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Interuniversity Center for Research in Hepatobiliary Disease, Institute of Surgical Clinics, University of Siena, 53100 Siena; Department of Pathology, University of Chieti (M.C.C., A.C., P.B.), Chieti; and Institute of Surgical Clinics, Second University of Naples (A.B.), Naples, Italy
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. E-mail: cetta{at}unisi.it
Abstract
Papillary thyroid carcinoma (PTC) is a rare extracolonic manifestation of familial adenomatous polyposis, determined by germline mutations of the adenomatous polyposis coli (APC) gene. The aim of this study was to assess the presence of loss of heterozygosity of APC in the thyroid tumoral tissue. Specimens from six female patients, aged 20–36, were analyzed for germline and somatic mutations of the APC gene by restriction enzyme analysis and sequence analysis. Five of the six also had analysis for ret/PTC, a chimeric gene, the activation of which is restricted to papillary TC. Because a previous study showed that germline mutations in familial adenomatous polyposis-associated thyroid carcinoma were located between codons 140 and 1513, the search for somatic mutations of the APC gene was restricted to this genomic area. Three of the six patients, belonging to the same kindred, had a germline mutation at codon 1061. The remaining three, one per kindred, had germline mutations at codons 1061, 1061, and 1309, respectively. None of the six patients had loss of heterozygosity for APC or somatic mutation in the explored genomic area (codon 545 and codons 1061–1678). Four of five had activation of ret/PTC in the thyroid tumoral tissue, as ret/PTC1 isoform.
Either APC has a tissue-specific dominant effect in the thyroid gland or the germline mutation confers a generic susceptibility to cancer development, but other factors (sex-related factors, environmental radiation, modifier genes) are also required for TC development. This usually involves ret/PTC activation, suggesting a possible cooperation between altered function of APC and gain of function of ret.
FAMILIAL ADENOMATOUS polyposis (FAP) is an inherited autosomal dominant syndrome typically characterized by the development of hundreds to thousands of colorectal adenomas that, if left untreated, turn into colon carcinoma after the third to fifth decade. FAP is caused by germline mutations of the adenomatous polyposis coli (APC) gene, which maps on chromosome 5q21. Many FAP kindreds also show various extracolonic manifestations. Benign FAP-associated extracolonic lesions include congenital hypertrophy of the retinal pigment epithelium (CHRPE), desmoid tumors, epidermoid cysts, and dental abnormalities. In addition to gastric adenomas or carcinomas, duodenal or periampullary neoplasms, and hepatoblastomas, extraintestinal cancers also have been reported, including tumors of the thyroid gland (1). The majority of germline mutations occur in the first half of the APC gene. Specific genotype-phenotype correlations have been established concerning an attenuated adenomatous polyposis coli phenotype (<100 polyps) (2) and a diffuse polyposis phenotype (>5000 polyps) (3), as well as for CHRPE (4) and desmoids (5).
The exact incidence of FAP-associated thyroid carcinoma (TC) has not yet been established. Analysis of the largest polyposis registries yields a 1–2% rate (1, 2, 3, 4, 5, 6). The Leeds Castle Polyposis Group has recently reported an incidence of 1.2% of TC in FAP patients (7). In a recent review of the literature, we collected 97 patients with FAP-associated TCs and added 15 new cases, for a total of 112 subjects (8). There was a striking female predominance (17:1) in patients with at least 2 siblings affected by TC in the same kindred. Six kindreds with at least 2 siblings and 2 kindreds with 3 members affected by TC have been reported (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). This unequivocally demonstrates that thyroid carcinoma is integral to the FAP syndrome (8). FAP-associated thyroid tumors showed age at tumor diagnosis of less than 30 yr in about 90% of cases, papillary histotype in more than 95%, and frequent multifocality (9, 10). It has recently been suggested that FAP-associated TCs show some peculiar histological findings, namely an unusual frequency of the so-called cribriform pattern, with flat cells arranged back to back, which could facilitate proper diagnosis of this type of papillary TC, and an increased detection of FAP-associated thyroid tumors (17).
Most reports on FAP-associated TC are case studies without molecular genetic investigations and/or specific genotype-phenotype correlations. However, there have been some recent papers (9, 10, 11, 12, 16), including three from our institution, that have described APC germline mutations in these patients (9, 10, 11, 12, 16). Somatic inactivation of the residual allele of the tumor suppressor APC gene occurs early in most tumors of the FAP syndrome (19). Therefore, if lack of the APC function also plays a basic role in the development of TC, it is important to determine 1) the incidence of biallelic inactivation, and 2) whether somatic mutations cluster in a specific genomic area. Due to the rarity of the manifestation, a systematic search for loss of heterozygosity (LOH) and screening of the entire gene for somatic mutations in the thyroid tumoral tissue of FAP patients have never been performed. There are only two recent studies in which somatic mutations of the APC gene have been investigated. The former (12) showed absence of LOH in three patients with FAP-associated TC, in the mutation cluster region (MCR; codons 1286–1513), where 65% of somatic APC mutations have been located (19). The latter reported two somatic mutations at codons 1060–1063 and 886, respectively (20).
The aim of the present study was to investigate both germline and somatic APC mutations in the thyroid tumoral tissue of 6 patients with this rare extracolonic manifestation of FAP. Because a previous study showed that most germline mutations in FAP-associated TC (22 of 24) were in the 5'-portion of exon 15, and all of them were between codons 140 and 1513 (8), the search for somatic mutations of the APC gene was restricted to this genomic area. In addition, because two other studies also showed that in FAP-associated TCs there is frequent activation of ret/papillary TC (PTC) (11, 12), a chimeric gene that is restricted to the papillary histotype of thyroid carcinoma (21, 22), a search for activation of ret/PTC was also performed in the same patients.
Materials and Methods
The study includes 6 FAP patients with associated TCs. These patients belong to a series of 51 FAP kindreds and are part of a cooperative study among various European countries including different FAP registries (8, 9). Only patients with both detailed clinicopathological data and genetic analysis were included. None of these 6 patients had gross thyroid abnormality. Three of the 6, all females, aged 20, 22, and 36 yr, respectively, 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, including CHRPE, desmoid in one man, hepatoblastoma, osteomas, dental abnormalities, and duodenal and gastric polyps (23). The remaining 3 patients belonged to 3 different kindreds. The first was a 22-yr-old girl belonging to a small kindred with only 2 affected siblings (the proband and her mother). The second patient, a 24-yr-old girl, had her mother and 1 of 3 brothers affected by FAP. The last patient was a 26-yr-old female belonging to a large FAP kindred. Her mother had colectomy for FAP. Her sister as well as 3 female cousins also had colectomy for polyposis. Interestingly, there was 1 female child with hepatoblastoma. All available living patients underwent colonscopy, 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. Fundus oculi was examined in all 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.
All grossly identifiable nodules, as well as normal thyroid areas, were extensively sampled. Histological techniques in these patients have previously been described (24).
Search for germline mutations of the APC gene
To extract genomic DNA (gDNA), 1 mL whole fresh blood from each sample was spun in a microcentrifuge, and peripheral blood lymphocytes were washed twice with phosphate-buffered saline (1x). Then, gDNA was isolated using the QIAmp Blood Kit 50 (QIAGEN, Chatsworth, CA). One microliter of DNA was used to set up 10-µL PCR reactions.
Search for somatic mutations of the APC gene
Paraffin-embedded sections from thyroid tumor tissue were collected on microscope slides. Areas representative of tumor and normal tissue were identified within single deparaffinized sections lightly counterstained with hematoxylin and microdissected into 1.5-mL polypropylene vials using a hematoxylin/eosin-stained step section from the same block as a guide. The samples were incubated in xylene for 15 min and pelleted at full speed in a microcentrifuge. The xylene was then removed, and the pellet was washed in ethanol. One hundred microliters of digestion buffer containing 1 mol/L Tris-HCl, 0.5 mol/L ethylenediamine tetraacetic acid (EDTA), 0.02% Tween-20, and 100 mg/mL proteinase K were added to each tube. After an incubation of 3 h at 55 C, the samples were pelleted, and the supernatant was stored at -20 C until use (25, 26).
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. The various segments were amplified by PCR, according to Cama et al. (26), using primer pairs previously reported (27). 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 µL of a mixture containing 10 mmol/L Tris (pH 8.3), 2.5 mmol/L MgCl2, 50 mmol/L KCl, 200 mmol/L of each deoxynucleotide triphosphate, 10 pmol of each primer, 0.1 µg gDNA, and 0.25 U Taq polymerase (Perkin-Elmer Corp./Cetus, Norwalk, CT). Samples were denatured at 94 C for 5 min and processed through 30 cycles of amplification at appropriate annealing temperature for 90 s, at 72 C for 90 s, and at 94 C for 1 min, followed by one cycle at 72 C for 10 min. One microliter of the resulting PCR product was used as DNA template in a 10-µL reaction containing the internal pair of primers. PCR products were denatured, cooled on ice, and electrophoresed overnight through a 6% polyacrylamide gel under two conditions: 4 C (25 watts) in a buffer containing 45 mmol/L Tris-borate and 1 mmol/L EDTA, 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 (28). Sequence variants were always confirmed using DNA from independent blood samples.
Heteroduplex analysis on agarose minigel
This technique, previously described (28), was performed to evaluate the presence of the mutation at codon 1061 in exon 15 of the APC. Primer pairs for amplification were designed (5'-GTCCTTCACAGAATGAAAGATG and 3'-ATGTGGTTGGAACTTGAGGTG). The fragment of PCR-amplified DNA, spanning codons 1042–1092, was tested for heteroduplex formation by electrophoresis on a 3% NuSieve and 1% SeaKem (FMC BioProducts, Rockland, ME) agarose minigel stained with ethidium bromide in 90 mmol/L Tris-borate (pH 8.0) and 2 mmol/L EDTA.
Restriction enzyme analysis for the detection of intragenic loss of APC
The silent polymorphism at codon 1678 in exon 15 of the APC gene recognized by the AspHI restriction enzyme was selected for further analysis. DNA was amplified using primers complementary to nucleotides 4960 (5'-GCTACATCTCTAAGTGATCT) and 5145 (3'-GTCATCCAATTCAGGTATGG). The length of the amplified region is 185 bp. The polymorphism creates a new restriction site and consequently two amplified regions of 113 and 83 bp.
Analysis of LOH
We used PCR amplification of polymorphism at the following loci to assess LOH on chromosome arm 5q: four dinucleotide repeats (CA) at D5S644 (5q14–15), at D5S82 and D5S299 (5q15–23) proximal to the APC gene, and at D5S346 (30–70 kb downstream from the APC gene) (30, 31, 32). A two-step protocol was used, consisting of a nonradioactive external PCR, followed by a radioactive internal PCR (nested PCR), and using a 1:104 dilution of primary PCR products as template. The radioactively amplified PCR products were then run on a formamide denaturing acrylamide gel electrophoretic system and analyzed by autoradiography. Loss of a chromosomal marker was considered to be present when the PCR assay showed the absence or more than 50% loss of intensity of a heterozygous band from a tumor sample compared with the corresponding nontumor sample.
Search for ret/PTC activation
The ret gene, which is also involved in multiple
endocrine neoplasm 2A and B, encodes a receptor-type tyrosine kinase
for neurotropic molecules belonging to the glial cell-line derived
neurotropic factor family. After ribonucleic acid extraction from
paraffin-embedded samples, RT-PCR, as described previously (23, 24), was used for subsequent identification of
ret/PTC expression as ret/PTC1, -2, or -3. In
fact, the ret/PTC oncogene derives from the fusion of the
tyrosine kinase domain of the ret protooncogene with the
5'-terminal region of other genes. ret/PTC1 is fused with
another gene, named H4, also located on the long arm of chromosome 10,
whereas the 5'-portions of the ret/PTC2 and ret/PTC3 are represented,
respectively, by the regulatory subunit RI
of the
cAMP-dependent protein kinase A and the RFG/ELE1 gene. The
sequences of the forward primers used were: ret/PTC1,
5'-ATTGTCATCTCGCCGTTC-3' (nucleotides 196–214), ret/PTC2,
5'-TATCGCAGGAGAGACTGT-3' (nucleotides 483–503), and
ret/PTC3, 5'-AAGCAAACCTGCCAGTGG-3' (nucleotides 697–714).
The sequence of the reverse primer, synthesized accordingly to the ret
tyrosine kinase sequence, was 5'-TGCTTCAGGACGTTGAAC-3'
(24). Further details of this method were reported
previously (11, 21, 22).
Results
Data describing genotype-phenotype correlations in the present
series of 6 patients with FAP-associated TC are reported in Table 1
. All patients were females. The mean
age was 25 yr (range, 20–36). None of these 6 patients had gross
thyroid abnormalities. Therefore, there were 4 of 51 FAP kindreds
(7.8%) that had at least 1 member with thyroid carcinoma (1 kindred
had 3 affected members). Three patients had single nodules; 3 had
multiple tumors. All patients had histological diagnosis of papillary
carcinoma. However, an unusual histological pattern, the so-called
cribriform pattern, that has been considered typical for FAP-associated
tumors was found in 3 of the 6 subjects. Two patients had some areas
with the so-called follicular variant of the papillary histotype. A
trabecular pattern was present in one patient, and a solid pattern in
another patient. Capsular invasion and infiltration of local lymph
nodes were present in 2 patients.
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Four patients had unilateral lobectomy, and two patients had total thyroidectomy.
Follow-up
All patients are presently alive and well without local or distant recurrence 5–7 yr after surgery.
Genetic analysis
Three patients belonged to the same kindred and had a germline
mutation of the APC gene at codon 1061. In the three remaining
patients, germline mutations were at codons 1061, 1061, and 1309,
respectively (Table 1
). To investigate LOH of the APC gene, gDNA from
normal and tumoral tissue of the six patients underwent various DNA
analyses in the genomic area of the APC gene between codons 1061 and
1678 and in exon 13. SSCP analysis of exon 13 was performed to
discriminate the heterozygosity of the DNA of the patients
vs. the silent polymorphism at codon 545 of the APC gene
before performing sequence or enzymatic digestion analysis (Fig. 1).
Either normal or tumoral tissues from patient 1329 were heterozygotes
for the polymorphism, showing both bands representative of the two
alleles, whereas patient 8743 was homozygote for one form of the
polymorphism in either normal and tumoral tissue (data not shown).
Therefore, DNA from patient 8743 was not informative for the analysis
of LOH. These data are in agreement with those obtained by sequence
analysis (Fig. 1). After SSCP, the intragenic restriction fragment
length polymorphism at codon 1678 in exon 15 of the APC gene was used
for patient 1329. DNA from patient 1329 was thus digested with a
restriction enzyme, AspHI, which recognizes the silent
polymorphism at codon 1678. Figure 2
shows that both normal and tumoral tissues were heterozygotes for the
GGG/GGA polymorphism, in agreement with data obtained by sequence
analysis (data not shown) demonstrating lack of LOH for patient 1329.
To confirm further the lack of LOH for patients who resulted
homozygotes in the previous analysis, direct mutation analysis of codon
1061 of the APC gene was also carried out by heteroduplex analysis on
agarose minigel. In the three DNA samples, from patients 2934, 1329,
and 8743 carrying APC mutation at codon 1061, in addition to the
wild-type PCR product, heteroduplex bands became visible in normal and
tumoral tissues, confirming the presence of both alleles in the tumoral
tissue (data not shown). In addition to analysis of the APC gene using
intragenic markers, chromosome 5 in areas proximal or distal to the APC
gene was analyzed for LOH. Four highly polymorphic flanking
microsatellite markers surrounding the APC gene were used: D5S299,
D5S82, D5S644, and D5S346. Markers were selected on the basis of
previous information showing high rates of LOH (30, 31, 32).
D5S299 and D5S82 are proximal to the APC gene; D5S644 is distal to the
APC gene. For D5S346, a sequence was identified that contained CA
repeats, located 30–70 kb downstream from the APC gene within the
3'-untranslated message of a nearby gene, DP1. Among these markers,
only tumor tissue from sample 8743 showed LOH in the D5S644 locus (Fig. 3), suggesting that LOH involved regions
of chromosome 5 that were unrelated to the APC gene. Five patients had
their tumoral tissue scanned for ret/PTC activation. Four of
the five had activation of the ret/PTC1 isoform (Table 1). A
detailed description of data concerning ret/PTC activation
was reported previously (11).
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Previous reports have suggested that regions within chromosomes 3p, 11q, 2p, 2q, 10q, and 1p may be frequently deleted in thyroid cell tumors (33). However, in most cases deletions were found in follicular carcinomas or adenomas, whereas no specific areas of chromosomal deletion have been identified in PTC (33). In particular, previous studies of sporadic TCs, including our own, have shown that alterations of the tumor suppressor gene APC, mapped on 5q21 (1, 2, 3), were very rare (2 of 120 cases) (34, 35, 36).
TC is a well known, if infrequent, extracolonic manifestation of FAP (7, 8), a multitumoral syndrome determined by germline mutations of the APC gene. Somatic inactivation of the residual allele of the APC gene is a frequent and early event in most tumors of the FAP syndrome: colorectal adenomas, hepatomas, hepatoblastomas, and desmoids (19, 37, 38). The present series together with Soravia’s series of four of five patients (12) is the only series analyzing specifically both LOH of the APC gene and ret/PTC activation in FAP-associated TCs. Due to the limited availability of thyroid tumor specimens, Soravia et al. restricted screening for somatic mutations to the MCR (codons 1286–1513), which includes over 60% of all somatic APC mutations (19). The analysis of nine different tumor sections from four thyroid tumors showed in only one specimen a deletion of 205 bp and a concomitant insertion of 160 bp between APC nucleotides 4366 and 4571 (codons 1455–1523). Interestingly, this occurred in an unusual patient with an aggressive tumor recurrent after 17 yr, which also had a p53 mutation (12). They found no somatic APC mutation in the other tumors. In particular, no LOH for APC was found (12). In a recent study, Iwama et al. (20) reported 1) a somatic mutation at codon 1060–1063 in the largest tumoral nodule of a 20-yr-old woman with a germline APC mutation at codon 1110 and multiple thyroid tumors; and 2) a somatic mutation at codon 886 in one of multiple thyroid nodules of another patient, a 26-yr-old woman with APC germline mutation at codon 175. Somatic mutations were found in very large nodules, but not in small tumoral nodules, of the same patients. However, this is the only report describing somatic APC mutations in the thyroid tumoral tissue of FAP patients.
In a previous study we have shown that most germline mutations of the APC gene in patients with FAP TC tend to cluster before codon 1230, out of the MCR (codons 1286–1513). Interestingly, the hot spot for APC germline mutations in patients with FAP TC was around codon 1061. In particular, 6 of the 24 patients had mutations at 1061, and 2 patients had mutations at codon 1105, which determined the same stop codon as mutation at 1061, i.e. the same truncated APC protein at 1125–1126 (11). Therefore, in the present series the search for somatic APC mutations was performed not only in the MCR, as in Soravia’s study, but also in the entire genomic area (codon 140-1309), where germline mutations of the APC gene have previously been detected in patients with FAP TC. No LOH or somatic mutation of the APC gene was found in the screened region. Therefore, the most relevant result of both the present study and Soravia’s series is that even in subjects with APC germline mutations, such as FAP patients, complete loss of function of the APC protein is not necessary for thyroid carcinogenesis. This is in accordance with the rare finding of APC somatic mutations in sporadic TCs (34, 35, 36) and shows an unusual behavior of TC compared with other phenotypic manifestations of multitumoral inherited diseases, usually showing LOH of the basic tumor suppressor gene in the tumoral tissue (37, 38). This could suggest 1) a dominant positive function of APC, varying from tissue to tissue, analogously to what occurs in some other multitumoral syndromes; and 2) that APC, which certainly does not play a major role in sporadic TC, even in FAP-associated TC, could simply give a generic susceptibility to TC that for full phenotypic expression requires other cofactors (10). These include modifier genes, sex-related genes (female to male ratio, 17:1 in our recent review of 112 cases) (8), or environmental factors, such as radiation (39).
Whereas LOH of APC is unusual or absent, ret/PTC activation is a very frequent finding in FAP-associated papillary TC (11, 12). Soravia et al. found ret/PTC activation in three of five patients with FAP PTC (100%) (12). We previously documented ret/PTC activation (always as the ret/PTC1 isoform) in four of five patients with FAP TC (11). This is the highest ratio of ret/PTC activation in a well defined subset of patients with TC, even higher than that reported in children from Belarus after the Chernobyl nuclear accident (67–87%) (39, 40). Interestingly, cumulative data confirm that, in contrast to children from Belarus, who usually showed the ret/PTC3 isoform (40), ret/PTC1 was the constant isoform in FAP TC and was detected in seven patients (three by Soravia and four by ourselves). Even more interestingly, the only subject who, in addition to ret/PTC1 positivity, also had focal positivity for ret/PTC3, was the patient, who also had nuclear positivity for p53 and TC recurrence 17 yr after removal of the TC by monolateral lobectomy (12).
Concerning long-term follow-up of subjects with FAP TCs who had detection of both germline mutation and ret/PTC activation, our patients do well without recurrence, but the maximum follow-up interval is presently 7 yr. The occurrence of local recurrence and/or distant metastases even after 17 yr, as in the two patients of Soravia’s series, deserves further evaluation. In particular, in these patients with tumor recurrence, it seems important to screen for p53 mutations and focal activation of ret/PTC3.
In conclusion, thyroid carcinoma occurs in patients with FAP in the absence of biallelic inactivation of the APC gene. The germline APC mutation probably confers a generic susceptibility to thyroid carcinogenesis, but other cofactors are usually required for TC development. This generally involves ret/PTC activation, suggesting a possible cooperation between altered function of APC and gain of function of ret. Further confirmation of lack of LOH for APC in TC is required. Due to the rarity of FAP-associated TC, only international multicentric cooperation, analyzing in detail both clinicopathological data and genetic alterations, will give a better insight into this intriguing extracolonic manifestation of FAP.
Footnotes
1 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
(Grants E611 and E1155). 
Revised September 1, 2000.
Accepted September 13, 2000.
References
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