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Clinicopathologic features and BRAFV600E mutation analysis in cutaneous metastases from well-differentiated thyroid carcinomas
Article first published online: 26 MAR 2007
Copyright © 2007 American Cancer Society
Volume 109, Issue 10, pages 1965–1971, 15 May 2007
How to Cite
Erickson, L. A., Jin, L., Nakamura, N., Bridges, A. G., Markovic, S. N. and Lloyd, R. V. (2007), Clinicopathologic features and BRAFV600E mutation analysis in cutaneous metastases from well-differentiated thyroid carcinomas. Cancer, 109: 1965–1971. doi: 10.1002/cncr.22641
- Issue published online: 25 APR 2007
- Article first published online: 26 MAR 2007
- Manuscript Accepted: 6 FEB 2007
- Manuscript Revised: 28 JAN 2007
- Manuscript Received: 15 DEC 2006
- papillary carcinoma;
- follicular carcinoma;
- cutaneous metastases
Cutaneous metastases from well-differentiated thyroid carcinomas are rare and are usually identified in patients with widely disseminated disease. Occasionally, thyroid carcinomas can present as cutaneous metastases for which the primary site needs to be determined. Papillary thyroid carcinomas (PTCs) commonly have BRAFV600E mutation. A series of 16 cutaneous metastases were analyzed from well-differentiated thyroid carcinomas to learn more about the clinicopathologic features and BRAFV600E mutation status.
Eleven cases of PTC and 5 of follicular thyroid carcinoma (FTC) metastatic to the skin were evaluated. All cutaneous metastases were studied histologically and with thyroglobulin and thyroid transcription factor immunostains. All tumor samples were analyzed for mutations at nucleotide 1799 in exon 15 of the BRAF gene.
Two patients with FTC presented with cutaneous metastases. Fourteen of 16 patients died of disease and 2 were alive with disease at follow-up. The histologic features of the cutaneous metastases were generally characteristic of the primary tumor; however, 2 of the 11 PTC metastases demonstrated cytoplasmic clearing not typical of classic PTC. BRAFV600E mutation (T1799A) was detected in 5 of 11 cases of PTC and in none of the 5 FTCs.
Cutaneous metastases from PTC may show prominent clear cell change requiring differentiation from clear cell hidradenoma, clear cell dermatofibroma, malignant melanoma with prominent clear cell change, and cutaneous metastasis from renal cell carcinoma. BRAFV600E mutation is identified in a subset of cutaneous metastases from PTC. Cutaneous metastases from PTC and FTC are associated with a very poor prognosis. Cancer 2007. © 2007 American Cancer Society.
Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy and is usually associated with an excellent prognosis even with regional lymph node metastases.1 However, a small number of patients with PTC die of their disease. Follicular thyroid carcinoma (FTC) is the second most common type of thyroid carcinoma. FTCs account for approximately 10% to 15% of thyroid malignancies.2 Although FTCs are less frequently associated with lymph node metastases than PTC, patients with FTC may have distant metastases at presentation in up to 20% of cases.2
Cutaneous metastases from well-differentiated thyroid carcinoma are rare, but there are a number of case reports and small series in the literature.3–17 In a study of 91 patients with PTC with distant metastases, only 6 had cutaneous metastases.18 Although FTCs are more commonly associated with distant metastases at diagnosis than patients with PTC,2 cutaneous metastases are rare. Cutaneous metastases are generally identified in patients with widely disseminated disease, but cutaneous metastases may be the initial manifestation of thyroid carcinoma.3–5, 10
BRAF belongs to the Raf-1 family of serine/threonine kinases, which also includes A-RAF and C-RAF. BRAF is involved in the RAS/RAF/MEK/ERK signal transduction pathway that is involved in mediating cell growth.19–21 RAS stimulates the activation of RAF, which phosphorylates and activates MEK, which then phosphorylates and activates ERK. The most common mutation is a thymine-to-adenine transversion in nucleotide 1799 (originally listed as 1796) that leads to a substitution of valine by glutamic acid at amino acid residue 600 (V600E) (originally designated V599E).19 PTCs and some poorly differentiated and anaplastic thyroid carcinomas have been found to have activating mutations in BRAFV600E nucleotide 1799.22–29 Although FTCs often show rearrangements of the PPAR gamma gene,30 they are not known to have BRAFV600E mutations.
We analyzed a series of 16 cutaneous metastases from well-differentiated thyroid carcinomas to learn more about the clinicopathologic features and BRAFV600E mutation status.
MATERIALS AND METHODS
Patients, Tissue Samples, and Cell Lines
Formalin-fixed paraffin-embedded tissues from 16 patients with cutaneous metastases of well-differentiated thyroid carcinoma, including 11 cases of PTC and 5 cases of FTC, were identified from the Tissue Registry of the Mayo Clinic from 1967–2005. Cutaneous metastases from Hurthle cell carcinoma, medullary thyroid carcinoma, poorly differentiated carcinoma, and anaplastic carcinoma were excluded. Tumors showing direct extension to involve the overlying skin in the neck or occurring in the surgical scar were also excluded. Clinical and follow-up information was obtained by chart review. Mayo Clinic Institutional Review Board (IRB) approval was obtained. The histologic features were evaluated with hematoxylin and eosin-stained sections in all cases. Two PTC cell lines were used for controls, including NPA cells known with BRAFV600E mutation and PTC1 cells with the known wildtype BRAF genotype.
Formalin-fixed, paraffin-embedded tissue sections were cut at 5 μm and treated with 0.1 mol/L citrate, pH 6.0, in an 800-W microwave oven for 15 minutes for antigen retrieval before immunostaining. The sections were then incubated with the primary antibodies overnight at room temperature. Antibodies to thyroid transcription factor (TTF; Dako, Carpinteria, Calif) were used at a 1:1000 dilution. Immunostaining was done with the elite avidin-biotin-peroxidase kit (Vector Laboratories, Burlingame, Calif) according to the manufacturer's specifications. Slides were counterstained with hematoxylin for 1 second. All cases were also immunostained for thyroglobulin (Dako) at a 1:800 dilution. Positive controls for TTF and thyroglobulin were utilized and normal mouse serum was substituted for the primary antibodies as a negative control.
DNA extraction was done as previously reported.25 Briefly, 4–6 10-μm thick paraffin sections were placed into a 1.5-mL nuclease-free tube. To avoid cross-contamination between samples, disposable microtome blades that were cleaned by xylene and 100% ethanol after each sample were used. The sections were deparaffinized in xylene for 10 minutes × 2, followed by 100% ethanol for 5 minutes × 2. Microcentrifugation was performed between each step to remove the solution. The tissue pellets were air-dried and resuspended in 480 μL of lysis buffer (20 mM Tris-HCL, pH 7.5; 20 mM EDTA; 1% sodium dodecyl sulfate) with 40 μL of 25 μg/μL proteinase K (Roche Diagnostics, Indianapolis, Ind), and incubated in a water bath at 55°C overnight. Genomic DNA was extracted and mixed gently. After microcentrifugation at 10,000g for 2 minutes, the aqueous phase (≈0.5 mL) was transferred to a new tube and mixed with 0.5 mL chloroform and 0.5 mL of isopropyl alcohol (Sigma, St. Louis, Mo). The samples were then centrifuged and the aqueous phase was transferred to a new tube. Total DNA was precipitated by adding 1.0 mL of isopropyl alcohol along with 100 μL of 3 M sodium acetate and 5 μL of 20 mg/mL glycogen (Roche) at −20°C for 2 hours, followed by centrifugation at 12,000g at 4°C for 20 minutes. The DNA pellets were washed in 70% ethanol and centrifuged for another 20 minutes. After removal of ethanol the pellets were air-dried, and reconstituted with 50–100 μL of DEPC-treated water. The concentration of DNA was measured by optical density with a spectrophotometer.
Polymerase Chain Reaction (PCR)
All tumor samples and controls were analyzed for the thymine (T) to adenine (A) missense mutation at nucleotide 1799 in exon 15 of the BRAF gene. This hotspot was chosen because the reported BRAFV600E-activating mutation in thyroid carcinoma occurs predominantly at this position. PCR primers to amplify a 224 basepair (bp) fragment of BRAF gene exon 15 (forward: 5′-TCATAATGCTTGCTCTGATAGGA; reverse: 5′-GGCCAAAAATTTAATCAGTGGA) as described.25 PCR was performed in 50 μL of reaction mixture containing 500 ng of genomic DNA, 0.3 μM of each primer, 0.2 mM of each deoxynucleotide, 2.5 mM MgCL2, 1× reaction buffer, and 1.25 U Platinum Taq DNA Polymerase (Invitrogen, La Jolla, Calif). The PCR reactions were carried out in a 96-well thermocycler (Gene Amp PCR system 9700, PE Applied Biosystems, Foster City, Calif). Cycling included a denaturation step at 94°C for 5 minutes, followed by 40 cycles of 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute, and a final extension at 72°C for 10 minutes.
BRAF Mutation Analysis
PCR-amplified fragments were separated on a 1% agarose gel and visualized by ethidium bromide staining. The amplified bands with expected molecular size were extracted with an ultra-free DA column and purified with a MicroconYM-100 column (Millipore, Bedford, Mass). Both strands of the PCR products were sequenced using the same PCR primers described above. The DNA direct sequencing was performed using an ABI Prism 377 DNA Sequencer (PE Applied Biosystems).
Clinical and follow-up information is presented in Table 1. Fourteen of the 16 patients died of disease and 2 were alive with disease at last follow-up. Fourteen patients developed cutaneous metastases after being diagnosed with thyroid carcinoma. The cutaneous metastases generally presented as nodules and radioactive iodine uptake was noted in lesions evaluated. The time period from diagnosis of primary thyroid carcinoma to the cutaneous metastases ranged from 0 to 14 years with a mean of 6 years. Two patients with FTC presented with cutaneous metastases, 1 with metastases to the skin of the neck and sternum and the other with a metastasis to the finger.
|Case||Diagnosis||Age at primary||Age at metastasis||Sex||Cutaneous metastases||Other metastases||Survival, y||BRAF status|
|1||Papillary carcinoma||61||70||Male||Neck||Lymph nodes, soft tissue||Alive with disease (9)||Wildtype|
|2||Papillary carcinoma||39||48||Female||Scalp*||Humerus, lymph nodes, lung, brain, pleura, ovary, pelvis||Died of disease (9)||Wildtype|
|3||Papillary carcinoma||53||60||Female||Supra-clavicular||Lymph nodes, subcutaneous||Died of disease (20)||Wildtype|
|4||Papillary carcinoma||50||57||Male||Neck||Lymph nodes, brain, lung, esophagus||Died of disease (17)||Wildtype|
|5||Papillary carcinoma||54||63||Male||Supra-sternal||Lymph nodes, lung||Died of disease (10)||Mutation†|
|6||Papillary carcinoma||72||74||Male||Neck||Lung||Died of disease (3)||Mutation†|
|7||Papillary carcinoma||77||79||Male||Neck||Lymph nodes, soft tissue||Died of disease (3)||Mutation†|
|8||Papillary carcinoma||56||67||Female||Scalp||Lung, vertebrae, brain||Died of disease (12)||Wildtype|
|9||Papillary carcinoma||56||63||Male||Scalp||Pleura, sternum, spine||Died of disease (7)||Mutation†|
|10||Papillary carcinoma||46||47||Male||Scalp, nose, temple*||Lymph nodes, femur, lung||Died of disease (3)||Wildtype|
|11||Papillary carcinoma||55||57||Male||Neck||Lymph nodes, sternum||Alive with disease (3)||Mutation†|
|12||Follicular carcinoma||64||75||Male||Neck||Lymph nodes||Died of disease (14)||Wildtype|
|13||Follicular carcinoma||68||68||Female||Chest (sternum)‡||Lung, skull||Died of disease (<0.5)||Wildtype|
|14||Follicular carcinoma||73||73||Female||Finger,‡ toe||Lung, spine||Died of disease (2)||Wildtype|
|15||Follicular carcinoma||63||77||Female||Eyebrow, toe||Lymph nodes, lung||Died of disease (16)||Wildtype|
|16||Follicular carcinoma||53||59||Male||Chest||Lymph nodes, chest wall||Died of disease (8)||Wildtype|
The histologic features in the cutaneous metastases were generally characteristic of the primary tumor; however, 2 of the 11 PTC metastases demonstrated cytoplasmic clearing not typical of classic PTC (Fig. 1). Thyroglobulin and TTF stains were positive in all cases. The primary thyroid carcinomas showed histologic features characteristic of PTC and FTC, respectively. The primary PTC ranged in size from 2.2 cm to 7.5 cm with a mean of 4.3 cm. Six of the 11 primary PTC showed extrathyroidal extension, 2 others were multifocal and bilateral, and 1 other was present at the margin. All of the cutaneous metastases were well-differentiated. One of the 11 primary PTCs was associated with foci of anaplastic carcinoma at the time of the primary diagnosis, and 1 case had cribriform and morular features. The patient with PTC with cribriform and morular features did not have a known history of familial polyposis. However, the cutaneous metastases were well-differentiated. The sizes of the primary FTC ranged from 2.5 cm (the largest nodule in a patient with bilateral tumors and extrathyroidal extension) to 11.5 cm with a mean of 7.3 cm. The sizes of 2 primary FTCs were not available. In 1 of the patients who presented with a cutaneous metastasis of FTC and was found to have disseminated disease the primary thyroid tumor was only biopsied.
Genomic DNA was extracted from all paraffin blocks and amplified by PCR with a 224-bp fragment of BRAF. BRAFV600E mutation (T1799A) was detected in 5 of 11 cases of PTC, but in none of the 5 FTCs (Table 1, Fig. 2). The PTC with cribriform and morular features and the PTC with foci of anaplastic carcinoma showed BRAFV600E mutation.
Papillary thyroid carcinoma is the most common thyroid carcinoma and is usually associated with an excellent prognosis.1 However, of the 11 patients with PTC metastases to the skin, 9 died of disease and 2 are alive with disease at last follow-up. All 5 patients with metastatic FTC to the skin also died of disease. Although patients with PTC are generally thought to have an excellent prognosis, patients whose tumors have metastasized to the skin have an extremely poor prognosis.3 In a small series and review of the literature of cutaneous metastases of thyroid carcinoma, 11 of 12 patients with cutaneous metastases from PTC or FTC died or were alive with disease.3 Only 1 patient was alive with no evidence of disease at follow-up of 1.5 years.3, 9 In well-differentiated thyroid carcinoma, cutaneous metastases indicate very poor prognosis.
Cutaneous metastases from well-differentiated thyroid carcinomas are rare, but the primary tumors showed features of increased risk for aggressive behavior. Six of the 11 primary PTC showed extrathyroidal extension, 2 others were multifocal and bilateral, and 1 was present at the margin and was incompletely excised. Additionally, 1 PTC was associated with foci of anaplastic carcinoma in the primary at diagnosis. One PTC had cribriform and morular features. The patients with FTC also had features suggesting increased risk for aggressive behavior, including large tumor size with a mean of 7.3 cm. The smallest FTC measured was 2.5 cm, but this reflected the size of the largest nodule in a patient with bilateral tumors and extrathyroidal extension. The average age at diagnosis of the primary PTC was 56.3 years and the primary FTC was 70.4 years. Overall, 10 of the 16 patients with cutaneous metastases were male, including 8 of the 11 patients with cutaneous metastases from PTC. Whereas cutaneous metastases of well-differentiated thyroid tumors are rare, clinical and pathologic features of increased risk were noted in these cases. Previous reports of cutaneous metastases have also described features of increased risk including areas of dedifferentiation within the primary tumor and extrathyroidal extension.3, 4
Cutaneous metastases from visceral tumors often present in the region of the skin near the primary tumor, and cutaneous metastases from PTC and FTC most commonly occur in the head and neck, with the scalp as a particularly common site.3, 8 The scalp was involved in 4 of the 16 cases in our study. However, metastases of thyroid carcinoma can involve a variety of diverse sites including the skin of the neck, scalp, supraclavicular region, suprasternal region, chest, nose, temple, eyebrow, fingers, and toes. Cutaneous metastases from thyroid carcinoma generally occur in patients with disseminated disease, but cutaneous metastases can be the presenting manifestation of thyroid carcinoma, as was seen in 2 of the 16 patients in this study. Thus, a high index of suspicion is needed in diagnosing these tumors, particularly at unusual sites and in cases in which the metastasis is the presenting manifestation of the disease.
Interestingly, the 2 patients whose well-differentiated thyroid carcinoma presented in the skin both had FTC. Whereas PTCs are more often associated with lymph node metastases at time of diagnosis, FTCs are more commonly associated with distant metastases at diagnosis than patients with PTC.2 All patients with cutaneous metastases from PTC were previously diagnosed with disease in the thyroid. Unfortunately, pathologists are not always provided with complete clinical histories when presented with unusual metastases. Additionally, cutaneous metastases can be the first evidence of recurrence of thyroid carcinoma.4 Cutaneous metastases from well-differentiated thyroid carcinomas are uncommon, but they must be considered when encountering an unusual cutaneous tumor.
Although the metastases generally show histologic features characteristic of the primary tumor, 2 of the 11 cases of cutaneous PTC metastases demonstrated cytoplasmic clearing uncommon in most PTC. Cytoplasmic clearing in well-differentiated thyroid carcinoma has been reported.8, 31 Pathologists need to be aware that cutaneous PTC metastases may also show this clear cell change and consider this entity in the differential diagnosis. These tumors need to be differentiated from clear cell hidradenoma, clear cell dermatofibroma, melanoma with clear cell change, and metastatic renal cell carcinoma. Clear cell hidradenomas can mimic cutaneous metastases of visceral tumors.32 The identification of ductular lumens and apocrine and squamoid change is helpful in recognizing these tumors.32 Clear cell dermatofibroma is also in the differential diagnosis of cutaneous metastases of clear cell tumors.33 Clear cell dermatofibroma is negative for epithelial markers, a feature that helps differentiate it from metastatic thyroid carcinoma and renal cell carcinoma. Malignant melanoma with prominent clear or balloon cell change usually has areas of conventional cutaneous melanoma and involvement of the overlying epidermis, which is helpful in recognizing the tumor. Also, this variant is more common on the extremities as compared with metastatic thyroid carcinoma and renal cell carcinoma, which are most common on the head and neck. Melanomas can also show BRAF mutation,19 but melanomas are positive for S100, Melan A, and HMB45 and negative for keratin. Cutaneous metastases of renal cell carcinoma are well known to occur, often on the head and neck, and tumors may present as a cutaneous metastasis.34 Renal cell carcinoma coexpresses keratin and vimentin, whereas well-differentiated thyroid carcinoma is positive for keratin, TTF, and thyroglobulin and negative for vimentin. This immunophenotype is very helpful in the differential diagnosis.
BRAFV600E mutation was detected in 5 of the 11 (45%) cutaneous metastases from PTC. BRAFV600E mutation is the most common genetic abnormality in papillary thyroid carcinoma.22–29BRAFV600E mutation has been associated with older patient age and extrathyroidal extension.26 All 5 patients with BRAFV600E mutation happened to be male, although the majority (8 of 11) patients with cutaneous PTC metastases were male. This mutation has been identified both with papillary thyroid carcinomas and in adjacent poorly differentiated and anaplastic carcinomas.26, 35 None of the 5 cutaneous FTC metastases showed BRAFV600E mutation. Mutations in BRAFV600E are not identified in FTC, but BRAF copy number gains can be seen, particularly in widely invasive tumors and in those presenting with distant metastases.36
Although the frequent identification of BRAFV600E mutation in patients with cutaneous metastases of PTC correlates with studies suggesting BRAFV600E mutation may be associated with more aggressive disease,37BRAFV600E mutations have been identified in PTC in patients who are cured and even in papillary thyroid microcarcinomas.27, 28 Thus, BRAFV600E mutation may be an early event in papillary thyroid tumorigenesis. Additional studies will be needed to identify other genes that contribute to the more aggressive behavior in the subset of PTC that behave aggressively and result in death.
In summary, cutaneous metastases from PTC are uncommon, but may show prominent clear cell change requiring differentiation from cutaneous clear cell tumors and from other metastases. BRAFV600E mutation is present in a subset of cutaneous metastases from PTC. Our study confirms that cutaneous metastases from PTC and FTC are associated with a very poor prognosis.
- 1Papillary carcinoma. In: DeLellisRA, LloydRV, HeitzPU, EngC, eds. World Health Organization Classification of Tumours Pathology & Genetics of Tumours of Endocrine Organs. Lyon, France: IARC Press; 2004: 57–66., , , et al.
- 2Follicular carcinoma. In: DeLellisRA,LloydRV, HeitzPU, EngC, eds. World Health Organization Classification of Tumours Pathology & Genetics of Tumours of Endocrine Organs. Lyon, France: IARC Press; 2004: 67–76., , , et al.
- 18Distant metastases in papillary thyroid cancer: a review of 91 patients. Cancer 1988; 177: 241–243., , , et al.
- 27The BRAFT1796A transversion is a prevalent mutational event in human thyroid microcarcinoma. Int J Oncol. 2004; 6: 1729–1735., , , et al.
- 30Genetic and biological subgroups of low-stage follicular thyroid cancer. Am J Pathol. 2003; 62: 1053–1060., , , et al.