通过高分辨率熔解曲线分析检测泰国恶性淋巴瘤的单克隆免疫球蛋白重链基因重排（FR3）。

UNLABELLED

Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan) has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR) followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM). The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma.

INTRODUCTION

Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population 1. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year 2. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal lymphoproliferation 34. Analyzing DNA extracted from formalin-fixed, paraffin-embedded tissues by multiplex PCR techniques is more rapid, accurate and highly sensitive. Measuring the size of the amplicon from PCR analysis could be used to diagnose malignant lymphoma with monoclonal pattern showing specific and distinct bands detected on acrylamide gel electrophoresis. However, this technique has some limitations and some patients might require a further confirmation test such as GeneScan or fragment analysis 56.GeneScan technique or fragment analysis reflects size and peak of DNA by using capillary gel electrophoresis. This technique is highly sensitive and can detect 0.5-1% of clonal lymphoid cells. It measures the amplicons by using various fluorescently labeled primers at forward or reverse sides and a specific size standard. Using a Genetic Analyzer machine and GeneMapper software (Applied Bioscience, USA), the monoclonal pattern revealed one single, sharp and high peak at the specific size corresponding to acrylamide gel pattern, whereas the polyclonal pattern showed multiple and small peak condensed at the same size standard. This technique is the most sensitive and accurate technique; however, it usually requires high technical experience and is also of high cost 7. Therefore, rapid and more cost effective technique are being sought.LightCycler PCR performs the diagnostic detection of amplicon via melting curve analysis within 2 hours with the use of a specific dye 89. This dye consists of two types: one known as SYBR-Green I which is non specific and the other named as High Resolution Melting analysis (HRM) which is highly sensitive, more accurate and stable. Several reports demonstrated that this new instrument combined with DNA intercalating dyes can be used to discriminate sequence changes in PCR amplicon without manual handling of PCR product 1011. Therefore, current investigations using melting curve analysis are being developed 1213.In this study, three different techniques were compared to evaluate the suitability of LightCycler PCR with HRM as the clonal diagnostic tool for IgH gene rearrangement in B-cell non-Hogdkin lymphoma, i.e. thermocycler PCR followed by heteroduplex analysis and PAGE, GeneScan analysis and LightCycler PCR with HRM.

未注明

恶性淋巴瘤，特别是非霍奇金淋巴瘤，是泰国最常见的血液恶性肿瘤之一。恶性淋巴瘤的诊断常常存在问题，尤其是在疾病的早期阶段。通过聚合酶链反应（PCR）检测抗原受体基因重排，包括 T 细胞受体（TCR）和免疫球蛋白重链（IgH），然后进行异源双链分析，目前已成为标准检测方法，而荧光片段分析（GeneScan）已被用于确认试验。在这项研究中，我们比较了三种技术：热循环 PCR 后异源双链和聚丙烯酰胺凝胶电泳、GeneScan 分析和实时 PCR 与高分辨率熔解曲线分析（HRM）。比较是用从石蜡包埋组织中提取的 DNA 进行的，这些组织被诊断为 B 细胞非霍奇金淋巴瘤。使用了针对 IgH 基因可变区 3 的特异性 PCR 引物序列，包括荧光标记的 IgH 引物，并与 HRM 进行了比较。总之，在 LightCycler 系统中通过 HRM 检测 IgH 基因重排显示出在 B 细胞非霍奇金淋巴瘤中区分单克隆性和多克隆性的潜力。

简介

恶性淋巴瘤，特别是非霍奇金淋巴瘤，是泰国最常见的血液恶性肿瘤之一。据公共卫生部报道，女性的发病率为每 10 万人中有 3.1 例，而男性的发病率为每 10 万人中有 4.5 例 1。在 Siriraj 医院，新诊断的恶性淋巴瘤病例为每年 214.6 例 2。恶性淋巴瘤的诊断常常存在问题，尤其是在疾病的早期阶段。因此，通过聚合酶链反应（PCR）检测抗原受体基因重排，包括 T 细胞受体（TCR）和免疫球蛋白重链（IgH），最近已成为区分反应性和恶性克隆性淋巴增生的标准实验室检测方法 34。通过多重 PCR 技术分析从福尔马林固定、石蜡包埋组织中提取的 DNA 更快速、准确和高度敏感。分析 PCR 分析的扩增子的大小可以用于诊断具有单克隆模式的恶性淋巴瘤，表现为在丙烯酰胺凝胶电泳上检测到特异性和独特的带。然而，这种技术有一些局限性，一些患者可能需要进一步的确认试验，如 GeneScan 或片段分析 56。GeneScan 技术或片段分析通过毛细管凝胶电泳反映 DNA 的大小和峰。该技术高度敏感，可检测 0.5-1%的克隆性淋巴细胞。它使用正向或反向侧的各种荧光标记引物和特定的大小标准来测量扩增子。使用遗传分析仪和 GeneMapper 软件（Applied Bioscience，美国），单克隆模式显示在与丙烯酰胺凝胶模式相对应的特定大小处的单个、尖锐和高的峰，而多克隆模式显示多个小峰聚集在相同的大小标准处。该技术是最敏感和准确的技术；然而，它通常需要高的技术经验，并且成本也很高 7。因此，正在寻找快速和更具成本效益的技术。LightCycler PCR 通过在 2 小时内使用特定染料进行扩增子的诊断检测，实现了熔解曲线分析 89。这种染料由两种类型组成：一种称为 SYBR-Green I，是非特异性的，另一种称为高分辨率熔解分析（HRM），是高度敏感、更准确和稳定的。有几个报告表明，这种新仪器与 DNA 嵌入染料结合使用，可以在不进行 PCR 产物手动处理的情况下，区分 PCR 扩增子中的序列变化 1011。因此，目前正在开发使用熔解曲线分析的研究 1213。在这项研究中，我们比较了三种不同的技术，以评估 LightCycler PCR 与 HRM 作为 B 细胞非霍奇金淋巴瘤中 IgH 基因重排的克隆诊断工具的适用性，即热循环 PCR 后异源双链分析和聚丙烯酰胺凝胶电泳、GeneScan 分析和 LightCycler PCR 与 HRM。