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Familial Adenomatous Polyposis-Associated Thyroid. Carcinoma: Results ... tion affecting about 1–2% of patients with familial adenomatous pol- yposis (FAP).
0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society

Vol. 85, No. 1 Printed in U.S.A.

Germline Mutations of the APC Gene in Patients with Familial Adenomatous Polyposis-Associated Thyroid Carcinoma: Results from a European Cooperative Study* F. CETTA, G. MONTALTO, M. GORI, M. C. CURIA, A. CAMA,

AND

S. OLSCHWANG

Interuniversity Center for Research in Hepatobiliary Disease, Institute of Surgical Clinics, University of Siena, 53100 Siena; and the Department of Pathology, University of Chieti (M.C.C., A.C.), 66013 Chieti, Italy; and Fondation Jean Dausset (S.O.), 75010 Paris, France ABSTRACT Papillary thyroid carcinoma (PTC) is one extracolonic manifestation affecting about 1–2% of patients with familial adenomatous polyposis (FAP). Ninety-seven patients with FAP-associated PTC have previously been reported, including 6 pairs of siblings. During a European collaborative study, 15 patients with FAP-associated PTC were collected. All 15 patients were females. The mean age at thyroidectomy was 24.9 yr (range, 19 –39 yr). In 13 subjects, APC germline mutations had been detected; they were at codons 140, 593, 778, 976, 993, 1061 (n 5 5), 1105 (n 5 1), and 1309 (n 5 2), respectively. A review of the literature added 11 other patients with FAP-associated PTC and detection of germline APC mutations; they were at codons 313 (n 5 2), 698 (n 5 3), 848 (n 5 2), 1209 (n 5 2), 1061 (n 5 1), and 1105 (n 5 1), respectively. The latter led to formation of the same stop codon (TAA) at 1125–1126 as the mutation at codon 1061. Therefore, 21 of 24 mutations were in exon 15 in the genomic area usually associated with congenital hypertrophy of the retinal pigment epithelium (CHRPE), i.e. codons 463-1387. Typical CHRPE was found

in 17 of 18 affected patients who had specific screening. Interestingly, 22 of the 24 patients had their mutation out of the mutation cluster region (codons 1286 –1513), which is currently considered the hot spot mutation area, in particular for extracolonic manifestations of FAP. The difference in the incidence of germline mutations before and after codon 1220 between PTC and non-PTC FAP patients was statistically significant (P , 0.05) for both patients and kindreds (P 5 0.005 and P 5 0.049, respectively). Even if most mutations were scattered throughout the entire 59-portion of exon 15, 8 of 23 patients (6 with mutation at 1061 and 2 with mutation at 1105; i.e. more than one third) had the same truncated protein product. The awareness that patients with PTC usually have APC mutations that cluster in a well defined genomic area, in addition to giving a deeper insight into gene function, could facilitate both earlier diagnosis and better treatment. In particular, intensive screening for thyroid nodules after age 15 yr is recommended when a single patient or an entire kindred have CHRPE and/or mutations in the 59-portion of exon 15. (J Clin Endocrinol Metab 85: 286 –292, 2000)

M

Thyroid carcinoma is integral to many multitumoral syndromes, even if different histotypes are specific for various syndromes (2–5). Familial adenomatous polyposis (FAP) is due to germline mutation of the adenomatous polyposis coli (APC) gene, mapped at 5q21 (6 –10). Papillary histotype was the most frequent histotype in this multitumoral syndrome (11–35). The first documented case of thyroid cancer in a patient with FAP was reported in 1949 (11), but the importance of this association was not fully appreciated until 1968, when Camiel et al. reported two sisters with papillary thyroid carcinoma (TC) and FAP, suggesting that this association was not fortuitous (12). Five additional pairs of sibling have subsequently been reported (13–17). The exact incidence of TCs in FAP patients has not been determined. Analysis of the largest FAP registries suggests that it affects about 1–2% of patients (18, 19). FAP-associated TCs exhibit a marked female preponderance (female to male ratio, .10:1) and are more common under the age of 30 yr (11–35). It has recently been suggested that FAP-associated thyroid tumors share some unusual and peculiar histological findings (namely an increased frequency of the so-called cribriform pattern), which could facilitate early detection (16, 24, 27, 31). Reports of patients with TC associated with FAP are very rare. In a review of world literature by Bell and Mazzaferri in 1993 (26), 49 cases with such association were found. A year later, a total of 63 patients were reported by Harach (27). Only a few of them had detection of the APC germline

OST OF THE inherited multitumoral syndromes are due to germline mutations of a tumor suppressor gene, which confer a selective growth advantage. In fact, tumor suppressor gene mutations are by definition accompanied by an immediate capability for territorial expansion and/or selective proliferation (1). The knowledge of the exact site of the germline mutation in patients with a given tumoral phenotype is of importance for both diagnostic and pathogenetic purposes. In fact, it could restrict multitumoral analysis to some codons or intensive clinical screening only to patients carrying some mutations, excluding patients with other mutations. In addition, because most mutations of tumor suppressor genes are inactivating mutations, leading to truncation of the protein product, knowledge of the site of truncation of the protein, thus more frequently determining a given tumoral phenotype, could yield a better insight into both the biological function of the protein and the multistep process of carcinogenesis (1). Received June 25, 1999. Revision received August 12, 1999. Accepted August 24, 1999. Address all correspondence and requests for reprints to: Francesco Cetta, M.D., Institute of Surgical Clinics, University of Siena, Nuovo Policlinico, Viale Bracci, 53100 Siena, Italy. * This work was supported in part by the National Research Institute (Grants 93.00239.CT04, 94.02376.CT04, and 95.00897.CT04), Regione Toscana (Grant 358/C, 1995), MURST 40%-MURST 60%, and TELETHON (Grant E611).

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mutation. Therefore, only multicentric studies can collect a sufficient number of patients with FAP-associated TC. We have recently reported a kindred with 3 siblings with FAPassociated TC (APC mutation at codon 1061) and a second kindred with APC mutation at codon 1309 (23–25). Here we report clinical, histological, and genetic findings in a cumulative series of 15 patients (including the 4 already mentioned) with FAP-associated TC, who were recruited in the course of an international cooperation. In particular, genotype-phenotype correlations were analyzed for both subjects of the present series and patients with FAP-associated TC from the world literature. Materials and Methods The 15 patients with FAP-associated TC were selected during an international cooperative study among various European countries including different FAP registers. Only patients available for genetic analysis were recruited. Six of these patients were observed in a single institution. In particular, 3 of them, all females, belonged to the same kindred. The extended pedigree of this kindred (23 siblings in 4 generations) has been reported previously together with a detailed list of all extracolonic manifestations (23). All living patients underwent colonoscopy, upper gastrointestinal endoscopy (supplemented by x-ray examination of the gastrointestinal tract in selected cases), and multiple biopsies. In addition, patients were screened for osteomas, dental abnormalities, and desmoid tumors. The fundus oculi was examined for congenital hypertrophy of the retinal pigmented epithelium (CHRPE) in 12 patients,. All patients underwent ultrasound examination of the thyroid gland. Fine needle aspiration (FNA) of nodules larger than 5 mm was performed. Some patients underwent multiple ultrasound and FNA procedures. Cytological examination of the FNA specimens was performed according to standard methods. In 5 patients, search for activation of the ret-papillary TC (PTC) oncogene was also performed.

Histological techniques All grossly identifiable nodules as well as normal thyroid areas were extensively sampled. Sections were routinely stained with hematoxylin and eosin. Immunohistochemistry was carried out using the following monoclonal antibodies: thyroglobulin (BioGenex Laboratories, Inc., San Ramon, CA; diluted 1:500), chromogranin A (Dakopatts, Glostrup, Denmark; diluted 1:200), carcinoembryonic antigen (Immunotech, Marseilles, France; diluted 1:10), and cytokeratin AE1/AE3 (Roche Molecular Biochemicals, Mannheim, Germany; diluted 1:1000). Color was developed using the APAAP method. A polyclonal antibody against calcitonin (BioGenex Laboratories, Inc. diluted 1:200) was also used, and the color was developed with 3,39-diaminobenzidine tetrahydrochloride (24). DNA extraction. Extraction of normal and tumor DNA from fresh samples was performed using standard methods. Formalin-fixed, paraffinembedded sections, 5–10 mm in thickness, were collected on glass slides and stained with hematoxylin. After pathological review, areas of normal tissue and tumor were marked and microdissected; if the areas of interest could not be clearly separated from the surrounding tissue,

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selective ultraviolet radiation fractionation was performed. To extract genomic DNA, microdissected samples were incubated in xylene, spun in a microcentrifuge, and washed twice with absolute ethanol. Pellets were resuspended in 100 ml digestion buffer containing 100 mg/ml proteinase K. After overnight digestion at 55 C, the samples were heated at 80 C for 10 min to inactivate proteinase K, rapidly cooled, and stored at 4 C. One microliter of DNA was used to set up 10-mL PCR reactions. Single strand conformation polymorphism (SSCP) and sequencing. The entire coding region (8532 bp) of the APC gene was analyzed by the PCR-SSCP method in all patients. All the amplified segments were 250 – 400 nucleotides long. PCR-SSCP analysis was performed as previously described (9, 10, 35, 36). To increase PCR specificity, a two-step protocol was used, consisting of a nonradioactive external PCR followed by a radioactive internal PCR that used a 1:10,000 final dilution of the primary PCR as a template. The external PCR was performed in 10 ml of a mixture containing 10 mmol/L Tris (pH 8.3), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 200 mmol/L of each deoxynucleotide triphosphate, 10 pmol/L of each primer, 0.1 mg complementary DNA, and 0.3 U Taq polymerase (Perkin-Elmer Corp./Cetus, Norwalk, CT). Samples were denatured at 94 C for 5 min and processed through 30 temperature cycles, consisting of 90 s at 58 C, 90 s at 72 C, and 1 min at 94 C, followed by 1 cycle at 72 C for 10 min. One microliter of the resulting PCR product was used as DNA template in a 10-mL reaction containing the internal pair of primers. PCR products were denatured, cooled on ice, and electrophoresed overnight through a 6% polyacrylamide gel under 2 conditions: 4 C (25 watts) in a buffer containing 45 mmol/L Tris-borate, 1 mmol/L ethylenediamine tetraacetate, and 24 C (7 watts) in the same buffer plus 5% glycerol. Gels were autoradiographed for 1–2 days without intensifying screens. PCR products corresponding to samples showing unique SSCP conformers were directly sequenced as previously described (9, 10). Sequence variants were also confirmed using DNA from independent blood samples.

Results

Table 1 shows patients with FAP-associated TC, reported in the world literature. Table 2 shows findings in the 15 patients collected as a result of our international cooperation. All patients were females. The mean age was 24.9 yr (range, 19 –39 yr). All patients had typical papillary carcinoma with at least some areas containing complex and branching papillae, slightly irregular nuclei with a ground glass appearance, and frequent grooving. An unusual histological pattern, the so-called cribriform pattern, that has been considered typical of FAP-associated tumors (16, 23, 24, 31) was found in 6 subjects. A solid pattern was found in 4. Three patients had some areas with the so-called follicular encapsulated variant of the papillary histotype (24). Interestingly, 3 siblings belonging to the same kindred showed 3 different histological patterns (24). The specific APC mutation was detected in 13 of 15 patients. Table 2 shows both the wildtype sequence and the mutant sequence of the APC gene in each patient. In particular, there were 5 patients with APC

TABLE 1. Summary of the review of the world literature on FAP-associated thyroid carcinoma No.

97

Age at diagnosis of TC (yr)

Age at diagnosis of FAP (yr)

M

Sex F

Mean age

Range

Mean age

Range

10

87

28

12– 62

24.84

11–50

FAP, Familial adenomatous polyposis; HTC, hyalinizing trabecular carcinoma; TC, thyroid carcinoma.

Histological type of TC (No.)

Papillary (42) Cribriform (13) Follicular (4) Adenocarcinoma (1) Adenoma (1) HTC (1) Not reported (28)

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TABLE 2. Genotype-phenotype correlations in 15 patients with FAP-associated thyroid carcinoma APC germline mutation

Patient no.

Sex

Age (yr)

Histotype of thyroid tumors

CHRPE

Exon

Codon

Wild-type sequence

Mutant sequence

1 2 3 4 5 6 7a 8a,b 9b 10b 11 12 13c 14 15

F F F F F F F F F F F F F F F

30 19 22 18 27 39 26 22 20 36 24 20 27 22 20

Papillary Papillary Papillary Papillary Papillary Papillary Papillary Papillary Papillary Papillary Papillary Papillary NA Papillary Papillary

2 1 1 1 1 1 1 1 1 1 1 1 NA NA NA

3 14 15 15 15 15 15 15 15 15 15 15 15 15 NA

140 593 778 976 993 1105 1061 1061 1061 1061 1061 1309 1309 NA NA

GAGAGgt TTA AATTAA AGA AAG CGGGGA AAACAAAGT AAACAAAGT AAACAAAGT AAACAAAGT AAACAAAGT AGAAAAGAT AGAAAAGAT NA NA

GAt TGA AATTTAA A AA CGGGA AAGT AAGT AAGT AAGT AAGT AGAT AGAT NA NA

NA, Not available; NP, not performed. Hepatoblastoma also associated in the same kindred. b Siblings belonging to the same kindred. c Mutation detected by Varesco et al. (35). a

FIG. 1. Sequence analysis in a FAP patient with APC germline mutation at codon 1061, which is the most frequent mutation in FAPassociated TC. Sequencing ladders were obtained by direct sequencing of PCR-amplified DNA derived from an unaffected individual and from the FAP patient with TC. The 5-bp deletion (AAACA) at codon 1061 of the APC gene causes a shift in the sequencing ladder of the FAP patient.

mutation at codon 1061 (Fig. 1). Three belonged to the same kindred (23). A fourth patient belonging to this kindred, a 15-yr-old girl, also had thyroid nodules that were negative for malignant transformation at fine needle biopsy (24). One patient had mutation at codon 1105. This mutation led to the formation of the same stop codon (TAA) at 1125–1126 as a mutation at codon 1061. Interestingly, 3 kindreds with germline mutation at codon 1061 had hepatoblastoma in addition to TC (37, 38) in one member of the kindred; the other 10 subjects had TC. Three patients had foci of solid growth with capsular infiltration. Two of the 3 also had micrometastasis in a neck lymph node. All patients are alive and disease free,

with a minimum follow-up greater than 5 yr. In particular, 3 patients belonging to the same kindred had monolateral lobectomy performed instead of total thyroidectomy because of personal preference. Eleven of 12 patients had CHRPE. Table 3 shows findings in the 11 patients with FAP-associated TC and sufficient genetic and clinical information from the world literature. In particular, 9 patients (belonging to 5 kindreds) had already had their APC mutations published in papers. The site of the APC mutation in the last 2 patients, who belonged to the same kindred (mother and daughter), was reported in a personal communication (Bulow, S., Copenhagen, Denmark). Additional germline mutations in patients with TC and insufficient details were at codons 302, 622, 1156, and 1597, respectively (39, 40). Taking together data from Tables 2 and 3, 20 of 24 patients had a mutation in exon 15, and 21 had a mutation in the genomic area, which is usually associated with CHRPE, i.e. codons 463-1387. Typical CHRPE was actually found in 17 of 18 affected patients, who had specific screening. In particular, 6 patients had mutations at codon 1061 (Fig. 1), and 2 patients had mutations at codon 1105, which produced the same biological effect as mutation at 1061, i.e. more than one third had the same truncated APC protein at 1125–1126. Figure 2 shows the prevalence of the various germline mutations of the APC gene in the cumulative series of 24 patients. Interestingly, 22 of 24 patients had their mutations out of the mutation cluster region (MCR; codons 1286 –1513), where 65% of somatic mutations and 23% of germline mutations were located (41). An arbitrary cut-off point was set at codon 1220. Comparing FAP patients with TC observed in the cumulative series (22 before codon 1220 and 2 39 to this codon) with FAP patients without TC, obtained from an international database (42) (201 with germline mutation before and 116 with mutation after codon 1220), a statistically significant was observed (x2 5 7.87; P 5 0.005; Fig. 3). When comparison was made among kindreds (14 with mutations before codon 1220 vs. 2 with downstream mutations), a sta-

MUTATIONS OF THE APC GENE IN FAP-ASSOCIATED TC

289

TABLE 3. Genotype-phenotype correlations in patients with FAP-associated thyroid carcinoma (literature review) Sex

Age (yr)

Histotype of thyroid tumors

CHRPE

Exon

15a,b

F

16

Papillary

1

15

848

15b

F

12

Papillary

1

15

848

34 32a Bulowb,c Bulowb,c 17b 17b 17b 17b 17b

F F F F F F M F F

18 30 40 19 38 24 23 49 36

Papillary NR Papillary Papillary Papillary Papillary Papillary Papillary Papillary

1 1 1 1 NR NR NR NR NR

15 15 15 15 15 15 15 9 9

1061 1105 1219 1219 698 698 698 313 313

Ref. no.

Codon

Wild-type sequence

Mutant sequence

AGATAGAAAA A AGATAGAAAA A AAACAAAGT CGGGGA TCCACA TCCACA GCATTATGG GCATTATGG GCATTATGG GTGGAAATG GTGGAAATG

AGAGAATAA AGAGAATAA AAGT CGGGA NR NR GCATGATGG GCATGATGG GCATGATGG GTGAATG GTGAATG

NR, Not reported. a These patients also had desmoids. b Siblings belonging to the same kindred. c Bulow, S., et al., personal communication.

FIG. 2. Prevalence of germline mutations of the APC gene in the cumulative series of 24 patients.

tistically significant difference was also obtained (x2 5 3.86; P 5 0.049). Discussion

A total of 112 patients with FAP-associated TC have been studied, including data from the literature (n 5 97) (11–35) and the present series of 15 patients. There were 11 men and 101 women. However, restricting the female to male ratio only to kindreds with at least a couple of siblings showing FAP-associated TC, i.e. kindreds in which TC cannot be considered a casual finding, there were 17 women and 1 man. The mean age at diagnosis of TC and/or thyroidectomy was 24.8 yr in our series, 28 yr in the 97 patients from the world literature, and 27.65 yr in the entire series of 112 patients. Age at diagnosis of FAP was slightly different (26.84 yr), with about one third concomitant diagnosis, one third first diagnosis of FAP, and one third first diagnosis of TC. The histological type of TC was almost always papillary TC (.95% of cases). This topic has been covered extensively (16, 24, 27,

31). A papillary pattern was usually detectable, but a very unusual pattern, the so-called cribriform pattern was very frequent in these tumors (16, 24, 27, 31). However, other patterns (solid, trabecular, etc.) also including follicular areas have been described in association with the classic papillary pattern. Therefore, the minority of follicular carcinomas (22) and/or adenomas (28) previously reported in the literature deserves histological reevaluation on the basis of current knowledge. In the present series, 13 of 15 patients with FAP-associated TC had detection of their germline APC mutations. Eleven had mutation in the 59-portion of exon 15 between codons 778 and 1309, whereas 1 patient had a mutation at codon 593 (exon 14), and the last patient had a mutation at codon 140 (exon 3). Twelve of 14 patients had their mutations in exon 15, and 12 had mutations in the genomic area between codons 463-1387 that is usually associated with CHRPE (40), Typical CHRPE was actually found in 11 of 12 affected patients who had specific screening. In particular, APC muta-

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FIG. 3. An arbitrary cut-off point was set at codon 1220 of the APC gene. Two hundred and one PTC-FAP patients had their germline APC mutations 59 to and 116 39 to this codon. On the contrary, 22 of 24 PTC1 FAP patients had their germline mutations 59 to codon 1220 (x2 5 7.87; P 5 0.005). PTC 2, FAP patients not affected by TC; PTC 1, FAP patients affected by TC.

tion at codon 1061 was a hot spot for TC and another rare extracolonic manifestation of FAP such as hepatoblastoma (37, 38). In the literature review, 11 additional patients were found with FAP-associated PTC, with detection of an APC germline mutation. In particular, a mutation at codon 1105 was reported in a 30-yr-old Japanese female with colorectal and gastric polyps, TC, and CHRPE. This mutation leads to the formation of the same stop codon (TAA) at 1125–1126 as mutation at codon 1061 (32). Most of these mutations also were in the 59-portion of exon 15. In particular, in the kindred with mutation at codons 848 (15) and 1105 (32), patients also had desmoid tumors. One male patient of our kindred with 3 siblings who had PTC and germline mutation at codon 1061 (23, 24) also had postcolectomy desmoid. The most frequent mutations were at codons 1061 and 1309, where most APC mutations are found. This could suggest that TC occurs in some siblings belonging to kindreds with the most frequent mutations or those usually determining the widest range of extracolonic manifestations. However, 22 of the 24 patients had their mutation out of the MCR (codons 1286 –1513), that is currently considered the hot spot mutation area, in particular for extracolonic manifestations of FAP (41). The difference in the incidence of germline mutations before and after codon 1220 between TC and non-TC FAP patients was statistically significant when comparison was made among individuals (P 5 0.005) or among kindreds (P 5 0.049). Even if most mutations were scattered throughout the entire 59-portion of exon 15, 8 of 23 patients (6 with mutation at 1061 and 2 with mutation at 1105; i.e. more than one third) had the same truncated protein product, determined by the same stop codon (TAA) at 1125–1126. Genotype-phenotype correlation is not easy to detect in patients with FAP and germline APC mutations (43, 44). Whereas a clear-cut correlation has been established for CHRPE, desmoids (45, 46), and number of colon polyps, no correlation was found for periampullary tumors (47). The present data strongly suggest a cluster of mutations in the 59-portion of exon 5 in FAP-associated TC. For the moment, no clear-cut relationship can be established between the cribriform histotype and the presence or site of germline APC

mutations. On the contrary, a wide range of histotypes has been observed not only in patients with the same germline mutation, but also in siblings belonging to the same kindred (17, 24). The molecular bases for the high prevalence of TC in FAP patients are still obscure. Familial aggregation is indisputable. In particular, the occurrence of papillary TC in six pairs of siblings (12–17) and two kindreds with three (or more) siblings (17, 24) confirms that TC is undoubtedly an aspect of the FAP syndrome. This view is given further support by present observation of three FAP kindreds with TC in association with hepatoblastoma, which is very rare as a sporadic tumor but extremely frequent in FAP (estimated greater risk, .1000:1). This suggests a role for APC in thyroid carcinogenesis. Somatic mutations of the APC gene have previously been searched for in sporadic thyroid tumors. However, at least three studies (48 –50) failed to demonstrate significant alterations in the MCR of the APC gene in sporadic tumors. The present data suggest that restriction of the mutational analysis of the APC gene in patients with TC to MCR will detect germline and/or somatic mutation in less than 20% of cases, even if there is a different aggregation between germline and somatic mutations (32). However, the absence of somatic APC mutations in thyroid tumors supports the view that alterations of the tumor suppressor gene alone do not represent a frequent event in thyroid tumorigenesis. Somatic inactivation of the residual allele of the APC gene occurs early in colonic polyps and cancers, hepatoblastomas, and desmoids tumors of FAP patients (34, 37, 38, 45, 46). In the present series, at least in the six patients who had this specific analysis, there was no loss of heterozygosity for the APC gene in the thyroid tumoral tissue (51, 52). On the contrary, in these same thyroid tumors, there was a very high rate of activation (80%) of the ret-PTC gene, a chimeric oncogene that is restricted to the papillary histotype (25). Similar findings have recently been reported by Soravia et al. (17). If the observations that the wild-type APC allele is not lost and there is a high rate of ret-PTC activation in FAPassociated thyroid tumors will be confirmed in larger series, this could suggest that whereas for colonic polyps, desmoids, and liver tumors double mutation is required, in the devel-

MUTATIONS OF THE APC GENE IN FAP-ASSOCIATED TC

opment of TC a single copy of the inactivated gene could be sufficient (tissue-specific dominant effect) (53). An alternative hypothesis is that the germline mutation of the APC gene confers only a generic susceptibility to thyroid cancer, but perhaps other factors, namely modifier genes, sex-related factors (female-male ratio, 17:1) or environmental factors, such as radiation, are also required for the phenotypic expression (54 –56). In conclusion, FAP-associated TC seems to be a particular subtype of TC with a predominant, even if not constant, histological aspect that probably reflects cooperation among carcinogenetic genes different from those occurring in sporadic tumors. Recent findings have contributed to highlighting some aspects, but several questions still remain unanswered. However, due to the rarity of this extracolonic manifestation of FAP, international cooperation is mandatory for an exhaustive analysis of the few available cases. The awareness that patients with PTC usually have APC mutations that cluster in a well defined genomic area in addition to giving a deeper insight into gene function could facilitate both early diagnosis and better treatment. In particular, intensive screening for thyroid nodules after age 15 yr is recommended when a single patient or an entire kindred has CHRPE and/or mutations in the 59portion of exon 15. If this finding is confirmed, it could restrict the range of patients at risk and be of major importance in terms of cost-effective screening.

19. 20. 21. 22. 23. 24. 25.

26. 27. 28. 29. 30. 31.

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