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Papillary carcinoma is the most common type of thyroid cancer, representing approximately 80% of all malignant thyroid tumors.1 Primary thyroid lymphomas, on the other hand, are rare and account for 1ā€“5% of all thyroid malignancies, and approximately 2% of all malignant extranodal lymphomas.2, 3, 4 Initial diagnosis of the epithelial thyroid neoplasms, and subsequent therapeutic planning, is often based on fine-needle aspiration biopsy and its cytological evaluation. To increase the diagnostic accuracy of thyroid fine-needle aspiration material, molecular testing for a panel of mutations is being more commonly used.5 Specifically, molecular analyses have focused on a set of somatic alterations of genes in the mitogen-activated protein kinase (MAPK) pathway, which are frequently present in carcinomas of the thyroid. These include point mutations of the BRAF and RAS genes, and RET/PTC and PAX8/PPARĪ³ chromosomal rearrangements.6, 7, 8 V600E mutation in the BRAF gene has been identified as the most common genetic event in papillary thyroid carcinoma occurring in 40ā€“45% of cases.9, 10 This mutation is also found in poorly differentiated and anaplastic thyroid carcinomas that typically have a component of residual well-differentiated papillary carcinoma.11 RAS mutations, on the other hand, are seen more commonly in thyroid tumors with a follicular pattern, including follicular carcinoma, follicular adenoma, and follicular variant of papillary carcinoma.5

Until the very recent discovery of BRAF V600E mutations in virtually all cases of hairy cell leukemia,12, 13, 14, 15, 16 the presence of these mutations in non-Hodgkin lymphomas had received little attention. The recent studies of BRAF mutations in hairy cell leukemia have also included a very large number of B-cell lymphomas and have identified only two cases of other chronic lymphoproliferative disorder, not fulfilling the criteria for hairy cell leukemia with BRAF V600E mutations. However, the presence of V600E mutation in these two chronic lymphoproliferative disorders could not be confirmed on Sanger sequencing.12, 13, 14, 16, 17 In an older study, Lee et al18 reported BRAF mutations in 6% (4/67) of diffuse large B-cell lymphomas involving various non-thyroid sites. However, all four of these BRAF mutations involved codons other than codon 600, where the most common mutation occurs in papillary thyroid carcinoma. Borie et al19 studied immunodeficiency-related non-Hodgkin lymphomas with microsatellite instability and found V600E BRAF mutations in three of their nine cases, which included one diffuse large B-cell lymphoma, one T-cell post-transplant lymphoproliferative disorder, and one primary central nervous system B-cell non-Hodgkin lymphoma. V600E BRAF mutations have also been reported in more than half of Langerhans cell histiocytoses,20 rare T-cell acute lymphoblastic leukemias,21 and few cases of multiple myeloma.22 In addition, NRAS and KRAS mutations have frequently been reported in plasma cell myeloma and plasma cell leukemias,23, 24 B-cell acute lymphoblastic leukemias,21 and some cutaneous T-cell lymphomas.25 The frequency of these mutations in lymphomas of the thyroid, where they might cause the greatest diagnostic confusion, is unknown.

We recently encountered a thyroid mass sampled by fine-needle aspiration, where a cytological diagnosis of malignant tumor was rendered with a differential diagnosis that included both carcinoma and lymphoma. Molecular analysis was positive for a BRAF V600E (c. 1799T>A) mutation, which was thought to support the diagnosis of thyroid carcinoma. The tumor was excised and found to represent a diffuse large B-cell lymphoma with no evidence of a papillary carcinoma in the thyroid gland. This case prompted us to perform a systematic study of BRAF and other common mutations activating the MAPK pathway (NRAS, KRAS, and HRAS) in 33 lymphomas of the thyroid. Eleven cases were also investigated for PAX8/PPARĪ³ rearrangement.

Materials and methods

Case Selection and Review

A total of 33 B-cell lymphomas presenting in the thyroid were obtained from the Department of Pathology at the University of Pittsburgh. The study was approved by the University of Pittsburgh Institutional Review Board. The following clinical features were recorded after review of the de-identified information from electronic medical records: gender, age, clinical presentation, treatment, and follow-up.

The routine histological sections and cytological preparations, together with all available immunohistochemical stains, flow cytometric data, and cytogenetic findings were reviewed (NA, SHS). When not already available, at least the following immunohistochemical stains for the following antigens were performed: CD20, CD3, kappa, lambda, CD10, BCL6, IRF4/MUM1, Ki-67, Cyclin D1, and PAX8 (Table 1). Lymphomas were classified using the WHO criteria,26 and diffuse large B-cell lymphoma further characterized using the Hansā€™ algorithm into germinal center (CD10+ or BCL6+ and IRF4/MUM1āˆ’) and non-germinal center (CD10āˆ’, BCL6āˆ’ or BCL6+, IRF4/MUM1+) types.27 Results of all available classical cytogenetic or fluorescence in-situ hybridization studies were also reviewed.

Table 1 Clones of antibodies used for the immunohistochemistry
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Microdissection and Nucleic Acid Isolation

Histological sections with the most dense lymphoma infiltration were identified, and 5-Ī¼m sections cut for BRAF and RAS mutation analysis. The targeted areas were manually microdissected to collect tumor tissue for DNA isolation. In one case, two different areas representing extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue type and diffuse large B-cell lymphoma were microdissected separately. Genomic DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. Total nucleic acid was isolated from snap-frozen tissue in 11 available cases (8 diffuse large B-cell lymphoma, germinal center type; 2 diffuse large B-cell lymphoma, non-germinal center type, and 1 extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue type) using the MagNA Lyser and MagNA Pure (Roche) for PAX8/PPARĪ³ rearrangement studies.

Detection of Point Mutations

First, mutational analysis was performed for hotspots typically found in thyroid cancer, ie, BRAF codons 600 and 601, KRAS codons 12 and 13, HRAS codon 61, and NRAS codon 61, using real-time LightCycler PCR followed by fluorescence melting-curve analysis as previously described.6 Briefly, for each gene, a pair of oligonucleotide primers flanking the mutation site was designed together with two fluorescent probes with the sensor probe spanning the codon of interest (TIB Molbiol, Berlin, Germany). Amplification was performed for 40 cycles. Post-amplification fluorescence melting-curve analysis was performed by gradual heating of samples at a rate of 0.1ā€‰C/sec from 45 to 95ā€‰C. For each mutation hotspot, DNA from a tumor or cell line known to carry a specific mutation was used as a positive control, and DNA from peripheral blood lymphocytes was used as a wild-type negative control. The sensitivity of mutation detection by the melting-curve analysis was 10% of cells with a mutant allele in the background of normal cells, as established by serial dilutions of the positive controls. Those samples that revealed no mutations in these hotspots were further analyzed for mutations in the entire exon 15 of the BRAF gene, exon 2 of NRAS and HRAS, and exon 1 of KRAS, using Sanger sequencing. Specifically, PCR amplification was performed using 25ā€‰ng of DNA and AmpliTaq Gold PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems).

Detection of PAX8/PPARĪ³ Rearrangement by RT-PCR

In those samples in which frozen tissue was available, PAX8/PPARĪ³ rearrangement was detected by RT-PCR as previously described.6 Briefly, reverse transcription and PCR amplification were performed in one step using QuantiTech Probe RT-PCR Kit (Qiagen), and gene-specific primers and probes. The reverse transcription was carried out at 50ā€‰C for 30ā€‰min, followed by 40-cycle PCR amplification. RNA from a tumor known to carry the PAX8/PPARĪ³ rearrangement was used as a positive control. The sensitivity of mutation detection by RT-PCR was 1% of cells carrying the rearrangement in the background of normal cells, as established by serial dilutions of the positive control.

Cytogenetic Studies

Classical G-banded cytogenetic karyotypes were obtained using previously described methods.28 Fluorescence in-situ hybridization was performed on interphase nuclei according to the manufacturer's protocol, using IGH/MYC dual-color translocation probe, IGH break-apart probe, and MYC break-apart probe (Vysis DNA probes, Abbott Molecular, Des Plains, IL, USA). Two hundred nuclei were analyzed for each of the probes. The cut-off value used for false-positive signals for the translocation probe was 1%, and for the break-apart probe was 3%.29

Results

The patients included 28 females and 5 males with a mean age of 65 years (range of 30ā€“94 years). All patients presented with a neck mass and a clinical diagnosis of a thyroid tumor. Biopsies/excisions showed 25 diffuse large B-cell lymphoma with 17 of germinal center type, 6 of non-germinal center type, and 2 unclassifiable (Hansā€™ algorithm), 6 extranodal marginal-zone lymphomas of marginal-zone lymphoid tissue, including 3 with extensive plasmacytic differentiation, and 2 follicular lymphomas (one grade 1ā€“2 of 3 and the other grade 3A of 3). None of the cases revealed an associated thyroid carcinoma based on histopathological and immunohistological studies. However, in addition to staining of thyroid epithelium, more than 80% neoplastic cells were positive for PAX8 in 26/33 lymphomas, consistent with a recent study reporting that this antibody cross-reacts with PAX5.30 Most of the plasmacytic areas of the tumors were PAX8-negative.

The index patient had a thyroid mass that was rapidly enlarging and was clinically suspected to be a carcinoma or lymphoma. Fine-needle aspiration biopsy of the lesion showed numerous large neoplastic cells with scant to moderate amount of cytoplasm, round to somewhat irregular nuclear contours, and conspicuous nucleoli in a background of small lymphocytes (Figure 1a). A part of the specimen was sent for flow cytometric immunophenotypic evaluation and another for molecular testing. The flow cytometric studies revealed polyclonal small B lymphocytes and admixed T cells; however, they did not characterize the large atypical cells. Molecular analysis revealed a BRAF V600E (c. 1799T>A) mutation (Figure 1b). The case was diagnosed as a malignant neoplasm favoring an anaplastic thyroid carcinoma. The thyroidectomy was performed and pathological examination revealed a CD20-positive diffuse large B-cell lymphoma of non-germinal center type (Figure 1cā€“e). The thyroid gland was entirely sectioned and submitted for microscopic examination, which revealed no papillary or anaplastic carcinoma. The mutation analysis was repeated on cells isolated from four different areas of the lymphoma, all of which confirmed the presence of a BRAF V600E mutation. The adjacent normal thyroid tissue revealed wild-type BRAF. The patient was subsequently treated with CHOP-R chemoimmunotherapy (cyclophosphamide, vincristine, adriamycin, and prednisone with rituximab) and radiation following his surgery. The patient is alive and had no evidence of disease on PET-CT scan 2 years post-diagnosis.

Figure 1
figure 1

Diffuse large B-cell lymphoma, index case. (a) The fine-needle aspiration biopsy demonstrates numerous large cells with scant cytoplasm, round to somewhat irregular nuclei and admixed small lymphocytes. (Papanicolaou stain, original magnification Ɨ 1000) (b) Sequencing of exon 15 of the BRAF gene revealed a 1799T>A, V600E mutation. (c) The excision demonstrated a diffuse proliferation of large transformed cells (hematoxylin and eosin, original magnification Ɨ 1000). (d) The neoplastic cells were CD20-positive (original magnification, Ɨ 1000) (e) with a relatively high Ki-67 proliferative index (original magnification, Ɨ 200).

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Molecular analysis of the 33 total thyroid lymphomas revealed 8 (24%) cases positive for one of the studied mutations. All mutations were identified in diffuse large B-cell lymphoma (8/25, 32%; Table 2). There were six BRAF mutations, including the BRAF V600E (c.1799T>A) mutation found in the diffuse large B-cell lymphoma, non-germinal center index case, three D594G (c.1781A>G) mutations (Figure 2a), and two K601N (Figure 2b) mutations, all in diffuse large B-cell lymphoma, germinal center type. Two of the non-germinal center type diffuse large B-cell lymphoma had NRAS mutations, both at codon 61, Q61K, and Q61H (Figure 3). There was no statistically significant difference between the frequencies of mutation in germinal center vs non-germinal center diffuse large B-cell lymphoma. One case of diffuse large B-cell lymphoma also showed a low-grade area of extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue type, which was separately analyzed for the mutations. Both areas (diffuse large B-cell lymphoma and extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue type) were negative for mutations. None of the cases showed mutations of KRAS codons 12/13 or HRAS codon 61(0/33) or PAX8/PPARĪ³ rearrangement (0/11).

Table 2 Summary of mutations identified in diffuse large B-cell lymphoma of the thyroid
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Figure 2
figure 2

Sequencing analysis of BRAF exon 15. (a) Diffuse large B-cell lymphoma of germinal center type with D594G (c.1781A>G) mutation. (b) Diffuse large B-cell lymphoma of germinal center type with K601N (c.1803 A>C).

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Figure 3
figure 3

Sequencing of NRAS exon 2. (a) Diffuse large B-cell lymphoma of germinal center type with Q61K (c.181C>A) mutation. (b) Diffuse large B-cell lymphoma of germinal center type with Q61H (c.183A>C) mutation.

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Classical cytogenetic studies demonstrated complex structural and numerical abnormalities in 7/12 diffuse large B-cell lymphoma cases tested (Table 3). No translocation t(2;3)(q13;p25) that corresponds to PAX8/PPARĪ³ rearrangements was observed. No apparent abnormalities were seen in 7q34 (BRAF), 1p13.2 (NRAS), 12p12.1 (KRAS), or 11p15.5 (HRAS). Two cases showed IGH/MYC translocation by fluorescence in-situ hybridization, one of which also had classical cytogenetics that showed a t(8;14) translocation.

Table 3 Cytogenetic abnormalities found in diffuse large B-cell lymphoma of the thyroid and their correlation with mutational status and lymphoma type
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Treatment information was available for 30 cases. Twenty-five patients received definitive surgery (nine received surgery alone, five received surgery with chemotherapy, eight received surgery with radiation, and three received surgery, radiation, and chemotherapy), four patients received chemoimmunotherapy alone, and one patient received chemotherapy and radiation. Chemoimmunotherapy was CHOP-R in all cases except for one patient who was given a Vanderbilt regimen. Four patients had a past history of lymphoma. Two patients developed diffuse large B-cell lymphoma, germinal center type of the thyroid following a prior lymphoma of unspecified type (one renal, one gastric). The other two patients had a prior history of diffuse large B-cell lymphoma, one in the left lobe of thyroid, and the other of the mediastinum. Both underwent surgery and received chemoradiation, and then presented with an extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue type of the right thyroid lobe.

Eleven (11/33, 33%) patients died after their initial diagnosis of thyroid lymphoma. All patients diagnosed with either extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue type or follicular lymphoma were alive after a median follow-up of 3.5 years. In all, 4/8 (50%) patients with diffuse large B-cell lymphoma with mutations and 7/17 (41%) patients with diffuse B-cell lymphoma without mutations died. There was no statistically significant difference in overall survival between the two groups (P=0.77; Figure 4).

Figure 4
figure 4

Overall survival analysis of patients with diffuse large B-cell lymphoma with and without mutations (P=0.77).

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Discussion

We report here for the first time the occurrence of BRAF and NRAS mutations in thyroid lymphomas. None of the cases studied had a coexistent thyroid carcinoma. It is also important to recognize that at least one commonly used antibody for PAX8, which may be helpful in confirming the follicular thyroid cell origin,31 cross-reacts with PAX5, and thus stains many B-cell lymphomas.30

All eight of these MAPK signaling pathway-activating mutations were identified in diffuse large B-cell lymphoma, with none found in the extranodal marginal-zone lymphoma of the mucosa-associated lymphoid tissue type or follicular lymphoma, although the number of these more indolent lymphomas was relatively small. There was no difference in the incidence of these mutations between germinal center and non-germinal center subgroups of diffuse large B-cell lymphoma. However, these two subgroups have been shown to be different, genetically and clinically, and the germinal center group has been reported to have a better prognosis.32

The MAPK cascade controls a major signaling network involved in various cellular functions. The signal is propagated through the RAS-RAF-MEK-ERK proteins into the nucleus regulating cell proliferation, survival, and differentiation.33 Constitutive activation of the effectors of this signaling pathway has a critical role in thyroid carcinogenesis. RET/PTC, RAS, and BRAF mutations appear to be mutually exclusive in thyroid carcinomas, suggesting that activation of this pathway at one level is sufficient for carcinogenesis.10

Molecular testing for alterations in the MAPK pathway genes is becoming an important part of the routine evaluation of fine-needle aspiration samples obtained from thyroid nodules. It is considered reliable and feasible, and has been shown to improve the overall accuracy of fine-needle aspiration cytology.6, 34, 35 A prospective evaluation of 470 fine-needle aspiration samples showed that molecular testing was particularly informative for cases in the indeterminate cytology category, where molecular testing increased the probability of carcinoma from 40% to close to 100% in cases in which mutations were identified.6 Detection of BRAF mutation was reported to have close to 100% positive predictive value for papillary thyroid carcinoma, and the risk of malignancy is above 80% when RAS mutation is detected.5 The importance of the diagnostic use of molecular markers has been reflected in the revised American Thyroid Association's management guidelines, which recommends the use of the mutational panel for nodules with indeterminate fine-needle aspiration cytology to help guide clinical management.36 In view of the increasing use of molecular testing in thyroid fine-needle aspiration samples, the specificity of these mutations becomes an important diagnostic and clinical issue.

The BRAF gene encodes a cytoplasmic serine/threonine kinase that is regulated by binding of RAS and propagates signals downstream to MEK kinase along the mitogen-associated protein kinase signaling pathway. Virtually, all BRAF mutations reported to date are located within the activation segment domain or G-loop, with the most common mutation being V600E (c.1799T>A).37 V600E BRAF mutation is the most common genetic event in papillary thyroid carcinoma, where it is found in 40ā€“45% of cases.38 It is also found in poorly differentiated and anaplastic thyroid carcinomas, but not in medullary carcinoma, follicular tumors, or benign hyperplastic nodules.11

Although screening of human tumors for BRAF mutations has been widely performed, the data concerning the frequency and type of mutations in lymphomas are very limited. Lee et al18 detected BRAF mutations in 4/164 (2.4%) non-Hodgkin lymphomas. All four cases were diffuse large B-cell lymphoma, and no mutations were identified in other non-Hodgkin lymphoma analyzed (3 mantle cell lymphomas, 4 follicular lymphomas, 49 extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue type, and 34 T-cell lymphomas). Whereas most BRAF mutations in human cancers, including carcinomas of the thyroid, involve V600E, all four BRAF mutations reported in this study involved other amino acids. These included one G469A, two G469R, and one D594G (previously reported as G468A, G468R, and D593G, respectively). V600E BRAF mutation has been observed in immunodeficiency-related non-Hodgkin lymphoma with microsatellite instability. The BRAF mutation in this subset of patients was not restricted to diffuse large B-cell lymphoma, but was also seen in a T-cell post-transplant lymphoproliferative disorder and a primary central nervous system B-cell non-Hodgkin lymphoma.19 BRAF V600E mutations have also been identified in over half of Langerhans cell histiocytoses20 and in all hairy cell leukemias.12, 13, 14, 15, 16 Recent studies have also identified BRAF mutations, including V600E and non-V600E mutations, in a small number of plasma cell myelomas (9/238 with 4 V600E mutation),14, 22 and some acute lymphoblastic leukemias (2/3 T-acute lymphoblastic leukemias with 1 V600E and 4/25 B-acute lymphoblastic leukemias with no V600E).21 BRAF mutations other than V600E have also been identified in chronic lymphocytic leukemias17 and one splenic marginal-zone lymphoma.13 In contrast, however, other than the two cases of chronic lymphoproliferative disorder described by Arcaini et al12, several recent large studies of BRAF V600E mutations in hairy cell leukemia have not found any other B-cell lymphomas with V600E mutations.12, 13, 14, 15, 16

In the current study, BRAF mutations were found in 6/25 diffuse large B-cell lymphomas. The index case had a V600E BRAF mutation, the type typically associated with papillary thyroid carcinoma. The other mutations in the BRAF gene that were found (D594G and K601N) have not been found in thyroid carcinomas. The most commonly identified mutation, present in three cases was D594G (c.1781A>G). It has been previously identified in diffuse large B-cell lymphoma,18 sporadic colorectal adenocarcinoma,39 and malignant melanoma.40 A different mutation at the same site (D594N) has been reported in a plasma cell myeloma.14 The K601N mutation has been reported in colorectal adenocarcinoma and plasma cell myeloma.22, 39 A K601E mutation has been identified in one case of splenic marginal-zone lymphoma.13

RAS proteins are a molecular switch involved in propagating signals along several signaling pathways, including the MAPK and PI3K pathways. Approximately 10ā€“20% of all human tumors have mutated RAS proteins.41 Activating point mutations of the RAS genes occur predominantly in epithelial thyroid neoplasms with a follicular pattern, including follicular adenomas and carcinomas, and the follicular variant of papillary carcinoma.5 They are also seen in poorly differentiated and anaplastic thyroid carcinomas.42, 43, 44 RAS gene mutations are reported in a small number of non-Hodgkin lymphoma,45 cutaneous T-cell lymphomas,25 and in up to about half of plasma cell neoplasms.23, 24 In this study, we identified NRAS mutations in two diffuse large B-cell lymphomas, with the overall prevalence of this mutation of 8% in thyroid diffuse large B-cell lymphoma. Both mutations (Q61H and Q61K) involved codon 61, which is the most common mutation site in this gene.46 The Q61K mutation is commonly seen in thyroid follicular carcinoma46 and in other malignancies such as melanoma.47 The Q61H mutation has also been reported in some thyroid carcinomas48 and papillary carcinoma of the breast.49

In summary, our results indicate that among primary thyroid tumors, BRAF and NRAS mutations are not restricted to epithelial neoplasms and may also be seen in thyroid lymphomas, specifically in diffuse large B-cell lymphomas. Importantly, mutations of these genes may be identical to those frequently seen in thyroid carcinomas, specifically V600E BRAF and Q61K NRAS mutations. Hence, when these mutations are detected in thyroid fine-needle aspiration samples, the differential diagnosis must also include lymphomas. Additional morphological and immunophenotypic studies remain critical in this situation to determine the correct diagnosis. Furthermore, our findings highlight the potential importance of the MAPK pathway mutations in the pathogenesis of diffuse large B-cell lymphomas developing in the thyroid gland.