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Regular Articles |
From the Departments of Surgery*
and Pathology
and Laboratory Medicine
and the Familial
Gastrointestinal Cancer Registry,
Mount Sinai
Hospital, University of Toronto, Toronto, Canada
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The Leeds Castle Polyposis Group has reported an incidence of 1.2% of thyroid carcinoma in FAP patients.14 Although the relative risk of thyroid cancer has been estimated to be 7.6% (95% CL 2.5–17.7) in FAP,15 the absolute risk of developing thyroid cancer is only approximately 2%16 and hence routine screening is not recommended. FAP-associated thyroid cancer is typically characterized by female predominance (94%), age at tumor diagnosis < 30 years (78%), papillary differentiation (89%), and multifocal development.17 Most reports on FAP-associated thyroid cancer are case studies without molecular genetic investigations and/or specific genotype-phenotype correlations.6,17-21 However, three recent reports have described APC germline mutations at codons 848 and 1061.18-20
The molecular pathogenesis of sporadic thyroid carcinoma is still a subject of investigation. Rearrangements involving the ret proto-oncogene have been specifically implicated in the development of papillary thyroid cancer.22 ret/PTC-1 and ret/PTC-3 are formed by paracentric inversions of the long arm of chromosome 10 fusing the tyrosine kinase domain of ret to H4 and ele-1, respectively. ret/PTC-2 is formed by the fusion of the tyrosine kinase domain of ret to 5'-terminal sequences derived from the regulatory subunit RIa of cAMP-dependent protein kinase A.22 These ret/PTC rearrangements appear to be an early event in thyroid tumorigenesis.23 Alterations of p53 have been implicated as a late event that correlates with dedifferentiation. However, the factors that determine progression of intermediate forms of differentiation in thyroid cancer are not clear. Tumor suppressor genes like APC do not appear to be involved in the progression of sporadic thyroid cancer.24-27 However, interactions between ret/PTC1 activation and APC mutations have been postulated in the development of thyroid cancer in FAP patients.28
In the present study, we report two FAP kindreds harboring two distinct germline APC mutations with a variable expression of adenomatous polyposis and thyroid cancer. Further, we have investigated the respective roles of APC, ret/PTC, and p53 genes in the development of thyroid cancer in these FAP patients.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Two FAP kindreds with associated thyroid cancer were identified at
the Familial Gastrointestinal Cancer Registry at Mount Sinai Hospital,
Toronto, Canada. Clinical, endoscopic, operative, histological, and
follow-up data are summarized in Tables 1 and 2
. Molecular genetic testing was
offered with pretest and posttest genetic
counseling.29
Patient accrual, blood samples,
tissue specimen accrual, and predictive genetic testing were carried
out according to protocols approved by the Human Ethics Committee of
the University of Toronto.
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APC molecular screening was done using the protein truncation test (PTT) as previously reported.30 RNA and DNA extraction was performed using TRIzol and DNAzol according to the manufacturer's protocol (Life Technologies, Burlington, Ontario). Reverse-transcriptase polymerase chain reaction (RT-PCR) was carried out using standard techniques. Briefly, cDNA was generated from total RNA (2–5 µg) using random hexamers, 1x first-strand buffer, 0.5 mmol/L deoxynucleotide triphosphates, 10 mmol/L DTT, and 200 U Superscript II reverse transcriptase (Bethesda Research Laboratories, Burlington, Ontario). cDNA and genomic DNA were amplified under the following conditions: initial denaturation at 95°C for 2 minutes; 35–40 cycles, each consisting of denaturation (95°C, 30 seconds), annealing (63–65°C, 1 minute 30 seconds), and extension (70°C, 2 minutes), and a final extension (70°C, 5 minutes). Five-milliliter aliquots of PCR products were electrophoresed on a 1.5% agarose gel to confirm amplification.
The in vitro-synthesized PTT assay was performed using a commercial kit TNT T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI) according to the manufacturer's protocol. Briefly, APC exon 15 was amplified in four overlapping segments as previously described.30 Exons 1 to 14 were also amplified in two overlapping segments, 1A (exons 1–9) and 1B (exons 8–14).9 The 5' end of each forward primer had a T7 promoter sequence and a translation initiation site for coupled in vitro transcription and translation and these products were separated on 12.5% polyacrylamide gels. Positive PTT assays were confirmed by two independent PCR reactions.
For sequence determination, PCR products generated from the putative positive PTT samples were purified using the QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA). The dideoxy-mediated chain-termination method was used for DNA sequencing.31 For this purpose, we used a-33P dideoxynucleotide and the ThermoSequenase radiolabeled terminator sequencing kit (Amersham Life Science, Oakville, Ontario). Sequencing was carried out according to the manufacturer's protocol. Finally, the gel was autoradiographed using a BioMax MR film (Kodak) for 24–48 hours at room temperature.
Molecular Genetic Analysis of ret/PTC and Somatic APC Mutations in Thyroid Cancers
Tissue sections of 20 µm thickness were deparaffinized from archival specimens in 1 ml xylene at room temperature for 20 minutes and washed once with 100% ethanol. After centrifugation, the tissue pellet was air-dried and resuspended in 200 ml of solution containing 6 mg/ml proteinase K (Sigma Canada Ltd., Oakville, Ontario), 1 mol/L guanidinium isothiocyanate, 25 mmol/L ß-mercaptoethanol, 0.5% Sarcosyl, and 20 mmol/L Tris (pH 7.5) and incubated at 45°C for 6 hours. One equivalent volume of 70% phenol/30% chloroform was added and phase separation was carried out at 4°C for 20 minutes followed by centrifugation at 14,000 x g. Overnight precipitation at -20°C followed the addition of one volume of isopropanol and 2 µg of glycogen to the aqueous supernatant. The pellet formed after centrifugation at 14,000 x g was washed with 70% ethanol, air-dried, and resuspended in 10 µl of DEPC water containing RNase inhibitor.
RT was performed on one-fifth of the paraffin-extracted RNA samples. The reaction mixture contained 5 mmol/L MgCl2, 1 mmol/L dNTP, 2.5 mmol/L respective antisense primer, 1 U/µl ribonuclease inhibitor, and 2.5 U/µl Moloney leukemia virus reverse transcriptase (Perkin-Elmer, Branchburg, NJ). RT was performed under following conditions: 15 minutes at 42°C, followed by 5 minutes of denaturation at 99°C and cooled for 5 minutes at 5°C. The integrity of the RNA and efficiency of the RT reaction in each sample was confirmed by PCR for the housekeeping gene PGK-1.22 Each reaction mixture contained 1 µmol/L sense and 0.5 µmol/L antisense primers, 0.3 mmol/L dNTPs, 2 mmol/L MgCl2, and 5 U Taq polymerase (Perkin-Elmer). After an initial denaturation at 94°C for 2 minutes, amplification was performed over 35 cycles consisting of 94°C for 30 seconds, 57°C (PGK-1) or 55°C for 2 minutes (ret/PTC-1, -2, and -3), 72°C for 2 minutes, and a final extension at 72°C for 4 minutes. The products were resolved on a 1.2% agarose gel containing ethidium bromide.
DNA extracted from sections of archival thyroid tumor specimens were screened for somatic APC mutations in the mutation cluster region (MCR, APC codons 1286–1513) of the gene.32 The MCR was divided into 8 overlapping segments and screened for altered conformants by SSCP analysis.33 PCR conditions were as follows: initial denaturation at 94°C for 4 minutes, followed by 30 cycles, each consisting of denaturation (94°C, 1 minute), annealing (55–57°C, 1 minute), and extension (72°C, 1 minute) as previously reported.33
Southern Hybridization
PCR products were transferred to nylon membranes (Boehringer Mannheim, Laval, PQ) by upward capillary action and fixed by UV cross-linking. Digoxigenin labeled probes for ret/PTC-1 and -3 were generated by RT-PCR of thyroid tumors known to harbor ret/PTC rearrangements.22 A cDNA probe for ret/PTC-2 was kindly provided by Dr. Jhiang (Columbus, Ohio). The primers for each probe were identical to those used for RT-PCR (Boehringer Mannheim). Labeling, hybridization, and detection were performed according to manufacturer's protocol.
Histology and Immunohistochemistry
Paraffin blocks of thyroid tumor specimens were sectioned at 5 µm thickness for histological and immunohistochemical evaluation of ret/PTC.23 Immunostaining was performed using a rabbit polyclonal IgG antibody to the carboxyl terminus of ret (Santa Cruz Biotechnology, Santa Cruz, CA). Tissue sections were mounted on sialinized slides and pretreated with 45% formic acid for 15 minutes at room temperature. Endogenous peroxidase was blocked with 3% hydrogen peroxide and nonspecific binding was prevented by incubation with a protein blocker reagent (Signet, Dedham, MA). The primary antibody (1:1000 dilution) was incubated at room temperature overnight, followed by detection with the ultrastreptavidin system (Signet). Immunohistochemical stains for p53 protein were performed on paraffin sections (5 µm) of thyroid tumor specimens, using a streptavidin-biotin technique and a monoclonal antibody (DO-7, dilution 1/100, Novocastra Laboratories Ltd., Newcastle-On-Tyne, UK).26
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Two apparently novel APC germline mutations were found
in these two FAP kindreds.34,35
In kindred #1,
PTT assay demonstrated a truncated mutant protein band of ~40 kd in
APC segment 1B (Figure 1A)
. In
kindred #2, a truncated band of ~50 kd in size was detected in
APC segment 1A (Figure 1B)
. DNA sequence analysis revealed
the truncation in kindred #1 to be caused by a transversion T 
G,
resulting in a substitution of leucine by a stop codon (TTA TGA) at
nucleotide 2092 (codon 698 in exon 15). In kindred #2, the
APC mutation occurred at nucleotides 937–938 (deletion GA)
within exon 9 (codon 313), resulting in a frameshift leading to a stop
codon (TGA) at nucleotides 975–977.
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In individual III-1 (kindred #1), the right lobe of thyroid
contained a mass measuring 3.5 x 2.3 cm. The left lobe contained
multiple lesions; the dominant nodule in the lower lobe measured 2.1
cm, and multiple microcarcinomas measured from 5 to 9 mm. The tumors
had unusual morphological features. The predominant architectural
pattern was that of a solid spindle-cell lesion with whorls and
squamoid nests (Figure 2a)
. This was
punctuated by tubular structures and papillae lined by tall cells with
stratified nuclei (Figure 2b)
. The nuclear morphology throughout was
characterized by irregular nuclear contours, occasional clearing of
nucleoplasm, prominent nucleoli, and frequent nuclear grooves. Rare
psammoma bodies were identified. The large lesions were
surrounded by thick fibrous capsules but there was evidence of capsular
and vascular invasion. The tumor cells were immunoreactive for low and
high molecular weight cytokeratins and displayed weak but unequivocal
staining for thyroglobulin. There was focal nuclear positivity for
p53.
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. The crowded tumor cell nuclei exhibited clearing,
grooves, and cytoplasmic inclusions that are characteristic of
papillary carcinoma. Capsular invasion was seen in both specimens.
Immunoreactivity for thyroglobulin and keratins was strong. The p53
antibodies have a short half-life for normal protein, resulting in only
focal faint or negative staining, whereas mutant p53 usually
accumulates in the nucleus, producing intense positivity. In our tumor
specimens, p53 was identified only in nuclei of cells in solid nests
(Figure 2f)
.
In individual III-1 (kindred#2), the thyroid gland contained multiple
nodules in both lobes varying from 0.6 to 2.3 cm in diameter. Three
nodules, including the largest, had cytologic features of papillary
carcinoma; the remainder were considered hyperplastic nodules. The
carcinomas had the architecture of follicular variant type papillary
carcinomas with follicles storing hypereosinophilic colloid and
occasional papillae. They also contained areas of cribriform
architecture (Figure 2d)
or formation of solid nests. The lesions were
unencapsulated and infiltrated surrounding parenchyma but vascular
involvement was not seen. The tumors contained high molecular weight
cytokeratins and thyroglobulin but immunoreactive p53 was not
identified.
ret/PTC and Somatic APC Gene Analyses in Thyroid Cancers
Thyroid tumor blocks from 3 FAP patients were screened by RT-PCR
for ret/PTC gene rearrangements. In patient III-1
(kindred #1) the dominant tumor and multifocal microcarcinomas were
positive for ret/PTC-1. In patient III-11 (kindred #1) the
dominant tumor was positive for ret/PTC-1 and three of four
blocks showed focal positivity for ret/PTC-3 (Figure 3)
. In patient III-1 (kindred #2)
multifocal papillary carcinoma was found. The three largest tumors were
all positive for ret/PTC-1. No positivity for
ret/PTC-2 was observed in any tumor specimen.
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, which was confirmed by two
independent PCR and sequencing reactions. Further, in an independent
assay by PCR amplification of the same tumor we observed two PCR
products with sequences correponding to the wild-type and
the inserted sequence. GenBank search indicated that this sequence
shared a significant homology (75%) with long interspersed nuclear
element-1 (L1) sequences (AC 003012, human PAC clone DJ0169K13).
Somatic APC mutations in the MCR were not identified in the
remaining eight thyroid tumor sections.
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ret immunoreactivity was positive but focal in all
tumors (Figure 2e)
. The cytoplasmic staining was of variable intensity
in different areas of any individual lesion. Immunoreactivity
correlated with the results of RT-PCR; one of the four tissue blocks
from a single large thyroid tumor (III-11, kindred #1) was negative for
ret by immunohistochemistry and did not express
ret/PTC rearrangements by RT-PCR analysis.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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. Average age at
polyposis diagnosis was 25.4 years (range, 8–50 years). Two female
patients developed thyroid cancer at ages 24 and 38, respectively. An
additional male FAP patient was diagnosed with thyroid cancer at age
24. Both females had recurrent thyroid cancer, although it is important
to note that the initial surgical procedure was not a total
thyroidectomy. Interestingly, in individual III-1, endometrial cancer
was diagnosed at 54 years of age. Another remarkable clinical feature
of this kindred is the early onset of FAP in one branch of the family
(individuals IV-28, 30, 44, and 46). Recently, this aggressive
polyposis feature has been correlated with APC germline
mutations occurring at the proximal end of exon
15.36,37
Our findings of an APC
mutation at codon 698 of exon 15 further supports these observations.
With respect to the second kindred (Table 2)
, the germline mutation is
located within exon 9, one of the three regions of the APC
gene corresponding to the attenuated variant;9
and accordingly the phenotypic expression consists of mild polyposis.
AAPC kindreds are characterized by fewer colonic adenomas (<100),
frequent right-sided colonic tumors, later onset of colorectal cancer,
and rectal polyp sparing. In this kindred, two female patients
developed thyroid cancer at 36 and 49 years of age; individual III-2
was also diagnosed with carcinoma in situ of the endometrium
at age 35. In this AAPC family, mean age at polyposis diagnosis was
42.4 years (range, 25–68 years). Table 3
summarizes the reported APC germline mutations in FAP
kindreds associated with thyroid cancer. Of these, mutations at codons
1061–1063 comprise one of the hot spots for germline APC mutations and
may account for approximately 10% of all germline mutations in FAP
patients.38
Paraf et al20
reported three additional FAP kindreds with thyroid cancer harboring
mutations within exon 14, at the beginning of exon 15, and in the
noncoding region of APC, but without further specific
characterization of the APC
mutations.20
Results from our study and those
published previously suggest that there are no specific
genotype-phenotype correlation with respect to the location of the
APC mutation and the occurrence of thyroid cancer in FAP.
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Somatic APC mutation analysis of thyroid tumors identified an insertion
of L1-like sequence in individual III-11 (kindred #1). Other tumor
specimens from this individual did not show this L1 insertion, possibly
due to the heterogeneity of thyroid tumors. This frameshift insertion
may have caused a truncated APC protein. A similar case of
APC disruption by an L1 insertion has been reported in a
colon cancer, where a duplication of an AT-rich 8-bp consensus sequence
(GAATAATG) was observed at the target site.42
In
our thyroid tumor specimen, the APC sequence flanking the
insertion also harbors the same 8-bp consensus sequence (nucleotide
4571–4578, Figure 4
) although it was not duplicated. This sequence
insertion may be in the same mutant germline allele or the remaining
wild-type allele. We were unable to verify this because the
APC germline mutation in this patient is located upstream
from L1 insertion (at nucleotide 2092) and both mutations could not be
checked in a single PCR assay due to the limited availability of
thyroid tumor specimen. L1 repeats are consensus elements of 6–7 kbp,
consisting of three major regions: (1) at the 5' end, ~1 kb of
sequence containing numerous stop codons in all reading frames, (2) a
sequence consisting of several hundred base pairs at the 3' of element
with no coding potential, and (3) a region of 5 kbp between the 5' and
3' end capable of coding for one or more
proteins.43
L1 repeats vary in length and
insertions ranging from 60 bp to several kilobase pairs have been
reported. The biological function of L1 is not yet fully understood
but, because of its mobility, its insertion is predicted to cause
disruption of protein function. Insertions of truncated L1 repeats in
factor VIII and DMD genes have been identified as
disease-causing mutations.43
We did not find
somatic APC mutations other than this L1 insertion in the
other thyroid tumor specimens but we cannot exclude that some mutations
may exist outside the MCR in these tumors. In addition, because
of insufficient tumor sample availability, a search for loss of
heterozygosity and screening of the entire gene for somatic
APC mutations were not possible.
We have previously demonstrated that ret/PTC rearrangements represent an early event in papillary thyroid carcinoma, are not present in tumors with aggressive morphological features, and are found in young patients (<45 years) with small thyroid carcinomas showing a predisposition for lymphatic involvement.22 Furthermore, ret/PTC rearrangements are early and multifocal events in thyroid tumorigenesis, in particular in microcarcinomas;23 the diversity of ret/PTC profiles also suggest that individual tumors arise independently in a background of genetic and/or microenvironmental susceptibility.23 The immunoreactivity for ret correlated with the RT-PCR expression profile of the gene rearrangements that allow expression of the tyrosine kinase domain of ret in thyroid follicular epithelial cells. Taken together, these findings suggest that the loss of function of the APC gene is associated with gain of function of ret/PTC-1 and -3 in FAP-associated thyroid cancer. However, it is not clear if tumor progression is similar to the multistep colorectal tumorigenesis in these patients.44 Further investigations are needed to clarify the exact role of ret/PTC in FAP-associated thyroid carcinoma and its interactions with the APC gene. The role of other genes such as ß-catenin, implicated in cell adhesion and signal transduction, may also be relevant45,46 and requires further studies to clarify possible interactions of APC and ß-catenin in thyroid tumors. Moreover, the modifier gene(s) may contribute to the phenotypic variations of FAP; ie, in kindred #1, a branch of the family presents with a very early onset of adenomatous polyposis.
Finally, nuclear positivity for p53 was identified only in the recurrent tumor lesions. We have previously reported that p53 immunoreactivity is mainly detected in advanced and aggressive thyroid tumors, and is likely a useful prognostic index of clinical behavior.26 Taking these findings together, we suggest that p53 is a rather late event in thyroid tumorigenesis.
In summary, although specific APC mutation genotype may not correlate with manifestations of thyroid tumors in FAP kindreds, rearrangements in ret/PTC-1 and -3 have a more relevant effect in FAP-associated thyroid tumorigenesis.
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Acknowledgements |
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Footnotes |
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Accepted for publication September 24, 1998.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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