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EXTENSIVE CLINICAL EXPERIENCE |
Departments of Surgery (C.L., R.G., C.U., A.P., P.B., M.M., G.M., P.M., F.B.) and Endocrinology (R.E.), University of Pisa, 56126 Pisa, Italy; and Dipartimento di Biologia e Patologia Cellulare e Molecolare (M.S.), Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, University Federico II, 80131 Naples, Italy
Address all correspondence and requests for reprints to: Fulvio Basolo, Ph.D., M.D., Department of Surgery, Division of Pathology, Via Roma, 57, 56126 Pisa, Italy. E-mail: f.basolo{at}med.unipi.it.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: We examined BRAF mutations in a consecutive series of 500 PTC patients who underwent surgery in the Department of Surgery of the University of Pisa, and we correlated the presence of the mutation with clinicopathological parameters of the patients: age, gender, tumor size, presence of tumor capsule, extrathyroidal invasion, multicentricity, presence of node metastases, and tumor class.
Design: BRAF (exon 15) mutation was examined by PCR-single strand conformational polymorphism followed by DNA sequencing in laser-capture microdissected tissue samples.
Results: In this study, BRAF mutation was found in 219 of 500 cases (43.8%). In particular, we found the most common BRAF V600E mutation in 214 cases (42.8%), BRAF K601E mutation in three cases (0.6%), BRAF VK600–1E (0.2%) in one case, whereas in one case we found a new 14-bp deletion with concomitant 2-bp insertion, VKSR600–3del and T599I, respectively. BRAF V600E was associated with extrathyroidal invasion (P < 0.0001), multicentricity (P = 0.0026), presence of nodal metastases (P = 0.0009), class III vs. classes I and II (P < 0.00000006), and absence of tumor capsule (P < 0.0001), in particular in follicular- and micro-PTC variants. By multivariate analysis, the absence of tumor capsule remained the only parameter associated (P = 0.0005) with BRAF V600E mutation.
Conclusions: Our data suggest that BRAF V600E mutation is associated with high-risk PTC and in particular in follicular variant with invasive tumor growth.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Somatic point mutations in the BRAF gene have been identified as the most common genetic event in PTC (approximately 44% of PTC cases) (3, 4, 5, 6, 7). BRAF codes for a serine/threonine kinase, which functions in the RAS gene/MAPK cascade. The V600E mutation (T1799A) represents more than 90% of BRAF mutations in PTC. This mutation affects the conformation of the activation loop in the BRAF kinase domain and potentiates by more than 500-fold its catalytic activity. Other more rare BRAF mutations have been found. In particular, the K601E (G1801A transversion) mutation has been described in follicular variant (FV) cases (5, 7).
At present, no unequivocal correlations between genotype and clinicopathological features of PTC patients have been reported (7, 8, 9, 10, 11, 12, 13, 14). A few studies (5, 15, 16, 17, 18) have shown a correlation of BRAF V600E with more advanced stage, nodal/distant metastases at diagnosis and (or) tumor recurrence. However, other reports failed to demonstrate the same association (8, 9). These discrepancies might be due to the heterogeneity of the histological variants of PTC, epidemiological factors, or the small number of cases studied.
To clarify better whether BRAF mutations may relate to clinicopathological parameters of prognosis, we evaluated 500 consecutive cases who underwent surgery in the same department. We found that V600E mutation is associated with extrathyroidal invasion (P < 0.0001), multicentricity (P = 0.0026), presence of nodal metastases (P = 0.0009), class III vs. classes I and II (P < 0.00000006), and absence of tumor capsule (P < 0.0001). Interestingly, by multivariate analysis, only carcinomas without tumor capsule remained statistically associated with BRAF V600E mutation.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Archival formalin-fixed and paraffin-embedded thyroid specimens from 500 consecutive patients with PTCs, from January to September 2006, were retrieved retrospectively from the files of the Department of Pathology, University of Pisa (Table 1
). All patients were treated at the Institute of Endocrinology and underwent thyroidectomy in the Department of Surgery of the same university. The initial treatment was total thyroidectomy and, when necessary, central neck and/or laterocervical lymph node dissection. Tumor samples were obtained in accordance with protocols approved by the institutional review board, and informed consent was achieved 1 d before surgery together with the surgical one.
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Histological diagnosis was made independently, in a blinded fashion, by two pathologists (C.U. and F.B.). Tumors were classified according to the histopathological typing of the World Health Organization (19). A concordance rate of 98% was obtained between the two investigators. The few discordant cases were discussed with a third pathologist (A.P.).
Microdissection and DNA extraction
Serial 5-µm sections were taken from paraffin blocks for histological examination on glass slides (Fig. 1) and for DNA extraction on membrane slides (Nikon, Firenze, Italy). Presence of the tumor tissue was confirmed in the first and the last section for each section series. Unstained sections were deparaffinized with Bio-Clear (Bio-Optica, Milano, Italy), rehydrated in graded ethanol, and stained with hematoxylin and eosin. Microdissections were performed by using the laser-assisted SL microcut Microtest (MMI GmbH, distributed by Nikon). For each sample, three to five microareas of 5 µm were obtained. Each area contained 200–500 cells. Particular care was taken in microdissecting areas of the dominant tumor, additional foci, and node metastases. Samples from the nonneoplastic thyroid parenchyma, generally from the contra lateral lobe, were dissected as a control reference. The microdissected cells were placed on SL microcut transfer film (Nikon), and the DNA was extracted overnight in a humidified chamber at 56 C in 200 µl of tissue lysis buffer (ATL DNeasy tissue kit; QIAGEN GmbH, Hilden, Germany) containing 20 µl of proteinase K. DNA was isolated by QIAGEN spin column; carrier tRNA was added to improve DNA recovery. Finally, DNA was eluted in 40 µl Tris/EDTA buffer and immediately processed for PCR amplification. A mock control in which no tissue was added and processed in parallel with each sample was included.
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PCR-SSCP screening of BRAF mutations was performed by amplifying exon 15 according to a standard procedure (20). DNA was used as a template in a 20-µl PCR mixture containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3), 0.2 mM deoxynucleotide triphosphates, 8 pmol of amplimers, and 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). PCR primers for the BRAF exon 15 were as follows: forward, 5'-TCCTTTACTTACTACACCTCAGAT-3' and reverse, 5'-AGTGGAAAAATAGCCTCAAT-3'. The amplicon size was 167 bp. Cycling conditions were as follows: initial denaturation (94 C, 5 min) and then 35 cycles (denaturation, 94 C for 40 sec; annealing, 55 C for 40 sec; synthesis, 72 C for 40 sec), followed by a final extension of 5 min at 72 C. All PCR products were visualized by electrophoresis on a 2% agarose gel and purified using a PCR purification kit (QIAGEN, Crawley, West Sussex, UK). Purified products were then diluted 1:1 with denaturing solution (1% xylene cyanol, 1% bromophenol blue, 0.1 mM EDTA, and 99% formamide), boiled for 5 min, and immediately placed on ice to prevent annealing of single-strand products. SSCP screening was carried out on a GenePhor electrophoresis unit using GeneGel Excel 12.5/24 (12.5% T, 2% C), according to manufacturer’s instructions (GE Healthcare, Milano, Italy). Electrophoresis (600 V, 25 mA, 15 W) was performed at 18 C for 100 min. Gels were stained with PlusOne silver staining kit (GE Healthcare, Milano, Italy), according to manufacturer’s instructions. Altered migration patterns in two or three independent PCR-SSCP runs were indicative of DNA mutations. Purified PCR products were then sequenced by an ALF II automated sequencer (GE Healthcare) using the Thermo Sequenase Cy5 dye terminator cycle sequencing kit (GE Healthcare). DNA sequences were compared with those of the normal BRAF gene exon 15 by using the Basic Alignment Search Tool (BLAST) software available at the National Center for Biotechnology Information. As a control, two human thyroid cancer cell lines, ARO and TPC, heterozygous and negative for the BRAF mutation, respectively, were used.
Statistical analysis
Data were analyzed using both univariate and multivariate tests (STATISTICA software; StatSoft, Tulsa, OK). P < 0.05 denoted the presence of a significant difference.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In this study, BRAF alteration was found in 219 of 500 cases (43.8%). In particular, we found the most common BRAF V600E mutation in 214 cases (42.8%), BRAF K601E mutation in three cases, BRAF VK600–1E (TGA deletion causing replacement of a valine and a lysine by a glutamate in the BRAF activation segment) (21) in one case, whereas in another case we found a new 14-bp deletion (1798–1811) with concomitant 2-bp insertion (1798–1799), VKSR600–3del and T599I, respectively (Table 2).
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, a high frequency (80.0%) of BRAF V600E has been found in the TCVs and CVs of PTC (68.3%). As expected, a significant low frequency (18.8%) of BRAF V600E mutations has been observed in FVs. In the Micro PTCs and the group called "others," the percentage of the same mutations was 39.4 and 45.5, respectively. Two Micro PTCs, with FV pattern, measuring 0.8 and 0.7 cm, respectively, and one case of FV PTC of 1.9 cm showed a K601E mutation. Interestingly, BRAF VK600–1E was found in a trabecular variant of PTC, whereas the new alteration VKSR600–3del and T599I was identified in one case of FV PTC showing angiolymphatic invasion.
Association of BRAF V600E mutations with clinicopathological parameters
In Table 3, BRAF V600E mutation has been associated with clinicopathological parameters such as age, gender, extrathyroidal invasion, multicentricity, presence of tumor capsule, nodal metastases, and tumor class. By univariate analysis, no significant statistical correlation has been reported between BRAF V600E and age and gender. On the other hand, a significant statistical association has been found with extrathyroidal invasion (P < 0.0001), multicentricity (P = 0.0026), presence of nodal metastases (P = 0.0009), and class III vs. classes I and II (P = 0.00000006) and absence of tumor capsule (P < 0.0001). However, by multivariate analysis (Table 4), only the presence of the tumor capsule was highly statistically associated with low frequency of BRAF V600E mutation. Similar results were also obtained when tumor 1 cm or less (Micro PTCs) were removed from the total number of cases. In detail, the absence of the capsule in the CV PTC is not related to BRAF V600E mutations (P = 0.46); in Micro PTC, FV PTC, and others, the absence of tumor capsule and BRAF V600E is significantly associated (P = 0.0004, P = 0.02, and P = 0.04, respectively). In fact, it is interesting to observe that only 11 of 56 encapsulated Micro PTCs and only five of 51 encapsulated FV PTCs showed the mutation (Table 5).
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To evaluate the significance of the presence of the tumor capsule as a prognostic indicator, we evaluated the association between encapsulated forms and clinicopathological parameters in the group of FV PTCs and Micro PTCs. As shown in Table 6, in FV PTCs larger size seems to be associated (P = 0.04) with the presence of capsule, whereas only one encapsulated FV PTC of 52 developed node metastases, compared with those without capsule, which show metastatic disease in eight of 62 cases (P = 0.03). In Micro PTCs, only the size of the tumor is associated with the absence of the capsule (P = 0.009).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Large tumor size, old age, extrathyroidal invasion, male gender, multicentricity, distant metastasis, and probably lymph node metastasis are the main determinants of a poor outcome in PTC patients (22, 24, 25).
Although some of these classical features have been consistently and strongly associated with poor prognoses, the availability of novel informative genetic markers such as RET/PTC rearrangements, the RAS gene family, and more recently the BRAF mutation have the potential to improve risk stratification, recurrence prediction, and death risk in PTC patients.
Mutations in the BRAF gene were the most recently identified MAPK effector in thyroid cancer. BRAF protooncogene encodes a serine/threonine kinase that transduces regulatory signals through the Ras-Raf-MEK-ERK cascade and is implicated in several human cancers, including melanoma and colon carcinoma (26). BRAF V600E is the most common alteration in sporadic papillary carcinoma (5), whereas FV of papillary carcinomas show the BRAF K601E mutation, and the in-frame VK600–1E deletion (BRAF VK600–1E) has been detected in a solid variant papillary carcinoma, indicating possible phenotype-genotype correlations (5, 13, 21).
BRAF mutations in PTC correlate with distant metastasis and more advanced clinical stage (15) and occur at a significantly higher frequency in older patients, whereas a gender difference is still controversial (13, 16, 27, 28). There is a high prevalence of BRAF mutations in the aggressive TCV PTC (55–100%), whereas a relatively low prevalence has been reported in FV PTC (7–14%) (5, 13, 16, 20).
The above results are also confirmed by our study, in which the V600E mutation has been revealed in 80% of the TCV PTCs analyzed, whereas the percentage of the same genetic alteration was found in 68.3 and 39.4% of CV PTCs and tumors less than 1 cm, respectively; in FV PTC the frequency was 18.8%.
We did not find any association with age and gender, but a strong statistical association has been found in PTCs with extrathyroidal invasion, multicentricity, presence of node metastases, and higher tumor class, confirming that BRAF V600E is linked to the more aggressive phenotype.
However, the strongest association has been found between the V600E mutation and absence of tumor capsule. In fact, only 30 of 135 (22.2%) encapsulated tumor had a BRAF alteration in contrast to 184 of 360 (51.1%) of the nonencapsulated form.
Presence of the tumor capsule is a strong predictor of excellent prognosis in particular in the FV PTC. Kakudo et al. (29) found that tumors exhibiting invasive growth with no tumor capsule had a higher risk of recurrence, compared with tumor with expansive growth with the presence of capsule.
It is well known that FV PTC is distinct as two types (30), including the diffuse FV and the encapsulated form. In the diffuse FV, usually the gland is diffusely replaced by tumors and lymph node metastases are common. The prognosis appears to be poor in these patients, although only a few cases have been described (31). On the contrary, the encapsulated FV, characterized by the presence of a capsule around the tumors, is associated with rare node metastases and with excellent prognosis. Some authors have suggested that this tumor may be classified as tumors of undetermined malignant potential due to their excellent prognosis (32); however, others have shown that some cases belonging to this category can lead to distant metastasis (33).
As already mentioned above, we found a low percentage of V600E in completely encapsulated forms of PTC. However, the statistical association between the genetic alteration and the presence of the capsule was observed only in Micro PTCs (tumor less than 1 cm) (P = 0.0005) and FV PTCs (P = 0.02). The possible biological explanation that correlates BRAF mutation and infiltrative tumor growth may be due to metalloproteinase induction by BRAF, particularly matrix metalloproteinase-3, -9, and -13 (34).
By contrast, in CV PTCs no difference regarding BRAF mutation has been found between tumor with or without tumor capsule.
In addition, it is interesting to observe, as indicated by our data, that in FV PTCs and Micro PTCs, the presence of tumor capsule was associated with a very low frequency of node metastasis (only one of 52 encapsulated FV PTCs and none of 57 encapsulated Micro PTCs had node metastases). Taken together, these data indicate that the low frequency of BRAF V600E mutation and the presence of the tumor capsule are both indicators of low-risk tumors.
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Footnotes |
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Disclosure Statement: The authors have declared no conflict of interest.
First Published Online September 4, 2007
Abbreviations: CV, Classical variant; FV, follicular variant; Micro, microcarcinoma; PTC, papillary thyroid carcinoma; SSCP, single-strand conformation polymorphism; TCV, tall cell variant.
Received May 29, 2007.
Accepted August 24, 2007.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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