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Clonal analysis of bilateral, recurrent, and metastatic papillary thyroid carcinomas
Human Pathology (2010) 41, 1299–1309
www.elsevier.com/locate/humpath
Original contribution
Clonal analysis of bilateral, recurrent, and metastatic papillary thyroid carcinomas☆ Weibin Wang MD a , Haiyong Wang MDa , Xiaodong Teng MDb , Haohao Wang MD a , Chenyu Mao MD a , Rongyue Teng MDa , Wenhe Zhao MD a , Jiang Cao PhD c , Thomas J. Fahey III MDd , Lisong Teng MD, PhD a,⁎ a
Cancer Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China Department of Pathology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China c Key Laboratory of Biotherapy of Zhejiang Province, Sir Run Run Shaw Institute of Clinical Medicine, Zhejiang University, Hangzhou 310016, China d Department of Surgery, New York Presbyterian Hospital, and Weill Medical College of Cornell University, New York, NY 10021, USA b
Received 26 November 2009; revised 9 February 2010; accepted 25 February 2010
Keywords: Papillary thyroid carcinoma; Bilateral, BRAF mutation; X-chromosome inactivation; Clonal origin
Summary Papillary thyroid carcinoma usually presents as a multifocal disease; and tumors often recur in the contralateral thyroid lobe, raising questions concerning their clonal origins. The clonality of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe remains unknown. Accordingly, we examined 25 pairs of bilateral papillary thyroid carcinomas (synchronous or metachronous) and 15 matched metastatic lymph nodes. BRAF gene mutation analysis combined with X-chromosome inactivation was used to evaluate these tumors' clonal origins. Genomic DNA was prepared from paraffin-embedded tissues after microdissection. In total, 62 tumors yielded DNA of adequate quality. Eighteen (18/21, 85.7%) of 21 informative cases showed concordant BRAF status in tumors from both thyroid lobes, being either BRAF mutation positive (12 patients) or BRAF mutation negative (6 patients). Metastatic lymph nodes in 12 patients (12/15, 80%) had a complete concordance of BRAF state with their primaries. Of the 18 studied female patients, 11 were suitable for X-chromosome inactivation assay. Nine cases (9/11, 81.1%) showed the same pattern of inactivation in bilateral tumors. A close correlation was found between BRAF mutation and X-chromosome inactivation analysis. In conclusion, our data provide evidence that bilateral, recurrent, and metastatic papillary thyroid carcinomas often arise from a single clone and that intrathyroidal metastasis may play an important role in the development of bilateral tumors, as well as in the recurrence of this malignancy. © 2010 Elsevier Inc. All rights reserved.
1. Introduction ☆
Funding support: This study was supported by National Basic Research Program of China (973 Program, No. 2009CB521704) from the Ministry of Science and Technology of the People's Republic of China (Beijing, China). ⁎ Corresponding author. E-mail address: [email protected] (L. Teng). 0046-8177/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2010.02.008
Papillary thyroid carcinoma (PTC) is the most common histotype of thyroid cancer and accounts for about 1% of human malignancies [1]. Patients with PTC generally have a good prognosis, especially in those younger than 45 years at
1300 the time of diagnosis [2]. However, about 5% of patients with PTC experience a recurrence within 5 years after the initial treatment [3,4]. In patients with local or distant recurrence after lobectomy, a tumor is found in more than 60% of the time in the resected contralateral lobe [5,6]. In addition, PTC is often multifocal, with a reported frequency varying widely from 18% to 87%, among which 61% are bilateral [7,8]. The clonal origin of multifocal PTC is still under debate. There are 2 different theories to address this issue. One is that these tumors are of monoclonal origin, arising from intrathyroidal metastasis of a clonal population of cells. The other is that the multiple foci are of independent origin and arise separately in a background of field cancerization. Distinguishing between these 2 theories will improve our understanding of both the biological mechanisms of multifocal PTC, as well as the possible reasons why PTC recurs. Several studies have been performed to investigate the clonality of tumors located in the same thyroid lobe with varied results [9-13]. The clonality of tumors occurring simultaneously in both thyroid lobes or that of recurrent tumors in the contralateral lobe remains unknown. Previous studies have shown that PTC displays several highly prevalent genetic alterations, like mutations in BRAF or RAS, and rearrangements of the RET or NTRK1; among these alterations, mutations in the BRAF gene (exon 15BRAFV600E) is the most common [14-18]. Because of its high prevalence and presumed early place as a genetic event in thyroid carcinogenesis [19], BRAFV600E mutation has been used as a marker for clonality analysis and has shown some advantage in evaluating the clonal origin of multifocal PTCs [12,13]. However, few studied have combined this mutation with other methods based on changes established early enough to assess the clonality of tumors. Determination of X-chromosome inactivation (XCI) has also been used to study clonality in many human tumors, including thyroid cancers [20]. According to the Lyon [21] hypothesis, 1 of the 2 X-chromosomes in all female somatic cells is randomly deactivated by the DNA methylation at CpG islands during early embryogenesis. Once established, the inactivated X-chromosome is stably transmitted from a parent cell to its progeny. Therefore, a given group of cells resulting from clonal expansion of a single progenitor shows the same pattern of XCI. Distinguishing the patterns of XCI permits investigators to determine whether multiple tumors in female subjects arise from one or more progenitors. In this study, we investigated the clonality of bilateral PTCs (synchronous or metachronous) and metastatic lymph nodes by assaying for the presence or absence of the BRAF mutation. We also examined the XCI status in separate tumors in female patients. By combining the 2 strategies, genotypic clonality (BRAF mutations, involved in the malignant transformation) and phenotypic clonality (XCI, established in early embryogenesis), we reasoned that we could provide more definitive answers as to clonality in multifocal or recurrent PTCs.
W. Wang et al.
2. Materials and methods 2.1. Case selection and tumor specimen collection A computer-based database comprising 809 patients was established to select eligible cases. These patients were admitted to the First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China), from 1997 to 2006 and underwent thyroidectomy for the treatment of thyroid carcinoma, with or without neck lymph node dissection. A list of cases was generated according to the following criteria: (a) bilateral or recurrent PTCs (including both primary and recurrent tumors) were diagnosed and treated in the same hospital; (b) the intervals between the 2 diagnoses were either less than or equal to 1 month (synchronous) or greater than or equal to 3 months (metachronous); and (c) cases with a history of previous irradiation of the neck were excluded. Finally, after excluding patients whose tumors were only available for study in one thyroid lobe (therefore, unable to be analyzed as a pair), 25 cases were enrolled in this study. Paraffinembedded blocks were retrieved from the surgical pathologic files in the Department of Pathology, and patients were followed up in our Cancer Center. Tumors in individual cases were randomly numbered without the knowledge of the paired patient's ID and the molecular analysis results. All of the slides were reviewed to confirm the diagnosis and to locate the tumor's area for subsequent microdissection by a senior pathologist. The histologic subclassification of PTC into conventional variant (CVPTC), follicular variant (FVPTC), and tall cell variant was made according to the criteria of DeLellis et al [22]. A tumor stage was defined according to Tumor, Nodes, and Metastases system as described by the American Joint Committee on Cancer Staging Manual, Sixth Edition [23]. This study was approved by the Institutional Review Board of First Affiliated Hospital, Zhejiang University School of Medicine.
2.2. Microdissection and DNA extraction Genomic DNA was isolated from archival paraffinembedded specimens by microdissecting the tumor tissue. Briefly, 4 unstained 10-μm–thick sections were deparaffinized in xylene and rehydrated in graded ethanol. Tumor margins were marked using the hematoxylin and eosin (H&E)–stained section as a template, and the tumor tissue was dissected from the unstained slides. Finally, the dissected but unstained sections were stained by H&E to confirm that the harvested tumor cells had indeed been segregated from the surrounding normal cells. Wherever available, samples from distant nonneoplastic thyroid parenchyma were dissected as control reference. The harvested tumor cells were transferred into 1.5-mL Eppendorf tubes, and DNA was extracted overnight at 56°C
Clonal analysis of bilateral, recurrent, and metastatic PTCs in a 500-μL tissue lysis buffer (10 mmol/L Tris, 0.2 mmol/L EDTA, and 0.5% sodium dodecyl sulfate; pH 8.0) plus 0.1 mg proteinase K (Sigma, St Louis, MO). On the second day, Chelex-100 resin (Sigma) was added to each sample; and the mixture was incubated at 37°C for another hour to improve the quality of DNA. After extraction with phenol/chloroform, genomic DNA was precipitated with ethanol in the presence of acetate.
2.3. BRAF mutation analysis by polymerase chain reaction amplification and DNA sequencing Because most BRAF mutations are located in exon 15 of BRAF gene, we amplified this exon followed by direct DNA sequencing. A 224–base pair (bp) fragment was amplified by using the primer sequences as described previously [24]. Two hundred nanograms of genomic DNA was used as a template in a 25-μL polymerase chain reaction (PCR) mixture containing 1× PCR buffer, 2.0 mmol/L magnesium chloride, 0.2 mmol/L of each deoxynucleotide triphosphate, and 1 unit of HotStart Taq polymerase (Takara, Shuzo, Japan). Cycling conditions were as follows: initial denaturation at 95°C for 10 minutes and then 40 cycles with denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. All PCR products were visualized by electrophoresis in a 2% agarose gel and purified using PureLink Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA). The purified PCR products were then sequenced on ABI PRISM 3730XL DNA Analyze (Applied Biosystems, Foster City, CA) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing results were compared with the normal BRAF gene in GenBank database.
2.4. XCI analysis The X-chromosome linked human androgen receptor gene (HUMARA) was chosen for XCI study. In the first exon of the X-linked HUMARA gene, there is a high polymorphic trinucleotide (CAG) repeat; and approximately 90% of females are heterozygous with respect to the number of CAG repeats in this region [25]. In addition, there are 2 HhaI restriction sites located close to the CAG repeat polymorphism and selectively methylated in inactive X-chromosomes [26]. Consequently, digestion with methylation-sensitive endonucleases followed by PCR amplification with primers flanking these restriction sites and the heterozygous CAG repeats can be used to distinguish between active and inactive X-chromosomes. Ten-microliter DNA extract from each sample was divided into 2 aliquots, which were subsequently subjected to either digestion with 1 unit HhaI endonuclease (New England Biolabs, Inc, Beverly, MA) at 37°C for 16 hours or mock digestion with restriction buffer alone in a total
1301 volume of 10 μL. After incubation, the enzyme was heat inactivated at 65°C for 20 minutes. To evaluate the efficiency of the restriction reactions, a paraffin-embedded tissue-derived male control sample was included in each assay. Three microliters of digested or undigested DNA was amplified in a 25-μL PCR mixture containing 1× PCR buffer, 4% DMSO, 2.0 mmol/L magnesium chloride, 0.2 mmol/L of each deoxynucleotide triphosphate, 1 unit of HotStart Taq polymerase (Takara), and the following primers [11] at a concentration of 200 nmol/L: forward primer, 5′-TCTGTTCCAGAGCGTGCGCGAAGT-3′ bearing a fluorescent FAM tag on the 5′ end, and reverse primer, 5′-CTCTACGATGGGCTTGGGAGAAC-3′. Cycling conditions were as follows: initial denaturation at 95°C for 10 minutes and then 34 cycles with denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. We size separated the PCR products on an automated DNA sequencer (ABI-3730, Applied Biosystems) and analyzed the results using GeneMapper 4.0 software (Applied Biosystems). Allele intensity was determined from the allele peak height, which are proportional to the amount of PCR product from the given allele. To account for possible differences in fluorescence signal acquisition and gel loading of each allele, we modified to uniform the volume of internal size marker (Genescan-500Liz; ABI Perkin Elmer, Warrington, United Kingdom) and calculated a corrected allele ratio as follows: (peak 1 height of undigested sample ÷ peak 2 height of undigested sample ÷ Liz 200 bp height of undigested group)/(peak 1 height of digested sample ÷ peak 2 height of digested sample ÷ Liz 200 bp height of digested group). We defined tumor samples as nonrandom XCI if this corrected ratio was less than 0.5 or greater than 2 [27]. Tumors were considered to be of single clonal origin if the same inactivation pattern was detected in each separate tumor from individual patient.
2.5. Statistical analysis χ2 test and Spearman rank correlation test were used to analyze the concordance in comparison of the 2 methods. A P value b .05 was considered significant. Data analysis was performed using the Statistical Package for Social Sciences for Windows (SPSS, Inc, Chicago, IL).
3. Results 3.1. Clinical features of the study group A total of 66 tumors from 25 patients (female, 18; male, 7) were analyzed in this study, among which 51 were bilateral tumors and 15 were metastatic lymph nodes. Twenty-two pairs were synchronous, whereas 3 pairs were metachronous
1302 Table 1
W. Wang et al. Clinical characteristics of patients studied
Patient Sex Age at Categories (left/right) ID diagnosis (y)
Synchronous cases 1 F 39 2 M 36 3 F 36 4 F 20 5 M 26 6 F 39 7 F 38 8 F 42 10 F 14 11 F 48 12 F 36 13 F 53 14 F 45 15 F 67 16 F 31 17 M 75 18 M 38 19 F 17 21 F 41 22 M 52 23 F 70 25 F 64 Metachronous cases c 9 F 51 20 M 45 24 M 32
Disease extent at surgery Extrathyroidal Lymph node Distant Stage extension metastases metastases
a
Follow-up Last known time (mo) disease status
FV/FVPTC CV/CVPTC FV/CV + FVPTC b CV/FVPTC FV/FVPTC CV/CVPTC FV/CVPTC CV + FV b/CV + FVPTC b CV/CVPTC CV/CVPTC FV/CVPTC CV/CVPTC CV/CVPTC CV/CVPTC FV/CVPTC TCV/TCVPTC CV/CVPTC CV + FV b/CV + FVPTC b CV/CVPTC CV/CVPTC CV/CVPTC CV/CVPTC
No Yes Yes No No No Yes Yes No No No Yes No No No Yes No No Yes No No Yes
Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes No No Yes
No No No No No No No No No No No No No No No No No No No Yes No Yes
I I I I I I I I I IV I IV I IV I IV I I I IV II IV
41 72 25 37 63 17 21 42 28 32 67 25 57 29 69 9 92 105 18 15 74 6
AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD DOD AFD AFD AFD AWD AFD DOD
CV/CVPTC FV/CVPTC CV/CVPTC
Yes No No
Yes Yes Yes
No No No
IV IV I
93 78 89
DOD AFD AFD
Abbreviations: F, Female; M, Male; TCVPTC, tall cell variant papillary thyroid carcinoma; AFD, alive, free of disease; AWD, alive with disease; DOD, dead of disease. a Histopathologic staging was reviewed according to the 2002 American Joint Committee on Cancer Staging Manual. b Displayed a coexistence of 2 different histopathologic components in the same focus. c Showed the characteristics at the initial treatment.
(ie, recurrent, shown in Table 1). The mean age at diagnosis was 42.2 years (range, 14-75 years), and the median diameter of the studied tumors was 2.2 cm (range, 0.3-9.4 cm). Of the 3 recurrent PTCs, both the primary and recurrent tumors were studied; and the intervals between recurrences were 4, 5, and 5 years, respectively. A large majority of studied patients (19/25, 76%) displayed a similar microscopic appearance in pairs of bilateral tumors, whereas 6 cases showed a coexistence of different histopathologic subtypes in distinct PTC foci. At the time of this study, all the patients had a postoperative follow-up with a mean period of 47.9 months (rang, 9-105 months); and the outcomes were detailed in Table 1.
3.2. BRAF mutation in pairs of separate tumors Because some samples were not well preserved and some extracted DNA from formalin-fixed and paraffin-embedded
blocks was degraded, 4 tumor tissues failed to yield DNA of adequate quality for PCR amplification; and 62 tumors from 25 patients were screened for BRAFV600E mutation. Fifteen patients (15/25, 60%) were positive for this common mutation (the missense T to A transversion at nucleotide 1799). Occasionally, we detected a novel BRAFV599Ins mutation also in exon 15 (an in-frame insertion of 3 nucleotides [GTT] at position 599), which was first reported in 2006 [28]. BRAF mutations were not found in any normal tissues that were available in 8 BRAF-positive cases (data not shown). A summary of BRAF mutations in bilateral PTCs (synchronous or metachronous) at multiple sites is shown in Table 2. Four cases were not informative because one of their multiple foci in each patient failed to amplify in PCR. Eighteen (85.7%) of 21 patients showed concordant BRAF status in tumors from both the left and right thyroid lobes, being either BRAF mutation positive (12 patients) or BRAF mutation negative (6 patients), indicating that these tumors
Clonal analysis of bilateral, recurrent, and metastatic PTCs Table 2
1303
BRAF mutation in bilateral cases
Patient Tumor left Tumor right Concordance ID Age at Tumor Tumor Mutation Age at diagnosis (y) Tumor no. Tumor size (cm) Mutation diagnosis (y) no. size (cm) Synchronous cases 1 39 2 36 3 36 4 20 5 26 6 39 7 38 8 42 9 51 10 14 11 48 12 36 13 53 14 45 15 67 16 31 17 75 18 38 19 17 21 41 22 52 23 70 25 64 Metachronous cases 9a 51 20 45 24 37
10 13 32 16 54 14 39 84 68 23 36 25 35 49 2 52 4 86 70 20 6 19 81
1.1 1.5 2.0 0.5 2.3 1.2 1.0 1.8 4.5 0.8 1.0 0.3 4.0 1.0 1.3 0.5 3.5 0.7 3.5 4.0 NA 2.0 2.0
WT T1799A T1799A WT WT T1799A WT NA V599Ins WT T1799A T1799A T1799A T1799A T1799A WT T1799A NA WT WT T1799A WT T1799A
39 36 36 20 26 39 38 42 51 14 48 36 53 45 67 31 75 38 17 41 52 70 64
9 50 5 28 31 37 38 40 75 44 17 47 48 85 51 53 55 63 74 87 42 29 82
1.2 4.5 1.5 2.0 1.5 1.5 1.5 2.5 1.8 1.2 0.8 1.3 1.0 1.1 3.0 1.5 5.0 2.0 NA 3.5 4.0 3.0 0.8
T1799A WT T1799A WT WT T1799A WT WT V599Ins WT T1799A T1799A T1799A NA T1799A WT T1799A WT WT NA T1799A T1799A T1799A
No No Yes Yes Yes Yes Yes – Yes Yes Yes Yes Yes – Yes Yes Yes – Yes – Yes No Yes
68 71 80
4.5 3.5 0.6
V599Ins T1799A T1799A
55 50 32
43 62 60
9.4 1.5 2
V599Ins T1799A T1799A
Yes Yes Yes
Abbreviation: NA, data not available because of incomplete clinical information or DNA quality being unsuitable for PCR amplification and sequencing. a Patient 9 was a case that initially presented as a bilateral PTC and recurred 5 years later.
might be of monoclonal origin. Fig. 1 shows a representative case with BRAFV600E positive in both the left and right thyroid lobes; the nearly equal peak areas of A and T nucleotides suggest a high homogeneity of tumor cells in these microdissected samples. The other 3 patients (patients 1, 2, and 23) presented with a coexistence of BRAF mutation-positive and BRAF mutation-negative tumor in both lobes, suggesting that these foci might arise from distant progenitors. In lymph node metastatic PTC, 12 (80%) of 15 cases had complete concordance between the defects present in the primary and metastatic site (Table 3). In the remaining 3 cases, BRAF mutation was positive exclusively in the primary tumors but not in the lymph node metastases. In addition, there was no case in which the status of BRAF mutation in the metastasis was exclusively identical to that of the contralateral primary tumor. These results suggested that most lymph node metastases were clonally related to the primary tumors, particularly the one on the ipsilateral side.
3.3. XCI assay Of 18 studied female patients, 12 were informative for XCI assay because of adequate quality of DNA and polymorphism in exon 1 of HUMARA gene (Table 4). The corrected ratios in 11 pairs of tumors were more than 2.0 or less than 0.5, representing a preferential loss of 1 of the 2 alleles after digestion (ie, a pattern of nonrandom XCI); and therefore, these foci were suitable for further analysis. The remaining patient (patient 13) demonstrated a random pattern of XCI in bilateral tumors and was excluded. Normal tissues obtained from 3 cases also displayed a random pattern of XCI, serving as an important control to avoid aberrant results due to embryonic patch size [29]. When comparing the pattern of tumors in each pair, we found 9 cases (9/11, 81.1%) that shared the same pattern of nonrandom XCI in bilateral thyroid tumors (a representative case is shown in Fig. 2, right panels), whereas different inactivated alleles were observed in 2 patients. In 7 metastatic lymph nodes, 5 showed the same pattern of XCI
1304
W. Wang et al.
Clonal analysis of bilateral, recurrent, and metastatic PTCs Table 3
1305
Comparison of BRAF mutation status in primary tumor and matched metastatic lymph node
Patient ID
Tumor no. of MLN
BRAF mutation status
Concordance with
MLN
Ipsilateral PT
Contralateral PT
Ipsilateral PT
Contralateral PT
1 3 4 5 7 8 9 9a 10 11 18 19 20 21 24 25
24 22 56 21 26 30 73 3 57 46 67 76 72 11 61 78
T1799A T1799A WT WT WT WT WT V599Ins WT WT WT WT WT WT T1799A T1799A
T1799A T1799A WT WT WT WT V599Ins V599Ins WT T1799A WT WT T1799A WT T1799A T1799A
WT T1799A WT WT WT NA V599Ins V599Ins b WT T1799A NA WT T1799A b NA T1799A b T1799A
Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes
No Yes Yes Yes Yes – No Yes Yes No – Yes No – Yes Yes
Abbreviations: NA, data not available because of DNA quality being unsuitable for PCR amplification and sequencing; PT, primary tumor; MLN, metastatic lymph node. a Patient 9 had another metastatic lymph node at recurrent treatment. b Lymph node metastasis is metachronous with the primary tumor.
as both the primaries, one was exclusively identical to the ipsilateral primary and the other was distinct to neither primary tumor.
considering that multiple foci from independent clones can also share the same pattern of XCI coincidentally, we deemed that this metastatic lymph node was not clonally related to the primary tumor we studied.
3.4. Close correlation between BRAF mutation and XCI Eleven pairs of bilateral PTCs and 7 metastatic lymph nodes were examined for both BRAF mutation detection and XCI analysis. We found a complete concordance in 10 bilateral PTCs between these 2 methods in evaluating the clonality (10/11, 90.1%; Pearson χ2, 2-sided P = .026; Spearman rank correlation test, r = 0.671). Only one patient showed a different pattern of XCI but shared the same BRAF mutation negative status in bilateral tumors. Because 2 unrelated tumors might present the same mutation status by chance, we assumed this case as an independent clonal origin. Therefore, in all, 17 (81%) of 21 patients with bilateral tumors in our study were suggested to be of monoclonal origin, whereas 4 bilateral cases arose as independent tumors. In 7 lymph node metastases, 6 shared concordance between BRAF mutation status and XCI and the primary, whereas only 1 (in patient 11) showed the same inactivated allele as the primary but with diverse mutation results. When
4. Discussion Multifocality is frequently observed in patients of PTC, with an increased risk of lymph node metastases and regional recurrence [7,30]. However, as a specific subtype of multifocal PTCs, bilateral PTC has not been widely investigated. Studies of this subtype were just restricted to clinical observations [5,6,30], and the clonal origin of bilateral tumors remains unknown. Most clonality studies on PTCs were done on multiple tumors located in one thyroid lobe [10,12]. Only a few reports included some pairs of tumor samples present in both thyroid lobes. Shattuck et al [11] studied the clonal origin of multifocal PTC in 10 informative subjects. With consideration of tumor location, 7 were bilateral, of which 3 showed independent clonal origin with different patterns of XCI. Lack of such pairs of tumor samples may be the main
Fig. 1 Concordant pattern of BRAF mutation in bilateral PTC and matched metastatic lymph node. Shown in the left-hand panels are photomicrographs (stained with H&E, original magnification: ×100) of 3 tumor foci from patient 3. For each tumor, the corresponding plot (right-hand panels) is a sequence result of BRAF exon 15. Although the bilateral tumors do not have a complete concordance in microscopic appearances (left lobe–tumor 32, FVPTC; right lobe–tumor 5, FV + CVPTC), they are of the same BRAFV600E mutation status. The metastatic lymph node (tumor 22) is also positive for BRAFV600E mutation, indicating that these tumors are clonally related.
1306 Table 4
W. Wang et al. XCI analysis in bilateral cases
Patient ID and tumor no.
3 32 5 22 N 4 16 28 56 6 14 37 9 68 75 73 Recurrence 43 3 N 10 23 44 57
Tumor location
XCI Corrected allele ratio
Inactivated allele a
Left Right LN (L) Left
22.4 5 20.67 0.69
L L L Random
Left Right LN (L)
3.38 0.18 5.71
L S L
Left Right
0.27 0.23
S S
Left Right LN (R)
0.02 0.07 2.06
S S L
Right LN (R) Right
0.16 0.11 0.9
S S Random
Left Right LN (R)
0.34 0.19 0.29
S S S
Patient ID and tumor no.
11 36 17 46 12 25 47 13 35 48 N 15 2 51 16 52 53 23 19 29 25 81 82 78
Tumor location
XCI Corrected allele ratio
Inactivated allele a
Left Right LN (R)
9.49 86.55 2.14
L L L
Left Right
0.11 0.02
S S
Left Right Right
0.85 1.76 0.78
Random Random Random
Left Right
0.35 0.37
S S
Left Right
9.86 10.15
L L
Left Right
0.1 36.7
S L
Left Right LN (R)
8.75 7.79 8.12
L L L
Abbreviations: N, normal tissues obtained around tumor area; LN (L), lymph node metastasis in left side of neck; LN (R), lymph node metastasis in right side; S, small allele; L, large allele. a For each informative patient, 2 alleles were separated because of the polymorphism of CAG-repeat numbers in HUMARA gene. After digestion, one of them was preferentially lost in tumors of nonrandom XCI, either the smaller allele or the larger allele. A random pattern of XCI (random) was characterized by the corrected ratios between 2.0 and 0.5. Tumors with random pattern of XCI were excluded in further analysis, whereas in normal tissues, this pattern of XCI could serve as qualified control.
reason why bilateral PTC has never been characterized in a large population. In this study, 25 pairs of bilateral PTCs were enrolled based on a database spanning one decade. Our series is, to our knowledge, the first to investigate the clonal origin of bilateral PTCs with a larger sample size. We have traced the genealogies of bilateral, recurrent, and metastatic PTCs by combining BRAF mutation and XCI analysis. Our results provided evidence that bilateral tumors in individual patients
often arose from the same progenitor cell and represented a single primary with subsequent intrathyroidal metastases to contralateral lobe. Thus, bilateral PTC should be considered as a progressive state of this disease. BRAFV600E mutation in exon 15, which was first described in 2002 [24], represents the most common genetic alteration in thyroid cancer [19]. As this mutation was shown to induce goiter and invasive PTC in transgenic mice [31] and was detected in the microcarcinomas [32,33],
Fig. 2 Comparison of BRAF mutation and XCI in a bilateral PTC. This figure presents a bilateral case (patient 25) with tumors in left thyroid robe (upper, tumor 81) and right thyroid lobe (down, tumor 82). Shown in the left-hand panels are DNA sequencing results of each tumor. PCR products of BRAF gene exon 15 are sequenced bidirectionally with forward primer (forward) and reverse primer (reverse); BRAFV600E mutation was characterized as the transversion from T to A by forward primer and A to T by reverse primer. Shown in the right-hand panels are results of separation of fluorescence-labeled PCR products on an automated sequencer. For each tumor, the corresponding height and size of plot represent the quantity and size in base pairs of PCR products, respectively. Amplified DNA from tumors without digestion showed 2 alleles, a smaller allele and a larger allele, indicating the polymorphism of CAG-repeat numbers in HUMARA gene. Loss of PCR amplification for the larger allele (indicated by the arrow) after digestion in both the left thyroid tumor (upper, tumor 81) and right thyroid tumor (down, tumor 82) indicates that the bilateral tumors share the same pattern of allele inactivation and are probably clonally related. The internal size marker (marked by ☆) is included in each assay to account for possible differences in fluorescence signal acquisition and gel loading. Abbreviations: H, height; S, size.
Clonal analysis of bilateral, recurrent, and metastatic PTCs
1307
1308 BRAFV600E mutation was presumed as an early genetic event and was already used as a marker for clonal analysis in multiple PTCs [12,13]. However, because this mutation is common in PTC, it cannot be distinguished whether multiple tumors sharing the same status of mutation is a consequence of a single mutation, spreading throughout the gland, or of multiple, unrelated mutational events in separate tumors. Only those cases when a very rare mutation is found in both tumors (BRAFV599Ins) are probably of the same clonal origin. Therefore, the proportion of tumors with single clonal origin may be overestimated when only BRAF V600E mutation was analyzed. In our study, we further examined the pattern of XCI in informative female patients. Almost all the studied cases showed the same clonality results between XCI and BRAF mutation analysis. Only one patient demonstrated a different allele inactivation but shared the same BRAF mutation status in bilateral tumors. We believe this close correlation between these 2 methods can at least partially resolve the ambiguity in evaluating tumors of the same mutational state. However, similar concerns should apply to the interpretation of the XCI status, for the presence of the same allele inactivation pattern provides only a 50% probability that 2 tumors are of the same clonal origin. Thus, caution is still warranted in interpreting our data. In contrast to our findings, Giannini et al [12] showed that as high as 40% of multifocal PTCs had discordant patterns of BRAF mutation. Such findings are seemed to be consistent with the presence of an independent clonal origin. The different results of the current study and that of Giannini et al may be mainly attributed to differences in case selection. Because the frequency of mutation was relatively higher here than that in the study of Giannini et al (64% versus 62%) and the prevalence of lymph node metastasis was obviously high in our study (21/25, 84%), we infer that bilateral PTCs studied here were quite distinct from the common multifocal PTCs studied by others. Few studies have compared the molecular defects in metastatic sites with those of the primaries, especially in bilateral PTCs. Our results showed that most lymph node metastases were derived from the primary tumors, particularly the ipsilateral one, supporting the current notion that lateral neck dissection is adequate for biopsy-proven lymph node metastases in clinical practice. However, there were 3 cases that BRAF mutation were present exclusively in the primary tumors but absent in lymph node metastases. The XCI analysis also showed a different pattern of inactivation in the primary tumors and metastatic lymph nodes of patient 9. Because several reports had proposed different clonal origins of multiple PTCs [11,12] and our study also indicated that some bilateral PTCs may arise as independent tumors, we infer that another unrelated tumor was present simultaneously within the thyroid and that this non-BRAF mutated tumor metastasized to the lymph node. Because most bilateral PTCs arise from a single progenitor, how does this progenitor cell lead to contralat-
W. Wang et al. eral and recurrent PTCs? It is likely that a specific genetic alteration (perhaps mutations in BRAF gene) occurs in a thyroid follicular epithelium and provides a growth advantage, giving rise to a proliferative state in which all neoplastic cells have the same genetic trait. In this view, such alterations can be used as a valid marker for clonality analysis. During further development, daughters of this transformed cell could gradually migrate regionally through an abundant network of intralobar lymphatic vessels [34]. These vessels, which intercommunicate from lobe to lobe across the isthmus, would allow tumor metastases ready access to the contralateral lobe. Subsequently, late genetic events, such as the recently reported AKT1 mutation [35], may convert a small percentage of such cells into a more invasive state, eventually leading to clinically bilateral carcinomas. The micrometastases, located in the residual thyroid tissue after surgery, are not detected by imaging studies at the time of initial treatment and remain to become the source of recurrences. As recommended in the NCCN Clinical Practice Guidelines in Oncology: Thyroid carcinoma (v.1.2009), patients who present with bilateral PTCs are one of the indications to be treated by total thyroidectomy. However, little molecular data have been incorporated to support this clinical guideline. Our study provides molecular evidence that bilateral PTC should be classified as a more advanced stage and that total thyroidectomy is appropriate for bilateral PTCs.
Acknowledgment We thank Ruokuo Han, Ruopeng Weng, and Zonglin Lin for patients' clinical follow-up, and Qixing Chen and Feng Zhang for technical assistance. We are also grateful to Dr Robert Wohlhueter (Centers for Disease Control and Prevention, Atlanta, GA; retired) for his contributions to the preparation of the manuscript.
References [1] Franceschi S, Boyle P, Maisonneuve P, et al. The epidemiology of thyroid carcinoma. Crit Rev Oncog 1993;4:25-52. [2] Tsang RW, Brierley JD, Simpson WJ, et al. The effects of surgery, radioiodine, and external radiation therapy on the clinical outcome of patients with differentiated thyroid carcinoma. Cancer 1998;82:375-88. [3] Fernandes JK, Day TA, Richardson MS, et al. Overview of the management of differentiated thyroid cancer. Curr Treat Options Oncol 2005;6:47-57. [4] Bilimoria KY, Bentrem DJ, Ko CY, et al. Extent of surgery affects survival for papillary thyroid cancer. Ann Surg 2007;246:375-81. [5] Pasieka JL, Thompson NW, McLeod MK, et al. The incidence of bilateral well-differentiated thyroid cancer found at completion thyroidectomy. World J Surg 1992;16:711-6. [6] Pacini F, Elisei R, Capezzone M, et al. Contralateral papillary thyroid cancer is frequent at completion thyroidectomy with no difference in low- and high-risk patients. Thyroid 2001;11:877-81.
Clonal analysis of bilateral, recurrent, and metastatic PTCs [7] Katoh R, Sasaki J, Kurihara H, et al. Multiple thyroid involvement (intraglandular metastasis) in papillary thyroid carcinoma. A clinicopathologic study of 105 consecutive patients. Cancer 1992;70: 1585-90. [8] Iida F, Yonekura M, Miyakawa M. Study of intraglandular dissemination of thyroid cancer. Cancer 1969;24:764-71. [9] McCarthy RP, Wang M, Jones TD, et al. Molecular evidence for the same clonal origin of multifocal papillary thyroid carcinomas. Clin Cancer Res 2006;12:2414-8. [10] Jovanovic L, Delahunt B, McIver B, et al. Most multifocal papillary thyroid carcinomas acquire genetic and morphotype diversity through subclonal evolution following the intra-glandular spread of the initial neoplastic clone. Pathol 2008;215:145-54. [11] Shattuck TM, Westra WH, Ladenson PW, et al. Independent clonal origins of distinct tumor foci in multifocal papillary thyroid carcinoma. N Engl J Med 2005;352:2406-12. [12] Giannini R, Ugolini C, Lupi C, et al. The heterogeneous distribution of BRAF mutation supports the independent clonal origin of distinct tumor foci in multifocal papillary thyroid carcinoma. J Clin Endocrinol Metab 2007;92:3511-6. [13] Park SY, Park YJ, Lee YJ, et al. Analysis of differential BRAF (V600E) mutational status in multifocal papillary thyroid carcinoma: evidence of independent clonal origin in distinct tumor foci. Cancer 2006;107:1831-8. [14] Nikiforova MN, Nikiforov YE. Molecular genetics of thyroid cancer: implications for diagnosis, treatment and prognosis. Expert Rev Mol Diagn 2008;8:83-95. [15] Kimura ET, Nikiforova MN, Zhu Z, et al. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 2003;63:1454-7. [16] Hara H, Fulton N, Yashiro T, et al. N-ras mutation: an independent prognostic factor for aggressiveness of papillary thyroid carcinoma. Surgery 1994;116:1010-6. [17] Grieco M, Santoro M, Berlingieri MT, et al. PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell 1990;60:557-63. [18] Pierotti MA, Vigneri P, Bongarzone I. Rearrangements of RET and NTRK1 tyrosine kinase receptors in papillary thyroid carcinomas. Recent Results Cancer Res 1998;154:237-47. [19] Xing M. BRAF mutation in thyroid cancer. Endocr Relat Cancer 2005; 12:245-62. [20] Namba H, Matsuo K, Fagin JA. Clonal composition of benign and malignant human thyroid tumors. J Clin Invest 1990;86:120-5. [21] Lyon MF. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 1961;190:372-3.
1309 [22] DeLellis RA. Pathology and genetics of thyroid carcinoma. J Surg Oncol 2006;94:662-9. [23] Greene FL, Page DL, Fleming ID, et al, editors. American Joint Committee on Cancer staging manual. 6th ed. Philadelphia: Springer; 2002. p. 77-87. [24] Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949-54. [25] Tilley WD, Marcelli M, Wilson JD, et al. Characterization and expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci U S A 1989;86:327-31. [26] Allen RC, Zoghbi HY, Moseley AB, et al. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgenreceptor gene correlates with X chromosome inactivation. Am J Hum Genet 1992;51:1229-39. [27] Moniz S, Catarino AL, Marques AR, et al. Clonal origin of nonmedullary thyroid tumours assessed by non-random X-chromosome inactivation. Eur J Endocrinol 2002;146:27-33. [28] Carta C, Moretti S, Passeri L, et al. Genotyping of an Italian papillary thyroid carcinoma cohort revealed high prevalence of BRAF mutations, absence of RAS mutations and allowed the detection of a new mutation of BRAF oncoprotein (BRAF(V599lns)). Clin Endocrinol (Oxf) 2006;64:105-9. [29] Jovanovic L, Delahunt B, McIver B, et al. Thyroid gland clonality revisited: the embryonal patch size of the normal human thyroid gland is very large, suggesting X-chromosome inactivation tumor clonality studies of thyroid tumors have to be interpreted with caution. J Clin Endocrinol Metab 2003;88:3284-91. [30] Carcangiu ML, Zampi G, Pupi A, et al. Papillary carcinoma of the thyroid. A clinicopathologic study of 241 cases treated at the University of Florence, Italy. Cancer 1985;55:805-28. [31] Knauf JA, Ma X, Smith EP, et al. Targeted expression of BRAFV600E in thyroid cells of transgenic mice results in papillary thyroid cancers that undergo dedifferentiation. Cancer Res 2005;65:4238-45. [32] Trovisco V, Soares P, Preto A, et al. Type and prevalence of BRAF mutations are closely associated with papillary thyroid carcinoma histotype and patients' age but not with tumour aggressiveness. Virchows Arch 2005;446:589-95. [33] Sedliarou I, Saenko V, Lantsov D, et al. The BRAFT1796A transversion is a prevalent mutational event in human thyroid microcarcinoma. Int J Oncol 2004;25:1729-35. [34] Reinhoff Jr WF. Lymphatic vessels of thyroid gland in dog and man. Arch Surg 1931;23:783-804. [35] Ricarte-Filho JC, Ryder M, Chitale DA, et al. Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1. Cancer Res 2009;69:4885-93.
www.elsevier.com/locate/humpath
Original contribution
Clonal analysis of bilateral, recurrent, and metastatic papillary thyroid carcinomas☆ Weibin Wang MD a , Haiyong Wang MDa , Xiaodong Teng MDb , Haohao Wang MD a , Chenyu Mao MD a , Rongyue Teng MDa , Wenhe Zhao MD a , Jiang Cao PhD c , Thomas J. Fahey III MDd , Lisong Teng MD, PhD a,⁎ a
Cancer Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China Department of Pathology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China c Key Laboratory of Biotherapy of Zhejiang Province, Sir Run Run Shaw Institute of Clinical Medicine, Zhejiang University, Hangzhou 310016, China d Department of Surgery, New York Presbyterian Hospital, and Weill Medical College of Cornell University, New York, NY 10021, USA b
Received 26 November 2009; revised 9 February 2010; accepted 25 February 2010
Keywords: Papillary thyroid carcinoma; Bilateral, BRAF mutation; X-chromosome inactivation; Clonal origin
Summary Papillary thyroid carcinoma usually presents as a multifocal disease; and tumors often recur in the contralateral thyroid lobe, raising questions concerning their clonal origins. The clonality of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe remains unknown. Accordingly, we examined 25 pairs of bilateral papillary thyroid carcinomas (synchronous or metachronous) and 15 matched metastatic lymph nodes. BRAF gene mutation analysis combined with X-chromosome inactivation was used to evaluate these tumors' clonal origins. Genomic DNA was prepared from paraffin-embedded tissues after microdissection. In total, 62 tumors yielded DNA of adequate quality. Eighteen (18/21, 85.7%) of 21 informative cases showed concordant BRAF status in tumors from both thyroid lobes, being either BRAF mutation positive (12 patients) or BRAF mutation negative (6 patients). Metastatic lymph nodes in 12 patients (12/15, 80%) had a complete concordance of BRAF state with their primaries. Of the 18 studied female patients, 11 were suitable for X-chromosome inactivation assay. Nine cases (9/11, 81.1%) showed the same pattern of inactivation in bilateral tumors. A close correlation was found between BRAF mutation and X-chromosome inactivation analysis. In conclusion, our data provide evidence that bilateral, recurrent, and metastatic papillary thyroid carcinomas often arise from a single clone and that intrathyroidal metastasis may play an important role in the development of bilateral tumors, as well as in the recurrence of this malignancy. © 2010 Elsevier Inc. All rights reserved.
1. Introduction ☆
Funding support: This study was supported by National Basic Research Program of China (973 Program, No. 2009CB521704) from the Ministry of Science and Technology of the People's Republic of China (Beijing, China). ⁎ Corresponding author. E-mail address: [email protected] (L. Teng). 0046-8177/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2010.02.008
Papillary thyroid carcinoma (PTC) is the most common histotype of thyroid cancer and accounts for about 1% of human malignancies [1]. Patients with PTC generally have a good prognosis, especially in those younger than 45 years at
1300 the time of diagnosis [2]. However, about 5% of patients with PTC experience a recurrence within 5 years after the initial treatment [3,4]. In patients with local or distant recurrence after lobectomy, a tumor is found in more than 60% of the time in the resected contralateral lobe [5,6]. In addition, PTC is often multifocal, with a reported frequency varying widely from 18% to 87%, among which 61% are bilateral [7,8]. The clonal origin of multifocal PTC is still under debate. There are 2 different theories to address this issue. One is that these tumors are of monoclonal origin, arising from intrathyroidal metastasis of a clonal population of cells. The other is that the multiple foci are of independent origin and arise separately in a background of field cancerization. Distinguishing between these 2 theories will improve our understanding of both the biological mechanisms of multifocal PTC, as well as the possible reasons why PTC recurs. Several studies have been performed to investigate the clonality of tumors located in the same thyroid lobe with varied results [9-13]. The clonality of tumors occurring simultaneously in both thyroid lobes or that of recurrent tumors in the contralateral lobe remains unknown. Previous studies have shown that PTC displays several highly prevalent genetic alterations, like mutations in BRAF or RAS, and rearrangements of the RET or NTRK1; among these alterations, mutations in the BRAF gene (exon 15BRAFV600E) is the most common [14-18]. Because of its high prevalence and presumed early place as a genetic event in thyroid carcinogenesis [19], BRAFV600E mutation has been used as a marker for clonality analysis and has shown some advantage in evaluating the clonal origin of multifocal PTCs [12,13]. However, few studied have combined this mutation with other methods based on changes established early enough to assess the clonality of tumors. Determination of X-chromosome inactivation (XCI) has also been used to study clonality in many human tumors, including thyroid cancers [20]. According to the Lyon [21] hypothesis, 1 of the 2 X-chromosomes in all female somatic cells is randomly deactivated by the DNA methylation at CpG islands during early embryogenesis. Once established, the inactivated X-chromosome is stably transmitted from a parent cell to its progeny. Therefore, a given group of cells resulting from clonal expansion of a single progenitor shows the same pattern of XCI. Distinguishing the patterns of XCI permits investigators to determine whether multiple tumors in female subjects arise from one or more progenitors. In this study, we investigated the clonality of bilateral PTCs (synchronous or metachronous) and metastatic lymph nodes by assaying for the presence or absence of the BRAF mutation. We also examined the XCI status in separate tumors in female patients. By combining the 2 strategies, genotypic clonality (BRAF mutations, involved in the malignant transformation) and phenotypic clonality (XCI, established in early embryogenesis), we reasoned that we could provide more definitive answers as to clonality in multifocal or recurrent PTCs.
W. Wang et al.
2. Materials and methods 2.1. Case selection and tumor specimen collection A computer-based database comprising 809 patients was established to select eligible cases. These patients were admitted to the First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China), from 1997 to 2006 and underwent thyroidectomy for the treatment of thyroid carcinoma, with or without neck lymph node dissection. A list of cases was generated according to the following criteria: (a) bilateral or recurrent PTCs (including both primary and recurrent tumors) were diagnosed and treated in the same hospital; (b) the intervals between the 2 diagnoses were either less than or equal to 1 month (synchronous) or greater than or equal to 3 months (metachronous); and (c) cases with a history of previous irradiation of the neck were excluded. Finally, after excluding patients whose tumors were only available for study in one thyroid lobe (therefore, unable to be analyzed as a pair), 25 cases were enrolled in this study. Paraffinembedded blocks were retrieved from the surgical pathologic files in the Department of Pathology, and patients were followed up in our Cancer Center. Tumors in individual cases were randomly numbered without the knowledge of the paired patient's ID and the molecular analysis results. All of the slides were reviewed to confirm the diagnosis and to locate the tumor's area for subsequent microdissection by a senior pathologist. The histologic subclassification of PTC into conventional variant (CVPTC), follicular variant (FVPTC), and tall cell variant was made according to the criteria of DeLellis et al [22]. A tumor stage was defined according to Tumor, Nodes, and Metastases system as described by the American Joint Committee on Cancer Staging Manual, Sixth Edition [23]. This study was approved by the Institutional Review Board of First Affiliated Hospital, Zhejiang University School of Medicine.
2.2. Microdissection and DNA extraction Genomic DNA was isolated from archival paraffinembedded specimens by microdissecting the tumor tissue. Briefly, 4 unstained 10-μm–thick sections were deparaffinized in xylene and rehydrated in graded ethanol. Tumor margins were marked using the hematoxylin and eosin (H&E)–stained section as a template, and the tumor tissue was dissected from the unstained slides. Finally, the dissected but unstained sections were stained by H&E to confirm that the harvested tumor cells had indeed been segregated from the surrounding normal cells. Wherever available, samples from distant nonneoplastic thyroid parenchyma were dissected as control reference. The harvested tumor cells were transferred into 1.5-mL Eppendorf tubes, and DNA was extracted overnight at 56°C
Clonal analysis of bilateral, recurrent, and metastatic PTCs in a 500-μL tissue lysis buffer (10 mmol/L Tris, 0.2 mmol/L EDTA, and 0.5% sodium dodecyl sulfate; pH 8.0) plus 0.1 mg proteinase K (Sigma, St Louis, MO). On the second day, Chelex-100 resin (Sigma) was added to each sample; and the mixture was incubated at 37°C for another hour to improve the quality of DNA. After extraction with phenol/chloroform, genomic DNA was precipitated with ethanol in the presence of acetate.
2.3. BRAF mutation analysis by polymerase chain reaction amplification and DNA sequencing Because most BRAF mutations are located in exon 15 of BRAF gene, we amplified this exon followed by direct DNA sequencing. A 224–base pair (bp) fragment was amplified by using the primer sequences as described previously [24]. Two hundred nanograms of genomic DNA was used as a template in a 25-μL polymerase chain reaction (PCR) mixture containing 1× PCR buffer, 2.0 mmol/L magnesium chloride, 0.2 mmol/L of each deoxynucleotide triphosphate, and 1 unit of HotStart Taq polymerase (Takara, Shuzo, Japan). Cycling conditions were as follows: initial denaturation at 95°C for 10 minutes and then 40 cycles with denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. All PCR products were visualized by electrophoresis in a 2% agarose gel and purified using PureLink Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA). The purified PCR products were then sequenced on ABI PRISM 3730XL DNA Analyze (Applied Biosystems, Foster City, CA) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing results were compared with the normal BRAF gene in GenBank database.
2.4. XCI analysis The X-chromosome linked human androgen receptor gene (HUMARA) was chosen for XCI study. In the first exon of the X-linked HUMARA gene, there is a high polymorphic trinucleotide (CAG) repeat; and approximately 90% of females are heterozygous with respect to the number of CAG repeats in this region [25]. In addition, there are 2 HhaI restriction sites located close to the CAG repeat polymorphism and selectively methylated in inactive X-chromosomes [26]. Consequently, digestion with methylation-sensitive endonucleases followed by PCR amplification with primers flanking these restriction sites and the heterozygous CAG repeats can be used to distinguish between active and inactive X-chromosomes. Ten-microliter DNA extract from each sample was divided into 2 aliquots, which were subsequently subjected to either digestion with 1 unit HhaI endonuclease (New England Biolabs, Inc, Beverly, MA) at 37°C for 16 hours or mock digestion with restriction buffer alone in a total
1301 volume of 10 μL. After incubation, the enzyme was heat inactivated at 65°C for 20 minutes. To evaluate the efficiency of the restriction reactions, a paraffin-embedded tissue-derived male control sample was included in each assay. Three microliters of digested or undigested DNA was amplified in a 25-μL PCR mixture containing 1× PCR buffer, 4% DMSO, 2.0 mmol/L magnesium chloride, 0.2 mmol/L of each deoxynucleotide triphosphate, 1 unit of HotStart Taq polymerase (Takara), and the following primers [11] at a concentration of 200 nmol/L: forward primer, 5′-TCTGTTCCAGAGCGTGCGCGAAGT-3′ bearing a fluorescent FAM tag on the 5′ end, and reverse primer, 5′-CTCTACGATGGGCTTGGGAGAAC-3′. Cycling conditions were as follows: initial denaturation at 95°C for 10 minutes and then 34 cycles with denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. We size separated the PCR products on an automated DNA sequencer (ABI-3730, Applied Biosystems) and analyzed the results using GeneMapper 4.0 software (Applied Biosystems). Allele intensity was determined from the allele peak height, which are proportional to the amount of PCR product from the given allele. To account for possible differences in fluorescence signal acquisition and gel loading of each allele, we modified to uniform the volume of internal size marker (Genescan-500Liz; ABI Perkin Elmer, Warrington, United Kingdom) and calculated a corrected allele ratio as follows: (peak 1 height of undigested sample ÷ peak 2 height of undigested sample ÷ Liz 200 bp height of undigested group)/(peak 1 height of digested sample ÷ peak 2 height of digested sample ÷ Liz 200 bp height of digested group). We defined tumor samples as nonrandom XCI if this corrected ratio was less than 0.5 or greater than 2 [27]. Tumors were considered to be of single clonal origin if the same inactivation pattern was detected in each separate tumor from individual patient.
2.5. Statistical analysis χ2 test and Spearman rank correlation test were used to analyze the concordance in comparison of the 2 methods. A P value b .05 was considered significant. Data analysis was performed using the Statistical Package for Social Sciences for Windows (SPSS, Inc, Chicago, IL).
3. Results 3.1. Clinical features of the study group A total of 66 tumors from 25 patients (female, 18; male, 7) were analyzed in this study, among which 51 were bilateral tumors and 15 were metastatic lymph nodes. Twenty-two pairs were synchronous, whereas 3 pairs were metachronous
1302 Table 1
W. Wang et al. Clinical characteristics of patients studied
Patient Sex Age at Categories (left/right) ID diagnosis (y)
Synchronous cases 1 F 39 2 M 36 3 F 36 4 F 20 5 M 26 6 F 39 7 F 38 8 F 42 10 F 14 11 F 48 12 F 36 13 F 53 14 F 45 15 F 67 16 F 31 17 M 75 18 M 38 19 F 17 21 F 41 22 M 52 23 F 70 25 F 64 Metachronous cases c 9 F 51 20 M 45 24 M 32
Disease extent at surgery Extrathyroidal Lymph node Distant Stage extension metastases metastases
a
Follow-up Last known time (mo) disease status
FV/FVPTC CV/CVPTC FV/CV + FVPTC b CV/FVPTC FV/FVPTC CV/CVPTC FV/CVPTC CV + FV b/CV + FVPTC b CV/CVPTC CV/CVPTC FV/CVPTC CV/CVPTC CV/CVPTC CV/CVPTC FV/CVPTC TCV/TCVPTC CV/CVPTC CV + FV b/CV + FVPTC b CV/CVPTC CV/CVPTC CV/CVPTC CV/CVPTC
No Yes Yes No No No Yes Yes No No No Yes No No No Yes No No Yes No No Yes
Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes No No Yes
No No No No No No No No No No No No No No No No No No No Yes No Yes
I I I I I I I I I IV I IV I IV I IV I I I IV II IV
41 72 25 37 63 17 21 42 28 32 67 25 57 29 69 9 92 105 18 15 74 6
AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD AFD DOD AFD AFD AFD AWD AFD DOD
CV/CVPTC FV/CVPTC CV/CVPTC
Yes No No
Yes Yes Yes
No No No
IV IV I
93 78 89
DOD AFD AFD
Abbreviations: F, Female; M, Male; TCVPTC, tall cell variant papillary thyroid carcinoma; AFD, alive, free of disease; AWD, alive with disease; DOD, dead of disease. a Histopathologic staging was reviewed according to the 2002 American Joint Committee on Cancer Staging Manual. b Displayed a coexistence of 2 different histopathologic components in the same focus. c Showed the characteristics at the initial treatment.
(ie, recurrent, shown in Table 1). The mean age at diagnosis was 42.2 years (range, 14-75 years), and the median diameter of the studied tumors was 2.2 cm (range, 0.3-9.4 cm). Of the 3 recurrent PTCs, both the primary and recurrent tumors were studied; and the intervals between recurrences were 4, 5, and 5 years, respectively. A large majority of studied patients (19/25, 76%) displayed a similar microscopic appearance in pairs of bilateral tumors, whereas 6 cases showed a coexistence of different histopathologic subtypes in distinct PTC foci. At the time of this study, all the patients had a postoperative follow-up with a mean period of 47.9 months (rang, 9-105 months); and the outcomes were detailed in Table 1.
3.2. BRAF mutation in pairs of separate tumors Because some samples were not well preserved and some extracted DNA from formalin-fixed and paraffin-embedded
blocks was degraded, 4 tumor tissues failed to yield DNA of adequate quality for PCR amplification; and 62 tumors from 25 patients were screened for BRAFV600E mutation. Fifteen patients (15/25, 60%) were positive for this common mutation (the missense T to A transversion at nucleotide 1799). Occasionally, we detected a novel BRAFV599Ins mutation also in exon 15 (an in-frame insertion of 3 nucleotides [GTT] at position 599), which was first reported in 2006 [28]. BRAF mutations were not found in any normal tissues that were available in 8 BRAF-positive cases (data not shown). A summary of BRAF mutations in bilateral PTCs (synchronous or metachronous) at multiple sites is shown in Table 2. Four cases were not informative because one of their multiple foci in each patient failed to amplify in PCR. Eighteen (85.7%) of 21 patients showed concordant BRAF status in tumors from both the left and right thyroid lobes, being either BRAF mutation positive (12 patients) or BRAF mutation negative (6 patients), indicating that these tumors
Clonal analysis of bilateral, recurrent, and metastatic PTCs Table 2
1303
BRAF mutation in bilateral cases
Patient Tumor left Tumor right Concordance ID Age at Tumor Tumor Mutation Age at diagnosis (y) Tumor no. Tumor size (cm) Mutation diagnosis (y) no. size (cm) Synchronous cases 1 39 2 36 3 36 4 20 5 26 6 39 7 38 8 42 9 51 10 14 11 48 12 36 13 53 14 45 15 67 16 31 17 75 18 38 19 17 21 41 22 52 23 70 25 64 Metachronous cases 9a 51 20 45 24 37
10 13 32 16 54 14 39 84 68 23 36 25 35 49 2 52 4 86 70 20 6 19 81
1.1 1.5 2.0 0.5 2.3 1.2 1.0 1.8 4.5 0.8 1.0 0.3 4.0 1.0 1.3 0.5 3.5 0.7 3.5 4.0 NA 2.0 2.0
WT T1799A T1799A WT WT T1799A WT NA V599Ins WT T1799A T1799A T1799A T1799A T1799A WT T1799A NA WT WT T1799A WT T1799A
39 36 36 20 26 39 38 42 51 14 48 36 53 45 67 31 75 38 17 41 52 70 64
9 50 5 28 31 37 38 40 75 44 17 47 48 85 51 53 55 63 74 87 42 29 82
1.2 4.5 1.5 2.0 1.5 1.5 1.5 2.5 1.8 1.2 0.8 1.3 1.0 1.1 3.0 1.5 5.0 2.0 NA 3.5 4.0 3.0 0.8
T1799A WT T1799A WT WT T1799A WT WT V599Ins WT T1799A T1799A T1799A NA T1799A WT T1799A WT WT NA T1799A T1799A T1799A
No No Yes Yes Yes Yes Yes – Yes Yes Yes Yes Yes – Yes Yes Yes – Yes – Yes No Yes
68 71 80
4.5 3.5 0.6
V599Ins T1799A T1799A
55 50 32
43 62 60
9.4 1.5 2
V599Ins T1799A T1799A
Yes Yes Yes
Abbreviation: NA, data not available because of incomplete clinical information or DNA quality being unsuitable for PCR amplification and sequencing. a Patient 9 was a case that initially presented as a bilateral PTC and recurred 5 years later.
might be of monoclonal origin. Fig. 1 shows a representative case with BRAFV600E positive in both the left and right thyroid lobes; the nearly equal peak areas of A and T nucleotides suggest a high homogeneity of tumor cells in these microdissected samples. The other 3 patients (patients 1, 2, and 23) presented with a coexistence of BRAF mutation-positive and BRAF mutation-negative tumor in both lobes, suggesting that these foci might arise from distant progenitors. In lymph node metastatic PTC, 12 (80%) of 15 cases had complete concordance between the defects present in the primary and metastatic site (Table 3). In the remaining 3 cases, BRAF mutation was positive exclusively in the primary tumors but not in the lymph node metastases. In addition, there was no case in which the status of BRAF mutation in the metastasis was exclusively identical to that of the contralateral primary tumor. These results suggested that most lymph node metastases were clonally related to the primary tumors, particularly the one on the ipsilateral side.
3.3. XCI assay Of 18 studied female patients, 12 were informative for XCI assay because of adequate quality of DNA and polymorphism in exon 1 of HUMARA gene (Table 4). The corrected ratios in 11 pairs of tumors were more than 2.0 or less than 0.5, representing a preferential loss of 1 of the 2 alleles after digestion (ie, a pattern of nonrandom XCI); and therefore, these foci were suitable for further analysis. The remaining patient (patient 13) demonstrated a random pattern of XCI in bilateral tumors and was excluded. Normal tissues obtained from 3 cases also displayed a random pattern of XCI, serving as an important control to avoid aberrant results due to embryonic patch size [29]. When comparing the pattern of tumors in each pair, we found 9 cases (9/11, 81.1%) that shared the same pattern of nonrandom XCI in bilateral thyroid tumors (a representative case is shown in Fig. 2, right panels), whereas different inactivated alleles were observed in 2 patients. In 7 metastatic lymph nodes, 5 showed the same pattern of XCI
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W. Wang et al.
Clonal analysis of bilateral, recurrent, and metastatic PTCs Table 3
1305
Comparison of BRAF mutation status in primary tumor and matched metastatic lymph node
Patient ID
Tumor no. of MLN
BRAF mutation status
Concordance with
MLN
Ipsilateral PT
Contralateral PT
Ipsilateral PT
Contralateral PT
1 3 4 5 7 8 9 9a 10 11 18 19 20 21 24 25
24 22 56 21 26 30 73 3 57 46 67 76 72 11 61 78
T1799A T1799A WT WT WT WT WT V599Ins WT WT WT WT WT WT T1799A T1799A
T1799A T1799A WT WT WT WT V599Ins V599Ins WT T1799A WT WT T1799A WT T1799A T1799A
WT T1799A WT WT WT NA V599Ins V599Ins b WT T1799A NA WT T1799A b NA T1799A b T1799A
Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes No Yes Yes Yes
No Yes Yes Yes Yes – No Yes Yes No – Yes No – Yes Yes
Abbreviations: NA, data not available because of DNA quality being unsuitable for PCR amplification and sequencing; PT, primary tumor; MLN, metastatic lymph node. a Patient 9 had another metastatic lymph node at recurrent treatment. b Lymph node metastasis is metachronous with the primary tumor.
as both the primaries, one was exclusively identical to the ipsilateral primary and the other was distinct to neither primary tumor.
considering that multiple foci from independent clones can also share the same pattern of XCI coincidentally, we deemed that this metastatic lymph node was not clonally related to the primary tumor we studied.
3.4. Close correlation between BRAF mutation and XCI Eleven pairs of bilateral PTCs and 7 metastatic lymph nodes were examined for both BRAF mutation detection and XCI analysis. We found a complete concordance in 10 bilateral PTCs between these 2 methods in evaluating the clonality (10/11, 90.1%; Pearson χ2, 2-sided P = .026; Spearman rank correlation test, r = 0.671). Only one patient showed a different pattern of XCI but shared the same BRAF mutation negative status in bilateral tumors. Because 2 unrelated tumors might present the same mutation status by chance, we assumed this case as an independent clonal origin. Therefore, in all, 17 (81%) of 21 patients with bilateral tumors in our study were suggested to be of monoclonal origin, whereas 4 bilateral cases arose as independent tumors. In 7 lymph node metastases, 6 shared concordance between BRAF mutation status and XCI and the primary, whereas only 1 (in patient 11) showed the same inactivated allele as the primary but with diverse mutation results. When
4. Discussion Multifocality is frequently observed in patients of PTC, with an increased risk of lymph node metastases and regional recurrence [7,30]. However, as a specific subtype of multifocal PTCs, bilateral PTC has not been widely investigated. Studies of this subtype were just restricted to clinical observations [5,6,30], and the clonal origin of bilateral tumors remains unknown. Most clonality studies on PTCs were done on multiple tumors located in one thyroid lobe [10,12]. Only a few reports included some pairs of tumor samples present in both thyroid lobes. Shattuck et al [11] studied the clonal origin of multifocal PTC in 10 informative subjects. With consideration of tumor location, 7 were bilateral, of which 3 showed independent clonal origin with different patterns of XCI. Lack of such pairs of tumor samples may be the main
Fig. 1 Concordant pattern of BRAF mutation in bilateral PTC and matched metastatic lymph node. Shown in the left-hand panels are photomicrographs (stained with H&E, original magnification: ×100) of 3 tumor foci from patient 3. For each tumor, the corresponding plot (right-hand panels) is a sequence result of BRAF exon 15. Although the bilateral tumors do not have a complete concordance in microscopic appearances (left lobe–tumor 32, FVPTC; right lobe–tumor 5, FV + CVPTC), they are of the same BRAFV600E mutation status. The metastatic lymph node (tumor 22) is also positive for BRAFV600E mutation, indicating that these tumors are clonally related.
1306 Table 4
W. Wang et al. XCI analysis in bilateral cases
Patient ID and tumor no.
3 32 5 22 N 4 16 28 56 6 14 37 9 68 75 73 Recurrence 43 3 N 10 23 44 57
Tumor location
XCI Corrected allele ratio
Inactivated allele a
Left Right LN (L) Left
22.4 5 20.67 0.69
L L L Random
Left Right LN (L)
3.38 0.18 5.71
L S L
Left Right
0.27 0.23
S S
Left Right LN (R)
0.02 0.07 2.06
S S L
Right LN (R) Right
0.16 0.11 0.9
S S Random
Left Right LN (R)
0.34 0.19 0.29
S S S
Patient ID and tumor no.
11 36 17 46 12 25 47 13 35 48 N 15 2 51 16 52 53 23 19 29 25 81 82 78
Tumor location
XCI Corrected allele ratio
Inactivated allele a
Left Right LN (R)
9.49 86.55 2.14
L L L
Left Right
0.11 0.02
S S
Left Right Right
0.85 1.76 0.78
Random Random Random
Left Right
0.35 0.37
S S
Left Right
9.86 10.15
L L
Left Right
0.1 36.7
S L
Left Right LN (R)
8.75 7.79 8.12
L L L
Abbreviations: N, normal tissues obtained around tumor area; LN (L), lymph node metastasis in left side of neck; LN (R), lymph node metastasis in right side; S, small allele; L, large allele. a For each informative patient, 2 alleles were separated because of the polymorphism of CAG-repeat numbers in HUMARA gene. After digestion, one of them was preferentially lost in tumors of nonrandom XCI, either the smaller allele or the larger allele. A random pattern of XCI (random) was characterized by the corrected ratios between 2.0 and 0.5. Tumors with random pattern of XCI were excluded in further analysis, whereas in normal tissues, this pattern of XCI could serve as qualified control.
reason why bilateral PTC has never been characterized in a large population. In this study, 25 pairs of bilateral PTCs were enrolled based on a database spanning one decade. Our series is, to our knowledge, the first to investigate the clonal origin of bilateral PTCs with a larger sample size. We have traced the genealogies of bilateral, recurrent, and metastatic PTCs by combining BRAF mutation and XCI analysis. Our results provided evidence that bilateral tumors in individual patients
often arose from the same progenitor cell and represented a single primary with subsequent intrathyroidal metastases to contralateral lobe. Thus, bilateral PTC should be considered as a progressive state of this disease. BRAFV600E mutation in exon 15, which was first described in 2002 [24], represents the most common genetic alteration in thyroid cancer [19]. As this mutation was shown to induce goiter and invasive PTC in transgenic mice [31] and was detected in the microcarcinomas [32,33],
Fig. 2 Comparison of BRAF mutation and XCI in a bilateral PTC. This figure presents a bilateral case (patient 25) with tumors in left thyroid robe (upper, tumor 81) and right thyroid lobe (down, tumor 82). Shown in the left-hand panels are DNA sequencing results of each tumor. PCR products of BRAF gene exon 15 are sequenced bidirectionally with forward primer (forward) and reverse primer (reverse); BRAFV600E mutation was characterized as the transversion from T to A by forward primer and A to T by reverse primer. Shown in the right-hand panels are results of separation of fluorescence-labeled PCR products on an automated sequencer. For each tumor, the corresponding height and size of plot represent the quantity and size in base pairs of PCR products, respectively. Amplified DNA from tumors without digestion showed 2 alleles, a smaller allele and a larger allele, indicating the polymorphism of CAG-repeat numbers in HUMARA gene. Loss of PCR amplification for the larger allele (indicated by the arrow) after digestion in both the left thyroid tumor (upper, tumor 81) and right thyroid tumor (down, tumor 82) indicates that the bilateral tumors share the same pattern of allele inactivation and are probably clonally related. The internal size marker (marked by ☆) is included in each assay to account for possible differences in fluorescence signal acquisition and gel loading. Abbreviations: H, height; S, size.
Clonal analysis of bilateral, recurrent, and metastatic PTCs
1307
1308 BRAFV600E mutation was presumed as an early genetic event and was already used as a marker for clonal analysis in multiple PTCs [12,13]. However, because this mutation is common in PTC, it cannot be distinguished whether multiple tumors sharing the same status of mutation is a consequence of a single mutation, spreading throughout the gland, or of multiple, unrelated mutational events in separate tumors. Only those cases when a very rare mutation is found in both tumors (BRAFV599Ins) are probably of the same clonal origin. Therefore, the proportion of tumors with single clonal origin may be overestimated when only BRAF V600E mutation was analyzed. In our study, we further examined the pattern of XCI in informative female patients. Almost all the studied cases showed the same clonality results between XCI and BRAF mutation analysis. Only one patient demonstrated a different allele inactivation but shared the same BRAF mutation status in bilateral tumors. We believe this close correlation between these 2 methods can at least partially resolve the ambiguity in evaluating tumors of the same mutational state. However, similar concerns should apply to the interpretation of the XCI status, for the presence of the same allele inactivation pattern provides only a 50% probability that 2 tumors are of the same clonal origin. Thus, caution is still warranted in interpreting our data. In contrast to our findings, Giannini et al [12] showed that as high as 40% of multifocal PTCs had discordant patterns of BRAF mutation. Such findings are seemed to be consistent with the presence of an independent clonal origin. The different results of the current study and that of Giannini et al may be mainly attributed to differences in case selection. Because the frequency of mutation was relatively higher here than that in the study of Giannini et al (64% versus 62%) and the prevalence of lymph node metastasis was obviously high in our study (21/25, 84%), we infer that bilateral PTCs studied here were quite distinct from the common multifocal PTCs studied by others. Few studies have compared the molecular defects in metastatic sites with those of the primaries, especially in bilateral PTCs. Our results showed that most lymph node metastases were derived from the primary tumors, particularly the ipsilateral one, supporting the current notion that lateral neck dissection is adequate for biopsy-proven lymph node metastases in clinical practice. However, there were 3 cases that BRAF mutation were present exclusively in the primary tumors but absent in lymph node metastases. The XCI analysis also showed a different pattern of inactivation in the primary tumors and metastatic lymph nodes of patient 9. Because several reports had proposed different clonal origins of multiple PTCs [11,12] and our study also indicated that some bilateral PTCs may arise as independent tumors, we infer that another unrelated tumor was present simultaneously within the thyroid and that this non-BRAF mutated tumor metastasized to the lymph node. Because most bilateral PTCs arise from a single progenitor, how does this progenitor cell lead to contralat-
W. Wang et al. eral and recurrent PTCs? It is likely that a specific genetic alteration (perhaps mutations in BRAF gene) occurs in a thyroid follicular epithelium and provides a growth advantage, giving rise to a proliferative state in which all neoplastic cells have the same genetic trait. In this view, such alterations can be used as a valid marker for clonality analysis. During further development, daughters of this transformed cell could gradually migrate regionally through an abundant network of intralobar lymphatic vessels [34]. These vessels, which intercommunicate from lobe to lobe across the isthmus, would allow tumor metastases ready access to the contralateral lobe. Subsequently, late genetic events, such as the recently reported AKT1 mutation [35], may convert a small percentage of such cells into a more invasive state, eventually leading to clinically bilateral carcinomas. The micrometastases, located in the residual thyroid tissue after surgery, are not detected by imaging studies at the time of initial treatment and remain to become the source of recurrences. As recommended in the NCCN Clinical Practice Guidelines in Oncology: Thyroid carcinoma (v.1.2009), patients who present with bilateral PTCs are one of the indications to be treated by total thyroidectomy. However, little molecular data have been incorporated to support this clinical guideline. Our study provides molecular evidence that bilateral PTC should be classified as a more advanced stage and that total thyroidectomy is appropriate for bilateral PTCs.
Acknowledgment We thank Ruokuo Han, Ruopeng Weng, and Zonglin Lin for patients' clinical follow-up, and Qixing Chen and Feng Zhang for technical assistance. We are also grateful to Dr Robert Wohlhueter (Centers for Disease Control and Prevention, Atlanta, GA; retired) for his contributions to the preparation of the manuscript.
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