利用石蜡包埋组织进行BIOMED-2标准化IG/TCR基因重排检测在淋巴瘤诊断中的意义

Significance of BIOMED-2 standardized IG/TCR gene rearrangement detection in paraffin-embedded section in lymphoma diagnosis

目的 探讨利用石蜡包埋组织（FFPE）标本进行BIOMED-2标准化IG/TCR基因重排检测在淋巴瘤诊断中的可行性以及提取DNA的扩增最大片段与IGH V-J不同引物区域阳性检出率之间的关系.方法 从50例患者FFPE标本中提取DNA.采用BIOMED-2系统引物,进行多重PCR扩增并应用PCR片段分析法进行IG/TCR基因重排的克隆性分析.结果 ①DNA由原浓度（100～500ng/μl）稀释至50～100 ng/μl后,30例弥漫大B细胞淋巴瘤患者蜡卷标本DNA扩增最大片段（300～400bp）含量由10.0％提高到90.0％ （P＜0.01）,IGH＋IGK阳性率由46.7％提高至83.3％,差异有统计学意义（P=0.006）;石蜡切片标本DNA扩增最大片段均在300 bp以上,IGH＋IGK阳性率为96.7％.蜡卷标本与石蜡切片标本IGH＋IGK阳性率差异无统计学意义（P=0.195）.②在DNA高浓度时,提取的DNA扩增最大片段以100或200 bp为主,短片段IGH FR3的检出率比长片段IGH FR1更加稳定.③13例外周T细胞淋巴瘤患者标本TCRG＋TCRB均为阳性,对照组7例反应性淋巴组织增生患者IG/TCR未见克隆阳性.结论 DNA浓度的稀释是唯一提高DNA最大片段扩增比例及克隆性检出率的方法;短片段IGH FR3的检出率几乎不受DNA浓度的影响.利用FFPE标本进行BIOMED-2标准化IG/TCR基因重排检测对于淋巴瘤的诊断有指导意义.

Objective To explore the feasibility of detecting lymphoma with the application of BIOMED-2 standardized immunoglobulin/T cell receptor （IG/TCR） gene rearrangement system in formalin fixed paraffin-embedded （FFPE） tissue samples,and to discuss the relationship between the longest amplification fragment of extracted DNA and positive detection rate of different IGH V-J primers.Methods DNA was extracted from 50 cases of FFPE tissue samples.Multiplex-PCR amplifications were performed and then the IG/TCR gene rearrangements were analyzed using BIOMED-2 standardized clonality analysis system.Results ①When the DNA concentration was diluted to 50-100 ng/μl from 100-500 ng/μtl,the proportion of the longest amplification fragment （300-400 bp） of DNA was improved from 10.0% to 90.0% in 30 cases of diffuse large B cell lymphoma （DLBCL） wax roll samples （P〈0.01）.The positive rate of IGH＋IGK was increased from 46.7% to 83.3%,the difference was statistically significant （P=0.006）.The lengths of the longest amplification fragments of DNA were all longer than 300bp in the paraffin section samples of DLBCL.The positive rate of IGH ＋ IGK of these samples was 96.7%.The difference of the positive rate of IGH＋IGK between the wax roll samples and the paraffin section samples had no statistical significance （P=0.195）.②When the concentration of DNA was high,most of the longest amplification fragments of extracted DNA were 100 bp or 200bp,and the detection rate of short fragment IGH FR3 was more stable than that of long fragment IGH FR1.③The clonality analysis of TCRG＋TCRB in all 13 cases of peripheral T cell lymphoma samples showed positive results,while no positive IG/TCR clones were found in 7 cases of reactive lymphoid tissue hyperplasia in control group.Conclusion Dilution of DNA is the only method to improve not only the proportion of longest fragment amplification but also the detection rate of clonality.The detection rate of IGH FR3 would not be affected by the concentration of DNA.The application of BIOMED-2 standardized IG/TCR gene rearrangement system in FFPE tissue samples plays an important role in the lymphoma diagnosis.