (ART). The interaction is mediated by a ligand specific co-receptor (e.g. the GFRa-1 is the co-receptor for the GDNF). The dimerization of RET protein induces autophosphorylation of the TK domain and the activation of downstream signaling pathways [127].

In 1987 genetic linkage analysis localized MEN2 to the centromeric region of chromosome 10 [128,129]. In 1993 two independent groups reported that activating germline point mutations of the RET proto-oncogene are causative events in MEN2A and in FMTC [8,9] (Figure 21.13). One year later, MEN2B was also associated with germline RET proto-oncogene mutations [130]. Since then, a large number of publications have addressed the relationship between RET mutations and the clinical pheno-type of MEN2 patients and the clinical implication of screening MEN2 family members for RET gene mutations.

About 98% of MEN2A cases are associated with RET mutations in the cystine-rich extracellular domain, in particular in codons 609, 611,618,620, and 634 of exons 10 and 11. Mutations at codon 634 of exon 11 (mainly TGC to CGC) are the most common, accounting for 85% of MEN2A cases [27-29,131]. Interestingly, mutation of cystine 634 significantly correlates with the presence of pheochromocytoma and parathyroid adenomas (Table 21.3).

A specific mutation in exon 16, at codon 918 (ATG to ACG) is almost invariably associated with MEN2B. The substitution of methionine with threonine causes alterations in the substrate recognition pocket of the catalytic probe determining the activation of the intra-signaling pathways. Other rare mutations of the intracel-lular domain have been reported in codon 883 of exon 15 [132]. A double RET mutation at codon 804 and 904 has also been described [133]. The Met918Thr mutation is associated with a very aggressive MTC that usually develops during childhood, often only a few years after birth.

In FMTC, the mutations are widely distributed among the five cystine codons 609, 611, 618, 620, and 634 but also in other non-cystine codons, such as codon 804 in exon 14, 891 in

Figure 21.13 Schematic representation of RET gene with the location of all known mutated codons in the three main regions of the gene and the relationship with the MEN syndromes.

CADHERIN-LIKE DOMAIN

CYSTEIN RICH DOMAIN

CADHERIN-LIKE DOMAIN

CYSTEIN RICH DOMAIN

MUTATED CODONS

EXONS

PHENOTYPE

609

10

FMTC-MEN 2A

611

FMTC-MEN 2A

618

FMTC-MEN 2A

620

FMTC-MEN 2A

630

11

FMTC

634

MEN 2A

Î —i

768

13

FMTC

i

790-1

TYROSINE KINASE DOMAIN

883

15

MEN 2B

891

MEN 2B

exon 15 and others (Figure 21.14). A different biological behavior, characterized by a lower aggressiveness and an older mean age at diagnosis, has been described for FMTC associated with mutations in non cystine codons with respect to both MEN2A and FMTC with mutations in cystine codons [28].

In about 4-10% of MEN2A or FMTC patients and in about 95% of those with MEN2B, the germline RET mutation is a "de novo" mutation,

Table 21.3 Correlation between phenotype and RET gene mutations

Most frequently involved codons3 609

611

618

620

634

768

804

918

MEN2A

MEN2A (1) (MTC +

6%

2%

92%

pheochromocytoma +

hyperparathyroidism)

MEN2A (2) (MTC +

3%

4%

13%

80%

pheochromocytoma)

MEN2A (3) (MTC +

8%

1S%

8%

69%

hyperparathyroidism)

MEN2B

97%

FMTC

7%

3%

33%

17%

30%

3%

Sporadic FMTC

9S%

a As a percentage of patients with somatic RET gene mutations. b Somatic mutations detectable in about 40% of sporadic MTC.

a As a percentage of patients with somatic RET gene mutations. b Somatic mutations detectable in about 40% of sporadic MTC.

coo i-CJ

coco

CO CO CO

00 CO CO Oi

coo i-CJ

coco

CO CO CO

00 CO CO Oi

Figure 21.14 Different MEN2 syndromes and corresponding germline RET mutations in an Italian series of MEN2 kindreds (n = 58). (Series of the Department of Endocrinology, University of Pisa, Italy.)

as demonstrated by the negative finding of the RET genetic analysis in the patients' parents. In these cases the mutation is usually located in the allele inherited from the patient's father [134].

Somatic RET mutations are found in about 40% of sporadic cases of MTC mainly consisting of a Met918Thr mutation in exon 16, which is the same mutation also occurring in MEN2B (Figure 21.15). Other RET somatic mutations and also some small deletions have been reported in other codons [135]. Several studies indicate that MTC patients with somatic RET mutations have a poorer prognosis than those with no evidence of RET mutation [136].

P 30

No somatic mutations

Exon 10 Del 48 bp

Exon 11 Cys 634

Exon 15 Ala 883

Exon 16 Met 918

No somatic mutations

Figure 21.15 Somatic RET gene mutations in an Italian series of 77 sporadic MTC. About 50% of cases do not harbor any known RET mutation. The most frequent somatic mutation is the Met918Thr substitution at exon 16. (Series of the Department of Endocrinology, University of Pisa, Italy.)

Several RET gene polymorphisms have been found both in MTC affected patients and in normal subjects. It is still controversial whether some of these polymorphisms have a higher prevalence in MTC with respect to normal individuals and if they play any role in the development of MTC [137-139].

Screening for RET Gene Mutations in MEN2 Family Members

The recognition of the role of RET mutation in MEN2 provided a reliable method to screen family members of an affected proband carrying a germline mutation. From a practical point of view, once the germline RET mutation of the index case has been recognized, blood is taken from all first degree family members. Informed consent and adequate genetic counseling are requested. This allows the identification of "gene carriers" at the time they are still clinically unaffected or at an early stage of the disease. It also has the advantage of excluding "non gene carriers" from further testing for the rest of their life. Although the presence of a germline RET mutation is diagnostic of MEN2 syndrome, gene carriers must be submitted to further clinical and biochemical evaluations to ascertain the actual development of the MTC and its extension, if already present. The involvement of other endocrine organs must also be assessed [29,140].

Screening for RET Gene Mutations in Apparently Sporadic Cases

All patients with MTC, independently from their apparent sporadic origin, should be submitted to genetic screening of the RET gene by analyzing their constitutional DNA derived from blood and, whenever possible, also from tumoral tissue. It is well known that from 5% to 10% of apparently sporadic MTC cases are found to harbor a germline RET mutation being "de novo" or misdiagnosed familial cases. This finding is of great relevance for the early discovery of the other gene carriers in the family who are unaware of their condition.

In sporadic MTC cases, RET gene analysis should be in any case performed also in the tumoral tissue, collected at surgery and kept at -80°C or in the paraffin-embedded tumoral tissue. There are at least three main reasons to justify this procedure: (a) the discovery of a somatic mutation, which usually occurs in 45% of cases, strongly supports the sporadic nature of the tumor; (b) the prognostic value of the presence/absence of the somatic mutation; (c) the future possibility for RET mutated patients to be treated with drugs specifically aimed at inhibiting the altered RET gene.

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