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Original Studies |
Departments of Pediatrics (C.L.F., C.A.D., G.L.F.) and Clinical Investigation (Y.L., D.N.), Walter Reed Army Medical Center, Washington, D.C. 20307; F. Edward Hebért School of Medicine, Uniformed Services University of Health Sciences (C.L.F., C.A.D., G.L.F.), Bethesda, Maryland 20814; and Endocrine Service, Memorial Sloan-Kettering Cancer Center (R.M.T.), New York, New York 10021
Address all correspondence and requests for reprints to: R. Michael Tuttle, M.D., Memorial Sloan-Kettering Cancer Center, Box 419, 1275 York Avenue, New York, New York 10021. E-mail: rmtuttle{at}hotmail.com
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Abstract |
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2 analysis) from that
reported for children with radiation-induced PTC. There was no
correlation between the presence or type of ret/PTC
mutation and patient age, tumor size, focality, extent of disease at
diagnosis, or recurrence. We conclude that ret/PTC
mutations are 1) common in sporadic childhood PTC, 2) predominantly
PTC-1, 3) frequently multiple, and 4) of different distribution than
that reported for children with radiation-induced PTC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The frequency of ret/PTC rearrangements has varied according to the geographic area. The highest prevalence was reported in Italy (33–35%) and the lowest in Saudi Arabia (2.5%) and Japan (0–9%) (2, 4, 5, 6, 7, 8). However, most of these studies examined only a small number of adult cases and made no reference to childhood tumors (5). Motomura et al. recently reported that 71% of sporadic PTC from children in the United States and 87% of PTC from children living in radiation-contaminated areas of Belarus contain rearrangements of the ret oncogene (2, 9). The majority of their cases (n = 57) were children with radiation-induced PTC that were found to contain predominantly PTC-3 mutations. There were only 17 children with spontaneous PTC, and in these PTC-1 was predominant. No correlation with clinical outcome was reported for either group, although it has been generally recognized that radiation-induced PTC following the Chernobyl nuclear accident is more aggressive (4, 6). These data indicate that mutations producing ret/PTC-3 rearrangements are common events in radiation-induced PTC in children (8). However, none of these previous studies correlated the presence of ret/PTC mutations to the clinical outcome of individual patients. Furthermore, the optimal control group of children who developed nonradiation-related, sporadic PTC remains small (n = 17). For these reasons, the current study was undertaken to examine the incidence of ret/PTC mutations, the type of ret/PTC mutations, and the clinical outcome for a cohort of 33 sporadic PTC that presented during childhood or adolescence.
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Materials and Methods |
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Clinical data
The centralized tumor registry for the Department of Defense (ACTUR) was searched for all individuals diagnosed with thyroid cancer before 21 yr of age. Original medical records were abstracted to construct a clinical database maintained by the principal investigator (10). The extent of disease at diagnosis was defined according to the classification system of DeGroot et al. (11). Disease class I was confined to the thyroid gland. Disease class II involved the regional lymph nodes. Disease class III had either direct extension beyond the thyroid capsule or inadequate surgical resection. Disease class IV involved distant metastasis to bone or lung. Recurrence was defined as the appearance of new disease (new radioactive iodine uptake or biopsy-proven disease) in any patient who had been free of disease (no disease detected by palpation or radioactive iodine scan) for at least 4 months. The clinical data for several of these patients [137 with PTC and 33 with follicular thyroid cancer (FTC)] have been previously reported (10).
The corresponding original paraffin-embedded tissue blocks were available for 53 of these patients. Slides were stained with hematoxylin and eosin and were reviewed to establish the diagnoses. We were unable to extract ribonucleic acid (RNA) from 9 tissue blocks, leaving a final number of 44 cases in this study. This included 33 patients (75%) with sporadic PTC, 1 patient (2.3%) with radiation-associated PTC, 8 patients (18.1%) with FTC, 1 patient (2.3%) with medullary thyroid cancer (MTC), and 1 patient (2.3%) with intrathyroidal lymphoma (LYM).
Only 1 patient (no. 10) had a history of previous radiation exposure. This patient was treated with external beam radiation for a diagnosis of Hodgkin’s lymphoma at the age of 8 yr. His thyroid cancer was first detected 11 yr later. A thorough review of the remaining cases revealed nothing to suggest radiation exposure in any other patient. Because the emphasis in this study is on sporadic PTC, the data from the single patient with radiation exposure are presented separately.
RNA extraction and complementary DNA (cDNA) preparation
Total RNA was isolated from five sections (1 µm thick) immediately adjacent to the diagnostic slides. Sections were pooled, deparaffinized using three extractions with xylene (1 ml, 65 C, 10 min), and collected by centrifugation (16,000 x g, room temperature, 5 min). The tissue was then rehydrated with graded ethanol washes (100%, 80%, and 50%, room temperature, 10 min) and diethyl pyrocarbonate (DEPC)-treated water. The DEPC-treated water was removed (16,000 x g, room temperature, 5 min), and the pellet was resuspended in 0.5 ml 0.1 mol/L Tris-HCl, 25 µmol/L ethylenediamine tetraacetate, and 1% SDS (pH 7.3) containing 500 µg proteinase K (Sigma, St. Louis, MO) and incubated with vigorous shaking (52 C, 24 h), with an additional 250 µg proteinase K added after 24 h (52 C, additional 24 h). After solubilization, 0.5 ml solution D [4 mol/L guanidinium isothiocyanate, 25 mmol/L sodium citrate (pH 7.0), 0.5% N-lauroylsarcosine, and 0.1 mol/L ß-mercaptoethanol], 25 µL sodium acetate (pH 4.0), 0.25 mL DEPC-treated water-saturated phenol, and 50 µL chloroform/isoamyl alcohol (49:1) were added. The aqueous phase was removed, and the RNA was precipitated (100% ethanol, -20 C, 1 h). The pellet was redissolved in 0.3 mL solution D, precipitated with 100% ethanol (-70 C, 30 min), and washed (70% ethanol). The RNA was then treated with deoxyribonuclease I, ribonuclease free (20 U/200 µL incubation volume; Roche Molecular Biochemical, Indianapolis, IN) and extracted with phenol followed by chloroform/isoamyl alcohol (24:1), and precipitated (100% ethanol, -70 C, 1 h). The pellet was finally resuspended in DEPC-treated water containing RNase inhibitor (1 U/22 µL; Ambion, Inc., Austin, TX).
One microgram of total RNA was reverse transcribed with random hexamers (2.5 µmol/L) using Moloney murine leukemia virus reverse transcriptase at a final concentration of 2.5 U/µL, deoxy-NTPs (1 mmol/L each), MgCl2 (5 mmol/L), PCR buffer II (1x), and RNase inhibitor (1 U) in a final volume of 20 µL and thermocycled (22 C for 10 min, 42 C for 15 min, 99 C for 5 min, and a 5 C soak) for one cycle (PE Applied Biosystems, Foster City, CA). PCR amplification for each ret/PTC rearrangement contained 1 µL cDNA template in a final volume of 5 µL of a Perkin-Elmer Corp. (Foster City, CA) PCR reaction buffer mixture using TaqGold polymerase. The reaction was cycled for 10 min at 95 C followed by 15 s at 95 C and 1 min at 60 C for a total of 43 cycles in a PE 9600 thermocyler (PE Applied Biosystems). The amplified product was separated either in a 4%, 3:1 NuSieve agarose gel (FMC, Rockland, ME) and detected using a 1:10,000 dilution of Syber Green stain (FMC) or a Novex (San Diego, CA) 20% Tris/borate/EDTA polyacrylamide gel and silver stained.
The primer pairs were designed to span the known breakpoints specific for each of the ret/PTC mutations in addition to the internal housekeeping gene glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). Template negative controls and RNA extracted from nonpapillary thyroid cancers were included as negative controls along with each amplification. Reverse transcriptase-negative controls were included as well. Positive control clones for ret/PTC-1, ret/PTC-2, and ret/PTC-3 were provided by Dr. C. Jhiang, Ohio State University (Columbus, OH).
Table 1
shows the primer sequences for
ret/PTC-1, ret/PTC-2, ret/PTC-3, and
GAPDH in both the sense and antisense directions. The three primer
pairs for the different ret/PTC mutations shared a common
antisense primer, but unique sense primers. These were synthesized by
Biotechnologies Ltd. (Laurel, MD) and were designed to amplify the
region across the fusion point of the chimera using information
reported by Klugbauer et al. (11). Based on this
ret/PTC primer design, the amplified sequence for
ret/PTC-1 would be 81 bp, that for ret/PTC-2
would be 108 bp, and that for ret/PTC-3 would be 134 bp. The
GAPDH primers were purchased from Perkin-Elmer Corp.
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Data analysis and statistical significance
After RT-PCR amplification, data were only included if at least
GAPDH or one of the three ret/PTC mutations was successfully
amplified (44 of 53 attempted samples). Statistical analysis was
performed using SPSS for Windows 95 (SPSS, Inc., Chicago,
IL). Correlation was performed using Pearson correlation, and
nonparametric analysis was performed using the
2 test.
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Results |
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. The median age at diagnosis was 18 yr
(range, 6–21 yr). Thirty-five percent of the patients were less than
17 yr of age, 18% were between the ages of 17–19 yr, and 47% were
between 19–21 yr. The majority of patients (82%) were defined as
having DeGroot disease class I or disease class II (12). The median
postoperative follow-up time was 3.6 yr (range, 0–13.4 yr). Forty-four
percent were followed for 5 yr or more. This cohort is similar to that
of most series, in which the majority of young patients with PTC are
female and have either class I or class II disease (9).
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Figure 1 shows the results of the PCR
amplification. The expected 226-bp GAPDH product was identified in 42
samples (Fig. 1A). Figure 1, B, C, and D, shows representative gel
electrophoretograms for the 3 different ret/PTC
oncogenes.
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As shown in Table 3, 15 subjects
had ret/PTC mutations (15 of 33, 45%). Eight of the 15
mutations (53%) were identified as ret/PTC-1, 2 (13%) were
ret/PTC-2, 2 (13%) were ret/PTC-3, and 3 (20%)
contained both ret/PTC-1 and ret/PTC-2. Nine
patients with ret/PTC mutations were classified as having
disease class I, and 6 were classified as having disease class II. None
was identified with either disease class III or IV. Two of the 15
patients with ret/PTC mutations (13.3%) developed recurrent
PTC within 12 months. The single patient (case 10) with
radiation-associated PTC contained both ret/PTC-1 and
ret/PTC-3, was classified as disease class I, and did not
develop recurrence. As expected, none of the patients with FTC or MTC
or the single patient with LYM was positive for ret/PTC
mutations.
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There was no correlation between the presence of ret/PTC mutations and patient age, tumor size, tumor focality (unifocal vs. multifocal disease), extent of disease at diagnosis, treatment (extent of surgery and/or use of adjunctive radioactive iodine ablation), recurrence, or disease-free survival.
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Discussion |
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The prevalence of ret/PTC rearrangements in childhood
thyroid cancer is not well known. Several researchers have studied
ret/PTC rearrangements in childhood thyroid cancers that
developed after the Chernobyl nuclear disaster (13). However, reports
of ret/PTC rearrangements in sporadic pediatric thyroid
cancers are rare. As shown in Table 4,
previous studies reported variable prevalence of ret/PTC
rearrangements in young patients with sporadic PTC. Bongazone et
al. examined 9 PTC from Italian children (4–19 yr of age) and
found that 67% contained ret/PTC mutations (2, 14).
Motomura et al. examined 8 Japanese children (9–14 yr of
age) and found that 37.5% contained ret/PTC mutations (2).
Finally, Nikiforov et al. examined 17 children (5–18 yr of
age) from the United States and found that 71% contained
ret/PTC rearrangements (2, 9). In all 3 studies
ret/PTC-1 was predominant. This pattern is similar to that
reported in adult populations (2, 15). None of these studies, however,
correlated the presence of ret/PTC rearrangements with the
clinical outcome for individual children.
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The distribution of ret/PTC rearrangements in childhood thyroid cancer is based predominantly on PTC that developed after exposure to the Chernobyl nuclear accident. Initial studies of Chernobyl-associated PTC identified ret/PTC-3 as the most common form of RET rearrangement in radiation-induced childhood PTC (3, 10, 11, 12, 14). However, Pisarchik et al. found that 29% of adult and childhood PTC in Belarus (n = 31) actually contained ret/PTC-1 rearrangements (3). Pisarchik hypothesized the increase in frequency of the ret/PTC-1 rearrangements could be related to the longer latency period in their cases, which developed PTC later than those in the initial reports. The patients who had ret/PTC-3 rearrangements had been detected much earlier after the Chernobyl incident (3).
The distribution of ret/PTC mutations in our series (53%
ret/PTC-1, 13% ret/PTC-2, and 13%
ret/PTC-3) is significantly different from the distribution
reported for radiation-associated PTC (P = 0.001, by
2 analysis) (8, 9, 11). There are several
possible reasons for this difference. First, it is possible that
ret/PTC-3 could be specifically induced by radiation
exposure. Only 1 patient in our series had prior radiation exposure,
and his tumor was found to contain both ret/PTC-1 and
ret/PTC-3. Second, previous reports contained a greater
number of children with extensive disease. For example, the series by
Pisarchik et al. included 15 children with disease class III
(3). In contrast, only 6 patients in our series had disease class III
or IV. It is therefore possible that ret/PTC-3 mutations
could be associated with more invasive disease. Finally, the children
in this study were all from the United States, and the differences seen
in the distribution of ret/PTC mutations may reflect a
difference in either ethnic background or geographic regions.
Overall, our data are most consistent with previous studies of spontaneous adult PTC, in which ret/PTC-1 mutations have been most common (2, 15). Sugg et al. found that ret/PTC-1 mutations were most frequent in adult PTC, followed by ret/PTC-3 and finally by ret/PTC-2 (1).
The clinical significance of ret/PTC mutations is debated, even in adults. Sugg et al. correlated the presence of several oncogenes with clinical outcome for 20 adults with PTC (16). They found ret/PTC mutations in 7 of 20 (35%) of their cases. Five of the 7 patients with metastatic disease had ret/PTC rearrangements. The researchers hypothesize that ret/PTC rearrangements may have a potential role in metastasis. In a follow-up to their initial study, Bongarzone et al. examined the clinicopathological features of adults and children with ret/NTRK1 rearrangements (6). They examined 76 patients (4–80 yr of age), of whom 34.2% (26 of 76) were positive for ret/PTC mutations. With respect to children less than 20 yr of age, 60% (6 of 10) had a ret/NTRK1 rearrangement. There was a positive correlation between ret/NTRK1 mutations and young age (<30 yr) as well as locally advanced disease (6). However, the study did not separate ret/PTC rearrangements from NTRK1 rearrangements for all age groups.
In contrast, Mayr et al. examined 58 PTC from patients aged 17–84 yr of age and found ret/PTC mutations in 13.8%, with ret/PTC-1 being most frequent (17). The patients with ret/PTC mutations commonly had lymph node involvement at diagnosis. In contrast, distant metastatic spread and death from disease were not observed in patients with ret/PTC mutations. Delvincourt et al. examined 16 PTC from patients aged 19–68 yr and found ret/PTC mutations in 12.5% (2 of 16). They found no correlation between the presence or absence of ret/PTC mutations and distant metastasis (7).
The data from our study reveal no correlation between the presence or absence of ret/PTC mutations and clinical outcome. Furthermore, there was no relationship between any specific type of ret/PTC mutation and clinical outcome. However, our median follow-up for this cohort is relatively short (3.6 yr) and may not be long enough to detect all recurrences. This is in direct contrast to the results reported by Sugg et al. (1), but is consistent with the findings of Mayr et al. (17).
Our study found double ret/PTC mutations in 12% of the PTC examined. This frequency is similar to that identified by Sugg et al. (1). They examined 36 PTC and found a total of 16 ret/PTC mutations. Two of the 16 samples contained multiple ret/PTC rearrangements (12.5%). In addition, 9 of 39 occult cancers (23%) contained 2 forms of ret/PTC mutations.
In summary, we found that ret/PTC mutations are present in spontaneous childhood PTC and that they occur more frequently than in adults. The distribution of mutations is similar to that seen in adults, with ret/PTC1 being the most common. However, the distribution is significantly different from that seen in PTC from the Chernobyl population. Double ret/PTC mutations were common, occurring in 12% of the samples examined. The presence of ret/PTC mutations does not correlate to the extent of disease at diagnosis or short term clinical outcome for children and young adults.
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Footnotes |
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Received July 16, 1999.
Revised October 20, 1999.
Accepted December 2, 1999.
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References |
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