Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0951
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 720-723
Copyright © 2005 by The Endocrine Society
Detection of Monoclonality of the Immunoglobulin Heavy Chain Gene in Thyroid Malignant Lymphoma by Vectorette Polymerase Chain Reaction
Toru Takano,
Akira Miyauchi,
Fumio Matsuzuka,
Hiroshi Yoshida,
Tsugunori Notomi,
Kanji Kuma and
Nobuyuki Amino
Department of Laboratory Medicine (T.T.), Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; Kuma Hospital (A.M., F.M., H.Y., K.K., N.A.), Hyogo 650-0011, Japan; and Eiken Chemical Co. Ltd. (T.N.), Tochigi 324-0036, Japan
Address all correspondence and requests for reprints to: Toru Takano, M.D., Department of Laboratory Medicine, Osaka University Graduate School of Medicine, D2, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ttakano{at}labo.med.osaka-u.ac.jp.
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Abstract
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Distinguishing between thyroid malignant lymphoma and lymphocytic thyroiditis (Hashimoto’s thyroiditis) is quite difficult and problematic. B cell lymphomas display clonal Ig heavy-chain (IgH) gene rearrangement, and Southern blot hybridization (SBH) is often used for detection of the monoclonality of the IgH gene. However, SBH is often problematic because it requires a large volume of samples. We examined the efficiency in detecting the monoclonality of IgH gene in thyroid malignant lymphomas by vectorette PCR, which we started with only 200 ng of genomic DNA. Monoclonality was detected in 36 of 47 (76.6%) malignant lymphomas, whereas it was not detected in 10 samples of tissue of Hashimoto’s thyroiditis. The sensitivity was almost the same as that with SBH in which monoclonality was detected in 33 of 45 (73.3%) malignant lymphomas. These results suggest that vectorette PCR may be a substitute for SBH, and because it requires only a small volume of samples, it may be used in the analysis of aspiration biopsies.
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Introduction
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PRIMARY THYROID LYMPHOMA (non-Hodgkin’s lymphoma) is a rare type of malignant tumor accounting for 2.2–5% of all thyroid cancers, typically occurring in elderly women with a history of lymphocytic thyroiditis (Hashimoto’s thyroiditis); it originates from B lymphocytes (1, 2, 3). Most patients with malignant lymphoma note a rapidly growing mass in the neck over a period of 2–3 months, and symptoms of thyroid lymphoma are mainly due to rapid growth of the tumor with compression of the adjacent organs (4). However, these symptoms often appear in patients with the aggressive inflammation caused by Hashimoto’s thyroiditis, and Hashimoto’s thyroiditis is believed to be one of the causes of malignant lymphoma. Thus, clinical differentiation between these two conditions is quite difficult, and in most cases, a histopathological method by open biopsy is used to confirm the diagnosis because the cytological differentiation of Hashimoto’s thyroiditis from a low-grade lymphoma is problematic due to the lack of marked nuclear atypia.
Molecular-based preoperative diagnosis is expected to reduce invasion for the patients. Southern blot hybridization (SBH) analysis is used for the assessment of Ig heavy-chain gene (IgH) rearrangement (B cell monoclonality) (5); however, it is not possible to perform SBH without surgery because it usually requires more than 10 µg of genomic DNA.
We developed a method of preoperative molecular-based diagnosis of malignant thyroid tumors, called aspiration biopsy nucleic acid diagnosis (ABND), which uses extracted nucleic acids from cells obtained by fine-needle aspiration biopsy (FNAB). In the thyroid, by detecting specific changes in nucleic acids in malignant thyroid tissues with ABND, an accurate and objective diagnosis can be made in papillary, anaplastic, and medullary carcinomas (6, 7, 8).
PCR has become a viable alternative to SBH (9, 10, 11). B cell monoclonality can be readily detected by PCR using a consensus IgH variable region (VH) framework (FR) III-directed primer along with a consensus IgH joining region primer. However, use of this method is not popular when analyzing the samples obtained by FNAB because the overall detection rate of monoclonality is consistently lower than that of SBH and requires a few micrograms of purified genomic DNA for a successful analysis, which cannot always be obtainable through the usual FNAB procedures.
In the previous study, we examined the sensitivity of RT-PCR analysis in the detection of B cell monoclonality. However, low sensitivity, probably due to a significant mismatch between the VH primer (FR3A) and the VH-FR III region of a particular B cell lymphoma, was an obstacle to clinical use (12). It was suggested that a modification of this method, in which the rearranged IgH gene is amplified without using a primer annealing to the VH sequence, might improve the sensitivity.
Vectorette PCR is a method for amplifying DNA fragments of which the 5' sequences are unknown, using a modified linker to facilitate specific amplification of the targeted sequence (13, 14), and it requires only 200 ng of genomic DNA to start with, which is easily obtainable by FNAB. In this study, we examined the sensitivity of vectorette PCR amplifying the J region of the IgH gene in detecting B cell monoclonality using tissues obtained surgically and evaluated the clinical usefulness of this method in the diagnosis of malignant lymphoma.
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Materials and Methods
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Tissue samples
The study protocol was approved by the institutional ethical committee. Informed consent was obtained from all patients. Twenty-six thyroid tissue samples from 47 malignant lymphomas and 10 cases of Hashimoto’s thyroiditis were obtained by open biopsy after the patients’ informed consent. All malignant lymphomas were B cell lymphomas. After hematoxylin-eosin staining, pathological diagnoses were made by a skillful pathologist, and they were confirmed by another pathologist. A portion of the tissue sample was immediately frozen in liquid nitrogen for the later use.
SBH
SBH was performed in 45 of 47 cases of malignant lymphoma in which a sufficient volume of the tissues were available. Genomic DNAs were extracted and purified with a QIAamp DNA midi kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s protocol. SBH was performed using a digoxigenin (DIG) DNA labeling kit and a DIG luminescent detection kit for nucleic acids (Roche, Tokyo, Japan) according to standard protocols that have been published previously (5). In brief, 10 µg of DNA extracted from a tissue sample were digested with BamHI and HindIII. The digested DNA was electrophoresed on a 0.8% agarose gel, transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia, Tokyo, Japan), and hybridized overnight with a 5.4-kb IgH joining region genomic DNA probe (Oncogene Science, Uniondale, NY) labeled with DIG. After probe hybridization, the membrane was incubated in a blocking solution for 2 h at room temperature. The blocking solution was discarded, and antidigoxigenin antibody conjugated to alkaline phosphatase was added. After appropriate washing, the signal was detected by adding CSPD (Roche), and the membrane was then exposed to a radiographic film.
Vectorette PCR analysis
A small leftover portion of tissues (approximately 5 mg) that was not used for SBH was cut out and genomic DNAs were extracted and purified with a QIAamp DNA minikit (QIAGEN) according to the manufacturer’s protocol. Genomic DNA was extracted from peripheral blood with a QIAamp DNA blood minikit (QIAGEN) according to the manufacturer’s protocol.
The primers used are listed in Table 1
. All primers were purchased from QIAGEN. The positions of the 3' primers and digestion sites of the restriction enzymes are summarized in Fig. 1. In most of the cases, there is no digestion site of BbrPI between the recognition site of Tsp509I and the annealing sequence of the J6E primer in the IgH gene after rearrangement, because the recognition site of BbrPI appears much less frequently than that of Tsp509I.
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FIG. 1. Representative positions of the 3' primers and digestion sites of the restriction enzymes in the IgH gene before (A) and after (B) rearrangement.
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A vectorette linker (VT) was prepared by annealing two oligonucleotides, TS and TL. The 5' end of TS (AATT) was designed to hybridize to the recognition sequence of Tsp509I. Two hundred nanograms of extracted DNA was digested with 5 U Tsp501I (Daiichi-Kagaku, Tokyo, Japan) for 60 min at 65 C. After phenol-chloroform extraction and ethanol precipitation, the digested DNA was dissolved in LoTE buffer [3 mM Tris-HCl and 0.2 mM EDTA (pH 8.0)]. Linker ligation was performed in a ligation mixture containing the whole DNA sample, 350 U of T4 DNA ligase (TAKARA, Shiga, Japan), 2 µl of 10 x T4 DNA ligase buffer (TAKARA), 0.5 µM of VT in a total volume of 20 µl for 16 h at 4 C. The ligated sample was purified with a QIAquick PCR purification kit (QIAGEN) according to the manufacturer’s protocol. The purified samples were treated with 10 U BbrPI (Roche) for 2 h at 37 C to digest the genomic DNA without rearranged IgH gene. After phenol-chloroform extraction and ethanol precipitation, the digested DNA was dissolved in 50 µl of LoTE buffer.
For the first PCR, each reaction mixture consisted of 20 µl of each sample, 0.2 µM of J6E and V1, 2.5 µl of 10 x Ex Taq buffer (TAKARA), 2 µl of 2.5 mM deoxynucleotide triphosphate mix (TAKARA), 0.5 U of Ex Taq Hot Start version (TAKARA), and nuclease-free water to a final volume of 25 µl. In the first cycle of the PCR, the second strand synthesis started with only the J6E primer because the V1 primer does not have an annealing site in the vectorette linker. This reaction allowed the highly specific amplification of the IgH gene. PCR products were purified with MultiScreen PCR (Millipore, Tokyo, Japan), and samples were dissolved in 50 µl of LoTE. For the second PCR, each reaction mixture consisted of 20 µl of each sample, 0.2 µM of J6I and V2, 2.5 µl of 10 x Ex Taq buffer, 2 µl of 2.5 mM deoxynucleotide triphosphate mix, 0.5 U of Ex Taq Hot Start version, and nuclease-free water to a final volume of 25 µl. In both reactions, the PCR conditions were 22 cycles of denaturation (98 C, 10 sec), annealing (65 C, 30 sec), and extension (72 C, 90 sec; autosegment extension, 5 sec). The final PCR products were separated by 1.5% agarose (Invitrogen, Tokyo, Japan). The gel was stained with SYBR Green I (TAKARA). The samples showing more than one clear bands that were not placed in 2.5 kb without a background smear were regarded as positive for B cell monoclonality. The determinations of monoclonality were performed without referring to the pathological diagnoses. These procedures are summarized in Fig. 2.
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FIG. 2. Procedures of vectorette PCR. The genomic DNA (blank bars) was digested with Tsp509I followed by ligation reaction with the VT (filled bars) (A and B). The genomic DNA without rearrangement was digested with BbrPI (C). In the first cycle of the PCR, the second-strand synthesis started with only the J6E primer (D). The rearranged gene was amplified by nested PCR (E and F).
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Results
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By vectorette PCR, the monoclonality of the IgH gene was detected in 36 of the 47 (76.6%) malignant lymphomas. In the 10 tissue samples of Hashimoto’s thyroiditis and the peripheral blood, no clear band was observed besides that derived from the genomic sequence without rearrangement, and the smear was observed as well. The representative data are shown in Fig. 3. There was no evident difference in sensitivity among pathological subtypes (Table 2). In the 45 malignant lymphomas in which SBH was performed, the results of both methods were compared. The representative data of SBH are shown in Fig. 4. The sensitivity of vectorette PCR and SBH were identical at 73.3% (Table 3). The results of the two methods agreed in 31 (68.8%) cases and disagreed in 14 (31.2%) cases.
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FIG. 3. Representative results of vectorette PCR. Lane 1, PHY markers (M) (TAKARA); lane 2, peripheral blood (PB); lanes 3–6, Hashimoto’s thyroiditis; and lanes 7–11, malignant lymphomas. Lanes 7–10 were diagnosed as monoclonal. The arrow shows the position of the PCR products derived from the remnants of genomes without rearrangement.
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TABLE 2. Detection of monoclonality of the IgH gene in Hashimoto’s thyroiditis and malignant lymphoma by vectorette PCR
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FIG. 4. Representative results of SBH. A, A genomic DNA from human peripheral blood cells as a negative control. B, A tissue sample from the case shown in lane 9 in Fig. 3 . Open arrows in B show the positions of rearranged bands.
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Discussion
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Because cytopathological diagnosis of some thyroid malignant lymphomas is difficult, histopathological confirmation by open biopsy is often performed. However, in most cases of thyroid malignant lymphoma, this should be avoided, if possible, because radiation or chemotherapy is recommended as an effective therapy but not surgical dissection (15). To perform molecular-based diagnosis in a less invasive way, it is necessary to start with a small volume of samples, such as FNABs. Nucleic acids obtained by FNAB usually consist of about 1 µg of DNA and 50 ng of RNA (16). Conventional methods such as SBH and PCR are not usually used preoperatively because they need a relatively large volume of samples for reliable diagnosis. In fact, we tested the PCR method of Brisco et al. (9) for the detection of B cell monoclonality using 200 ng of genomic samples of malignant lymphomas, but we detected monoclonality in only two of the 47 (4.3%) malignant lymphomas (data not shown). In the previous studies, immunohistochemical analysis of excess expression of 
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-light chains (light chain restriction), detection of DNA aneuploidy or increased proliferating activity in lymphomas by flow cytometry, or quantitative measurement of the expression of survivin was reported to be helpful in diagnosing malignant lymphoma. However, they are not widely used mainly because they do not show satisfactory sensitivity and specificity as diagnostic tests to distinguish Hashimoto’s thyroiditis from malignant lymphoma (3, 17, 18).
Vectorette PCR requires only 200 ng of genomic DNA, and it is possible to start with a small number of cells obtained by FNAB. The sensitivity of vectorette PCR was identical with that of SBH. Vectorette PCR has many advantages over SBH. It is less expensive, more rapid, and technically easier. It may be used both preoperatively and also postoperatively as a substitute of SBH.
The reason for the discordant results between vectorette PCR and SBH in some lymphomas is not clear. One possible explanation is the use of different restriction enzymes in the two methods. For example, the result of vectorette PCR is shown to be negative when the rearranged IgH gene does not have the digestion site of Tsp509I near the rearranged site. Use of multiple restriction enzymes may increase the sensitivity, although it will be more laborious. Another possibility to be considered is interference of the contaminated nontumorous lymphocytes with the detection of B cell monoclonality; they can produce background smear in vectorette PCR. This might be the most probable explanation because malignant lymphomas arise from long-lasting Hashimoto’s thyroiditis; thus, mixed pathological features of Hashimoto’s thyroiditis and malignant lymphoma are often observed (2). Due to the limitation in the obtainable sample volume by biopsy, we could not use the same DNA in both SBH and vectorette PCR. In SBH almost the entire biopsied sample is used, whereas in vectorette PCR only a very small portion is dissected. In some cases, two samples from the same patient obtained from different parts of the biopsied material showed discrepant results in vectorette PCR. It is suggested that the results were negative when we happened to cut out the places in which nontumorous cells were packed. On the contrary, the results could be positive only in vectorette PCR when we happened to cut out the places in which tumorous cells were packed. When applying vectorette PCR in ABND, it may be recommended to obtain samples by multiple aspirations to prevent false-negative results.
A clinical trial is ongoing to estimate the sensitivity of vectorette PCR. Vectorette PCR, which takes only 1.5 d, may help cytopathologists decide whether patients need further examinations such as open biopsy to confirm the diagnosis of malignant lymphoma.
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Footnotes
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First Published Online November 9, 2004
Abbreviations: ABND, Aspiration biopsy nucleic acid diagnosis; DIG, digoxigenin; FNAB, fine-needle aspiration biopsy; FR, framework; IgH, Ig heavy-chain; LoTE, buffer of Tris-HCl and EDTA; SBH, Southern blot hybridization; VH, IgH variable region; VT, vectorette linker.
This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan and Grants-in-Aid for Scientific Research C, 2004–2005 (16590456).
Received May 19, 2004.
Accepted November 3, 2004.
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Abstract
Introduction
