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Original Studies |
Department of Medicine (E.L.C., W.M.W., J.R.T., Q.D.), University of Sydney, Department of Anatomical Pathology (K.T.T., S.W.M.) and Head and Neck Surgery (N.P., C.J.O.), Royal Prince Alfred Hospital, and Dr. Lauer Pathology (C.S.L.), Sydney, New South Wales 2006, Australia; and Laboratoire d’Anatomie et de Cytologie Pathologiques (D.D.), Noumea, 98846 New Caledonia
Address correspondence and requests for reprints to: Dr. Elizabeth L. Chua, Department of Medicine, D06, University of Sydney, Sydney, New South Wales 2006, Australia.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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During the past few years, there have been significant advances
in our understanding of the molecular events associated with thyroid
cancer. From these studies, many genes (oncogenes and tumor-suppressor
genes) have been implicated in the pathogenesis of thyroid cancer. An
important candidate gene that has been extensively studied is the
ret protooncogene. This gene encodes a receptor tyrosine
kinase and is expressed in cells of neural crest origin (4). The ligand
for ret is the glial cell line-derived neurotrophic factor
(5) and neurturin (6). Normally, ret is not expressed in
thyroid follicular cells. In thyroid papillary carcinomas, however,
expression of the ret gene can be detected at both RNA and
protein levels. The mechanism for expression in cancer cells involves a
gross chromosomal rearrangement, resulting in the formation of a fusion
gene. There are 3 main types of ret rearrangements
identified in papillary carcinomas, designated as ret/ptc1,
2, and 3. In all types, the part of the ret gene coding for
the signal transducing tyrosine kinase is fused to a new upstream
region derived from different genes that are normally expressed in
thyroid follicular cells. The expression of ret is therefore
driven by the promoters of the fused genes (H4 for ret/ptc1,
RI
for ret/ptc2, and ele1 for ret/ptc3),
resulting in permanent activation of the tyrosine kinase activity of
ret (7, 8, 9). Two other types of ret rearrangements
(ret/ptc4 and 5) have also been described recently (10, 11).
Prompted by the high incidence of thyroid carcinoma in New Caledonia, the aim of this study was to investigate the prevalence and relative distribution of ret/ptc1, 2 and 3 in papillary carcinoma in this population and to compare the pattern with that of an Australian population whose incidence is much lower, at 4.5 females and 1.6 males per 100,000 population (12).
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Fresh-frozen thyroid tissues were obtained from the Laboratoire d’Anatomie et de Cytologie Pathologiques in New Caledonia, the Royal Prince Alfred Hospital, and Dr. Lauer Pathology in Sydney, Australia. After surgical removal, cancer specimens were immediately dissected from the center of nodules, by the respective pathologists; frozen in liquid nitrogen; and stored at -70 C until RNA extraction. After confirmation of papillary carcinoma (1988 WHO histological classification of thyroid tumors), the cancer specimens were cut into blocks of approximately 40 mg each. To determine the percentage of cancer tissue in each block, a section from each block was removed with a sterile surgical blade, fixed in formalin, and embedded in paraffin. Hematoxylin-eosin (H&E)-stained slides, prepared from these sections, were reviewed by two pathologists (K. T. Tran and S. W. McCarthy). Only those blocks confirmed to contain at least 70% cancer cells were included in the study.
Formalin-fixed paraffin-embedded thyroid tissues of patients with papillary carcinoma who had thyroidectomies between 1987 and 1998 were also included in the study. Consecutive macrocarcinoma cases from the anatomical pathology database were included, depending on the availability of H&E-stained slides and paraffin blocks. Two pathologists (K. T. Tran and S. W. McCarthy) reviewed the slides for confirmation of diagnosis and subclassification of papillary variants. Paraffin blocks with at least 70% cancer cells or with well-delineated cancer nodules were obtained. The selected blocks were then aligned with their corresponding H&E slides, and sterile surgical blades were used to obtain very thin slices from the confirmed cancer areas. Approximately 10 mg of tissue were collected in a 1.5-mL microfuge tube for each sample.
To avoid a possible confounding impact caused by differences in ethnicity within each study population, the New Caledonian cases were limited to Melanesians and the Australian cases limited to Caucasian Australians. Because the Melanesians comprise a majority of the population and have been shown to have a higher incidence of thyroid cancer than any other ethnic groups (Europeans, Polynesians, and Asians) living in New Caledonia (2), we have limited the New Caledonian samples to this homogeneous and representative group. In a similar manner, because Australia is a multicultural country with people of various ethnic backgrounds, we have limited our cases to a more homogeneous population of Caucasian Australians. In addition, none of the patients had a known history of radiation exposure. The study protocol was approved by the Central Sydney Area Health Services Ethics Review Committee.
RNA extraction
Total RNA was extracted from frozen thyroid tissues, after homogenization, using a pellet pestle (Kontes, Vineland, NJ) in the presence of TRI reagent (Sigma, Castle Hill, New South Wales, Australia), according to the manufacturer’s protocol. RNA was quantified by UV spectrophotometry (OD260).
For paraffin-embedded tissues, total RNA was extracted as described previously (13), with the following modifications. Briefly, tissue was deparaffinized with three 10-min incubations of 1 mL dipentene (Histolene, Fronine Pty Ltd, Riverstone, New South Wales, Australia) at room temperature, followed by graded (100%, 80%, 50%) ethanol washes and extraction with diethylpyrocarbonate-treated water. The tissue was solubilized by proteinase K digestion at 50 C for 16 h in 450 µL buffer with a final concentration of 500 mmol/L Tris, 20 mmol/L EDTA, 10 mmol/L NaCl, and 0.5% SDS (pH8.0). After solubilization, nucleic acid was extracted by following a standard phenol:chloroform protocol and was precipitated at -20 C overnight with 0.8 vol isopropanol and 20 µg glycogen. The nucleic acid pellet was washed in 75% ethanol, air-dried, and resuspended in 10 µL diethylpyrocarbonate-treated water.
RT-PCR
Total RNA (1 µg from frozen specimens or 1 µL from
paraffin-embedded samples) was reverse transcribed to complementary DNA
(cDNA) with Superscript II reverse transcriptase (Life Technologies, Melbourne, Victoria, Australia) primed with both
oligo-dT and random hexamers (Life Technologies) in a
final concentration of 20 ng/µL and 1 ng/µL, respectively. A
recombinant ribonuclease inhibitor (RNasin, Promega Corp.,
Sydney, New South Wales, Australia), in a final concentration of 1.6
U/µL, was also included in the reaction. All RT reactions were done
independently of the PCR reactions. Amplification of c-N-ras
with PCR, followed by hybridization with a c-N-ras probe,
was used as quality control for RNA integrity in both frozen and
paraffin-embedded samples. Only cDNA samples producing the
c-N-ras band with the expected size underwent amplification
with primers flanking rearrangements corresponding to
ret/ptc1, 2 and 3 (Fig. 1
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Briefly, 2.5 µL cDNA was amplified with 0.2 µmol/L primers, 0.4
mmol/L deoxynucleotide triphosphates, 1 x PCR buffer (PE Applied Biosystems, Foster City, CA) and 0.02 U/µL
Taq DNA polymerase (PE Applied Biosystems) in a
final vol of 25 µL. Samples were denatured at 94 C for 2 min,
followed by 40 cycles of denaturation at 94 C for 30 sec, annealing at
55 C for 30 sec, and extension at 72 C for 30 sec in a
Perkin-Elmer Corp. 9600 thermal cycler. Positive controls
of the rearrangements were diluted plasmids (500 pg each) harboring
ret/ptc1, 2 and 3 sequences (kindly provided by Dr. Y.
E. Nikiforov, University of Cincinnati). Water controls, which
consisted of all reagents except the cDNA templates, were used as
negative controls. Both positive and negative controls were included in
each batch of PCR. The RT-PCR was repeated at least twice for each
sample, using two batches of RNA extracted at different times from the
same tumor.
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The PCR products (25 µL) were electrophoresed on a 1.5%
agarose gel. In addition to the positive and negative controls from
PCR, at least one well was not loaded with PCR products and served as
an additional form of negative control for nonspecific hybridization.
PCR products were transferred to a nylon membrane (Hybond-N,
Amersham Pharmacia Biotech, Castle Hill, New South Wales,
Australia) by vacuum blotting (MilliBlot-V Transfer System,
Millipore Corp., Ryde, New South Wales, Australia) for
1.5 h. Probes specific for each of the fused genes (Table 1) were end-labeled with
[
-32P] ATP using T4 polynucleotide
kinase (Promega Corp.) and purified with Nuctrap columns
(Stratagene, Willoughby, New South Wales, Australia).
After overnight hybridization at 42 C in the hybridization solution
(ExpressHyb, CLONTECH Laboratories, Inc., Darra,
Queensland, Australia) with a probe concentration of 1–2 x
107 cpm/mL , membranes were subjected to 20-min
incremental stringency washes of 5 x SSC/0.1% SDS, 2 x
SSC/0.1% SDS, 1 x SSC/0.1%SDS, and 0.5 x SSC/0.1% SDS at
42 C. The membranes were then exposed to a storage phosphor screen
(Cyclone, Packard, Mount Waverly, Victoria, Australia) for 3 h
before hybridization signals were analyzed using
an image analysis software (OptiQuant, Packard).
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To generate enough amplicons of the ret
rearrangements for sequencing, 2.5 µL of the primary PCR products
were used as templates for nested PCR. The nested PCR mix and
conditions were the same as the primary PCR, except for
ret/ptc2, which was performed at an annealing temperature of
52 C. PCR products were separated by gel electrophoresis in 1%
low-melting-point agarose, excised, and purified using ß- Agarase
I (New England Biolabs, Inc., Arundel, Queensland,
Australia). PCR products were then sequenced unidirectionally using the
forward nested PCR primers. Sequences of the nested primers are shown
in Table
1.
Statistical analysis
The Number Cruncher Statistical System (Ness, Kaysville, UT) was used for statistical analysis. Data were analyzed by unpaired t test, two-tailed Fisher’s exact test, and discriminant analysis, as appropriate. A two-tailed P value < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Initially, 14 frozen and 40 paraffin-embedded papillary thyroid
carcinomas from 54 patients from the New Caledonian and the Australian
populations were examined. To check for RNA integrity, the
reverse-transcribed samples were amplified with c-N-ras
intron-spanning primers, followed by hybridization with a
c-N-ras specific probe. With these primers, PCR products
derived from RNA were clearly distinguishable, by size, from those
derived from genomic DNA. Only samples with the expected 236-bp band
were considered suitable for ret/ptc study. In 7 of the 40
formalin-fixed paraffin-embedded tissues tested, RNA extracted was of
insufficient quality, and these samples were subsequently excluded. The
remaining 47 cases were included in the study (Table 2). There was no significant difference
in age or gender between the New Caledonian and Australian study
populations. Classification of the morphological variants of papillary
carcinoma was based on the predominant growth pattern of the tumors. As
shown in Table 2, typical papillary carcinoma was the most prevalent
variant in both groups, followed by the follicular variant. Although
there were more follicular variants in the New Caledonian, compared
with the Australian samples, this was not statistically significant.
The solid subtype was not found in either group.
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ret/ptc was detected in 19 of the 27 (70%) cases of
New Caledonian papillary carcinoma. Representative results are shown in
Fig. 2. Interestingly, 8 of these showed
the presence of multiple rearrangements (Table 3). Seven cases had 2 types of
rearrangements, a combination of either ret/ptc1 and 2
(n = 1), 1 and 3 (n = 4), or 2 and 3 (n = 2). A sample
was randomly chosen for sequencing, and the presence of the 2
rearrangements was confirmed. One case was positive for
ret/ptc1, 2, and 3. On sequencing, the segment encompassing
the fusion points for the 3 different rearrangements was confirmed. The
sequence also revealed a T-to-G mutation in the H4 gene, the fused
gene in ret/ptc1, 7 bp from the fusion point. Overall, the
most prevalent type of rearrangement was ret/ptc1, followed
by ret/ptc3 and ret/ptc2 (Table 4).
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In the Australian study population, 17 of 20 (85%) had
ret/ptc. Eight cases had rearrangements of either
ret/ptc1 and 2 (n = 2), 1 and 3 (n = 4), or 2 and
3 (n = 2). Two of these were sequenced, and the rearrangements
were confirmed. In addition, 3 cases showed 3 types of rearrangements
(Table 3); and the presence of ret/ptc1, 2, and 3 was
confirmed by sequencing in all 3 samples. Surprisingly, 4 of the 5
samples sequenced contained the same T-to-G mutation in the
ret/ptc1 sequence. Fig. 3
shows the sequencing results from 1 of the samples with 3 types of
ret/ptc. Unlike the New Caledonian samples, which showed a
predominance of ret/ptc1, the Australian samples have an
equal distribution of ret/ptc1 and ret/ptc3. When
the distribution of the specific types of ret/ptc was
compared between these two populations (Table 4), ret/ptc2
was more prevalent in the Australian cases (Fisher’s exact test,
P = 0.04).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Methods that have been used for the detection of ret rearrangements include visualization of RT-PCR products by staining with ethidium bromide, hybridization of blotted genomic DNA or RT-PCR products with radioactive probes, transfection assays, and immunohistochemistry (16, 20, 21, 30). Among these methods, transfection assays, Southern blot analysis of genomic DNA, and visualization of PCR products by ethidium bromide staining were noted to have the same sensitivity for detection of ret rearrangements (21). A further 5-fold increase in the detection rate can, however, be achieved by hybridization of blotted RT-PCR products (30). It is clear that the highly sensitive RT-PCR method can be further enhanced if this is followed by hybridization. The importance of the method used in detecting ret rearrangements was clearly demonstrated when the same group of investigators obtained a higher rate of ret/ptc after modification of their method (17). The low incidence of 5%, initially reported based on RT-PCR alone (31), increased to 47% when RT-PCR was followed by hybridization (30). Furthermore, the improved sensitivity was noted not only in paraffin-embedded but also in fresh frozen samples. In our study, we also found that RT-PCR followed by hybridization was superior to RT-PCR alone for demonstrating ret rearrangements. Some samples with no detectable bands after PCR amplification were positive for ret rearrangement with hybridization, and the result was verified by sequencing. Some other technical aspects that could affect sensitivity are also worth considering. The amplicon size is an important factor for effective amplification, especially when paraffin-embedded tissues are used (32). In this study, the expected size of the PCR products ranged from only 151–242 bp, which likely contributed to the high rate of ret/ptc detection. The number of cycles used is another important variable. The cycle number of 40, used in this and other studies (15, 23, 29), had resulted in effective amplification of the genes of interest. To further increase the sensitivity of ret/ptc detection, 25 µL of PCR products were loaded onto the gels for membrane transfer, a volume that is larger than that used previously in most published studies.
In addition to the differences in the sensitivity of the methods used, the purity of the cancer samples could also have an effect on the detection rate. It is noteworthy that among the various studies, the higher prevalence rates of ret/ptc in the American and Japanese populations were reported by investigators who only used specimens that contained 70–100% cancer cells in addition to the highly sensitive method described above (24, 25). Although the purity of cancer specimens is a less critical issue for a qualitative detection of rearrangements, the sensitivity of detection could be compromised if the specimen is contaminated by a substantial amount of normal tissue. Furthermore, it has been noted that the expression of ret/ptc is not always homogeneous in papillary carcinoma (16, 30). For this reason, each specimen used in our study contained at least 70% cancer cells, as verified by pathologists, in case the expression of ret/ptc was low or absent in some cancer cells. It is possible that tissue preparation, in addition to a highly sensitive method, was an important determinant of the high prevalence rates.
To ensure that the improved sensitivity of ret/ptc detection
did not compromise the accuracy of the results, several steps were
included in our protocol. First, the primers used for PCR flank the
chimeric regions and, therefore, can only amplify rearranged sequences
and not c-ret messenger RNA. Second, taking into
consideration that plasmids used as positive controls during PCR are
potential sources of contamination, no RT reaction was performed in an
environment where the plasmids were present. Furthermore, because a
large amount of amplified sequences increases the chance of
contamination, the plasmids were substantially diluted to 500 pg/µL,
and only 1 µL was used for PCR. As shown in Fig. 2, the signals from
the plasmids were, in fact, sometimes even weaker than those from the
tissue samples. Third, two negative controls were included in every
experiment as a means of detecting contamination (a water control for
PCR and at least one unloaded well on the gel for hybridization).
Because the water control contains all the PCR reagents except the cDNA
template, any plasmid contamination would be amplified and detected.
Last, for each sample, RT-PCR was repeated at least twice, from two
batches of RNA extracted at different times from the same tumor.
An interesting finding in this study is the presence of multiple rearrangements in a relatively high percentage of tumors from both populations. This phenomenon has recently been reported where a subset of tumors showed 2 different types of rearrangements (16, 30, 33). In our study, however, besides 15 samples harboring 2 types of rearrangements, 4 tumors have 3 types of ret/ptc. To our knowledge, this is the first study to show the occurrence of 3 different types of ret rearrangements in the same tumor. Although the overall mechanisms underlying multiple rearrangements are, as yet, unknown, it certainly challenges the notion of monoclonality of thyroid neoplasms. The clonal origin of thyroid tumors has been investigated by several groups using X chromosome inactivation analysis in female patients (34, 35). In a study where the clonal composition of both benign and malignant thyroid tumors was investigated, most solitary thyroid nodules (anaplastic carcinoma, follicular carcinoma, and adenoma) were found to be monoclonal (34). However, 2 of the 3 papillary carcinomas had intermediate patterns. Although the authors proposed that the pattern could be attributable to the presence of a substantial amount of interspersed stromal tissue, the possibility of polyclonality was also hypothesized. In a recent study on multifocal papillary carcinoma, different foci of cancer within the same gland were shown to have different types of ret rearrangements. Furthermore, different types of ret/ptc were detected, even within the same focus (30). These 2 studies point to the possibility of polyclonality in papillary carcinoma, which is further attested by our study.
Because ret is not normally expressed in thyroid follicular cells, rearrangement affecting a single chromosome would be sufficient to result in the subsequent detectable expression of ret/ptc. To date, it is still not clear whether ret/ptc rearrangements could involve both paternal and maternal chromosomes. If ret rearrangement affects only one chromosome per cancer cell, the cancer can not be monoclonal when two types of rearrangements are present, because some cells would have one type of ret/ptc and some would have the other. On the other hand, if both chromosomes were involved, each harboring one type of ret/ptc, this would give rise to a monoclonal tumor, even when two types of ret/ptc are present. The occurrence of three types of ret/ptc in a single tumor, however, indicates strongly that at least some thyroid neoplasms have to be polyclonal. That is because the tumor will have three different types of cancer cells [each type harboring one of the three ret rearrangements if one chromosome was affected, or each type having one of the three combinations (ret/ptc1 and 2, ret/ptc1 and 3, or ret/ptc2 and 3) if two chromosomes were involved].
Correlation of ret/ptc with clinical outcome remains inconclusive (16, 31, 36, 37). Some of the specimens in our study were collected only a few months before the study and therefore precluded a reasonable length of follow-up. There is also some uncertainty regarding the correlation of ret/ptc with age. An earlier study showed that the frequency of ret activation is significantly higher in those whose age is 30 yr or less (21). Another group, however, reported that those who are 40 yr old or more had the highest prevalence of ret/ptc (16). More recently, it was shown that pediatric and adult papillary carcinoma had almost the same prevalence rates in a Japanese population (25). Similarly, we have found no correlation of patient’s age with the presence of ret/ptc or multiple rearrangements.
It has been suggested that radiation is an important trigger for ret/ptc formation, based on the high prevalence of ret rearrangement in the post-Chernobyl population and in patients who had a history of therapeutic irradiation. Post-Chernobyl tumors have a preponderance of ret/ptc3, compared with other types (23, 24), and ret/ptc3 has been strongly associated with the solid variant of papillary carcinoma (24). In this study, although ret/ptc3 was positive in 21 cases, none of the tumors was of the solid variant type. Though ret/ptc3 and the solid variant have been strongly associated with radiation-induced thyroid cancers, neither of them has been considered as a so-called radiation signature. As more studies on radiation-induced tumors are carried out, the relationship between ret/ptc and radiation does seem to be more complex. In contrast to the findings of earlier studies, a recent study showed equal distribution of ret/ptc1 and ret/ptc3 in a Belarusian population exposed to the Chernobyl nuclear accident (38). In a separate study on adults who had a history of therapeutic irradiation for benign or malignant conditions, the most common type was ret/ptc1 and not ret/ptc3 (33). More studies currently being conducted in different centers will hopefully give us further insights into the association between ret/ptc and radiation.
Four of five samples sequenced for ret/ptc1 had a T-to-G mutation in the H4 gene. This mutation, first described in a papillary thyroid carcinoma cell line TPC-1 (39), is silent and caused no change in amino acid. This same mutation has also been found in one of two samples and in three of four samples in two different studies (22, 19). Although it is likely that this mutation reflects a genetic polymorphism, a further study, to determine its prevalence in individuals with and without thyroid cancer, may be warranted.
In conclusion, this study shows the high prevalence of ret/ptc in both New Caledonian and Australian papillary thyroid carcinoma, determined by a highly sensitive and specific protocol. Because there are no significant differences in the ret/ptc prevalence and distribution between the two populations, ret/ptc rearrangements are probably not the underlying genetic defect for the uniquely high incidence of thyroid cancer in New Caledonia. This only highlights the need for further studies to characterize the important underlying molecular changes of thyroid cancer in this population. The striking finding is the high percentage of tumors that showed the presence of multiple rearrangements. The presence of multiple ret/ptc in the same tumors suggests that some thyroid neoplasms may indeed be polyclonal.
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Acknowledgments |
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Footnotes |
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Received February 1, 2000.
Revised April 13, 2000.
Accepted April 26, 2000.
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References |
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3 and -4 in German papillary
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