聚合酶链反应技术检测非霍奇金淋巴瘤抗原受体基因重排的局限性

病理形态学和免疫组织化学有效标记,结合分子病理学的检测是当前临床病理诊断的发展趋势.近年来这种三结合的诊断模式已在淋巴瘤的病理诊断中得到应用.各类淋巴瘤的病理形态学特征是病理诊断的基石,免疫组织化学有助于正确的诊断及分类,仅靠这二者,恶性淋巴瘤的误诊率仍较高,采用分子病理学技术检测病理标本中淋巴瘤特征性的分子标记具有重要的参考价值.其中运用聚合酶链反应(PCR)技术检测IgH/TCR基因的重排是目前常用的淋巴瘤的分子病理学的方法之一.当检测的结果为单克隆时,一般认为是肿瘤性增生;结果为多克隆时则提示反应性增生.近年来随着研究的深入及资料积累的日益增多,这一分子病理学诊断方法在临床病理上的应用出现了一系列亟待解决的问题,如检测结果的假阳性和假阴性,以及双重重排和寡克隆的现象常有报道.深入探讨这方面的问题有助于将分子病理学方法在淋巴瘤临床病理诊断中广泛开展,并提高分子病理诊断检测的可信度.

IgH基因重排在B细胞非霍奇金淋巴瘤诊断中的应用

正常淋巴细胞在发育中是多克隆性质,但恶性肿瘤表现为单克隆性基因重排.所以,通过基因重排检测不仅可以鉴别淋巴组织是肿瘤性增生还是反应性增生,而且使准确判断细胞起源,完善淋巴瘤的分型成为可能[1].我们拟了解在本地区不同病理类型B细胞非霍奇金淋巴瘤(NHL)IgH基因单克隆性重排的发生率,旨在为鉴别部分淋巴组织是反应性增生还是肿瘤性增生,推测肿瘤细胞起源,指导治疗及判断预后提供参考指标.

儿童急性T淋巴细胞白血病T细胞受体β链基因重排的特点及其在微小残留病定量检测中的意义

Objective To explore the characteristics of T-cell receptor beta (TCRβ) gene rearrangement in children with T-cell acute lymphoblastic leukemia (T-ALL) and establish a system for quantitative detection of minimal residual disease (MRD) by real-time quantitative PCR (RQ-PCR) targeting the TCRβ gene rearrangement. Methods Multiplex PCR designed by the BIOMED-2 was used to detect TCRβ gene rearrangement in the bone marrow samples of 26 children with T-ALL. Sequences of junctional region were then compared and analyzed in IMGT database. Allele specific oligonucleotide (ASO) upstream primers were designed complementary to the V-D-J or D-J junctional region of TCRβ gene rearrangements. Samples at diagnosis were serially diluted in DNA obtained from mononuclear cells (MNC) from a pool of 20 healthy donors to generate the patient-specific standard curves. Subsequently, TCRβ RQ-PCR was applied to six patients to quantify MRD with germline Jβ primer/probe combinations. To determine the quantity and quality of DNA, we also used RQ-PCR for the N-ras gene.Results Clonal rearrangements were identified in 92.3% of the children with T-ALL ( Vβ-Dβ-Jβ rearrangements in 84.6% and Dβ-Jβ rearrangements in 50% ). Comparative sequence analysis of 42 TCRβ recombination revealed that two downstream Vβ families (BV5, BV6) were preferentially used. The segment Jβ2. 7 was dominant in childhood T-ALL. Jβ1. 3, Jβ2.4, and Jβ2.6 were not detected. The slope of the standard curves was from - 3.54 to -3.37 with the intercepts between 19.35 and 20.51. The correlation coefficients of all the 6 standard curves were ≥0.98. None of the cases had a quantitative range of RQ-PCR lower than 10<'-4>. During the follow-up, an increased incidence of MRD was found before relapse.Conclusions RQ-PCR, which is a highly sensitive and specific method for detection of TCRβ gene rearrangements, will be of high value to study MRD in T-ALL. Close monitoring of MRD is of great importance for prognosis and follow-up of the patients with the disease.

T cell receptors (TCR) are a collection of several proteins located in the outer membrane of the T cell. Mature T cell has definitive TCR genes,which may be used as a clonal marker. To investigate whether TCR gene analysis may be useful for diagnosis of myasthenia gravis (MG) with thymoma, we used the cDNA of the β－ subunit of the TCR served as probes to analyze the structure of the TCR β gene and to determine the extent of diversity between MG with thymoma and MG without thymoma. Eighteen patients were clinically diagnosed generalized MG, eight of them had thymoma. Peripheral blood lymphocytes (PBL) were obtained at or shortly before thymectomy (except one, 2 years after thymectomy). When DNA was digested with EcoRI and hybridized with the 32P labeled probes, two germline bands of 10.5kb and 4.0 kb appeared in DNAs from all 18 patients. The 10.5 kb band contains the C β 1 region and the 4.0kb fragment the C β 2 region, respectively. The 4.0 kb band is usually reduced as compared with the 10.5kb band. This can be explained by the preferential rearrangement of the C β 2 gene in T cell with MG. We were able to demonstrate a rearranged band of 9kb in EcoRI－ digested DNAs of four patients. All the four patients had been found to have thymoma , of which 2 were lymphocyte and 2 mixed type. But rearranged bands could not be detected in the DNAs of another four patients with thymoma, of which 2 were epithelial and 2 mixed type. When DNA was digested with HindIII and BglII, germline bands appeared in DNAs from all patients. No rearranged bands could be observed . Single rearranged bands were detected in four patients with thymoma, this showed that specific TCR gene rearrangement may be related to certain types of thymoma . Detection of TCR gene rearrangement might be helpful to the early diagnosis of MG with thymoma . But we were unable to find gene rearrangement in another four thymoma (2 were epithelial and 2 mixed type). There are three possible explanations for the results. First, thymoma has heterogeneity. Second, TCR gene rearrangement only reflects the clonal origin of the T cell tumor. Third, in one patient, peripheral blood lymphocytes were obtained two years after thymectomy, neoplastic T cells maybe disappear. Thus, our results suggested that TCR gene rearrangement in patients with MG may be helpful for diagnosing certain type of thymoma.

亲毛囊性蕈样肉芽肿伴嗜酸粒细胞增多一例

患者男，69岁。3年来，头、躯干和四肢出现散在红斑、毛囊性丘疹及痤疮样皮损(如粟丘疹、囊肿等)和脱发，病程中伴外周血嗜酸粒细胞增多。皮损组织病理检查示真皮内灶性慢性炎细胞浸润伴毛细血管增生，毛囊周围慢性炎细胞浸润伴血管增生，伴少许嗜酸粒细胞，考虑为毛囊炎，予抗组胺药和抗生素治疗后皮损炎症消退，瘙痒减轻。3个月后，枕部出现斑块伴脱发，组织病理检查示真皮内密集淋巴样细胞、嗜酸粒细胞浸润，毛囊周围大量淋巴样细胞浸润伴较多嗜酸粒细胞，可见不典型淋巴细胞，部分侵入毛囊，毛囊上皮黏液样变性。阿新蓝染色阳性。免疫组化染色：CD20、CD79a、EB病毒(EBV)、CD56、磷酸葡萄糖变位酶-1(PGM-1)、髓过氧化物酶(MPO)、CD7、抗角蛋白单克隆抗体AE1/AE3均阴性，异形细胞CD3、CD4、CD5、CD2、CD43、泛素羧基末端水解酶-L1 (UCHL-1)均阳性。T细胞受体基因重排结果为阴性。诊断：亲毛囊性蕈样肉芽肿。予光化学疗法(PUVA)联合阿维A治疗，仍有新发皮损，目前患者在随访中。