普通甲醛固定石蜡包埋组织DNA提取方法的探讨

背景与目的:在国内,几乎所有的医院和科研机构均应用普通甲醛固定手术标本,但因为普通甲醛固定石蜡包埋组织中的DNA降解相对严重,从这些蜡块中提取高质量的DNA非常困难。本研究的目的是寻找从普通甲醛固定石蜡包埋组织中提取DNA的理想方法。方法:取我院2000年甲醛固定石蜡包埋的手术切除食管癌标本15例,分别以蛋白酶K消化和不同pH值下加热两种裂解方法裂解细胞,以酚氯仿抽提法提取其DNA;并在蛋白酶K消化法进行DNA提取的过程中,应用交叉设计对二戊烯或二甲苯脱蜡、37℃或56℃消化48h或72h、氯化钠盐析或酚氯仿抽提4种参数进行研究,所得DNA质量以电泳分析和PCR扩增结果判断。结果:蛋白酶K消化酚氯仿抽提法所得DNA产量(平均值为17.88μg)和质量均高于pH值为7～12条件下的加热提取法(P<0.05)。56℃消化所得的DNA质量明显优于37℃,而消化72h的DNA亦明显优于48h者。不同脱蜡方法和抽提方法对DNA的产量和质量影响不大,结论:以蛋白酶K消化氯化钠盐析法,从普通甲醛固定石蜡包埋组织中提取DNA质量较可靠。并且,应用蛋白酶K于56℃消化3天更能获得高质量和高产量的DNA。     

B-ALL病人TCR Vβ基因谱系和克隆性分析

 目的　了解B ALL病人外周血T细胞的TCRVβ基因谱系及其克隆性增殖情况。 方法　利用RT PCR方法扩增 13例初发未治B ALL病人外周血单个核细胞中 2 4个TCRVβ基因的互补决定区 3(CDR3) ,PCR产物进一步经荧光标记和基因扫描分析CDR3长度而确定T细胞的克隆性。结果　 13例B ALL病人分别表达 2～ 18个Vβ亚家族。 13例病人均存在 1个或多个Vβ亚家族的克隆增殖T细胞 ,增殖形式包括寡克隆及寡克隆增殖趋势、双克隆和单克隆。Vβ2 1和Vβ2 3亚家族出现克隆增殖性T细胞的频率较高。结论　B ALL病人外周血T细胞的TCRVβ谱系呈现优势利用的特点 ,并存在克隆性增殖的T细胞 ,这可能是机体对白血病相关抗原产生的特异性免疫反应 ;Vβ2 1和Vβ2 3亚家族T细胞发生克隆增殖改变的趋向性比较明显 ,可能与B ALL相关抗原刺激有关     

Real-time quantitative PCR analysis of amplified DNA on chromosomes 4p15.2 and 6q23-24 from formalin-fixed, paraffin-embedded breast cancer tissues.

This study was performed to detect amplification of DNA sequences on chromosomes 4p15.2 and 6q23-24, obtained from formalin-fixed, paraffin-embedded, breast-cancer tissues. The prognostic relevance of the amplification was also demonstrated. DNA from formalin-fixed, paraffin-embedded tumor and corresponding normal tissues of 53 patients with breast cancer was extracted and amplified by real-time quantitative PCR technique. Amplification of the DNA sequences on chromosomes 4p15.2 and 6q23-24 was detected in 23 (43%) and 32 (60%) cases, respectively. Thirty-six (68%) cases showed amplification on both or one of the chromosomes. These frequencies are similar to that obtained from fresh samples in our previous study. In addition, amplification of the DNA on chromosomes 4p15.2 and / or 6q23-24 was predominantly observed in tumors with invasive ductal carcinoma. The findings in this study demonstrate that DNA extracted from formalin-fixed, paraffin-embedded breast tumors can be used to determine amplification of DNA sequences on selective chromosomal regions. We also suggest that the amplified DNA on chromosomal regions 4p15.2 and 6q23-24 might be involved in the development and progression of breast cancer.     

T- and B-cell clonality and frequency of human herpes viruses-6, -8 and Epstein Barr virus in angioimmunoblastic T-cell lymphoma

Angioimmunoblastic T-cell lymphoma (T-AIL) is a peripheral T-cell lymphoma of unknown etiology. Previous clonality studies have shown a heterogeneous composition of this disease with varying restrictions of B- and T-cell populations in the tumour. For the first time in a single study and in the same pathological materials, we have analysed, lymphoid cell clonality and occurrence of human herpes viruses and Epstein Barr virus. Of 18 cases 12 (66.6%) had clonal T- and three (16.6%) had clonal B-cells. Presence of the lymphotropic viral genome of HHV6 was detected in four of 18 lymph node biopsies from T-AIL patients (22%), all were TCRgamma clonal. No HHV8 were found. Epstein Barr genome was found in 40% of cases. There was no significant association between T-cell clonality and HHV-6 or EBV infection, or between B-cell clonality and any virus infection. We conclude that T-AIL is a biologically and clinically heterogeneous entity whose true nature remains to be clarified.     

High Incidence of Clonal CD8+ T-cell Proliferation in Non-malignant Conditions May Reduce the Significance of T-cell Clonality Assay for Differential Diagnosis in Oncohematology

Polymerase chain reaction (PCR) analysis of rearranged T-cell receptor (TCR) genes is a valuable diagnostic tool for differential diagnosis of T-cell large granular lymphocytic (T-LGL) leukemia and reactive lymphocytosis. Age-related narrowing of T-cells repertoire and expansion of immune or autoimmune clones may lead to false-positive results. The objective of this study was to evaluate the specificity and positive predictive value of PCR-based clonality assessment for a differential diagnostics of T-LGL leukemia. Rearrangements of TCRG and TCRB genes using the BIOMED-2 protocol were assessed in healthy individuals including the elderly (n = 62) and patients with rheumatic diseases (n = 14), transitory reactive CD8+ lymphocytosis (n = 17), and T-LGL leukemia (n = 42). Monoclonal TCRG/TCRB rearrangements in blood were identified in 11.3%/4.8% (7/3 of 62) of healthy individuals; 21.4%/14.3% (3/2 of 14) of patients with rheumatic diseases, and 17.6%/11.8% (3/2 of 17) of patients with reactive lymphocytosis. Immunomagnetic selection of lymphocytes in healthy individuals (31 of 33) revealed that clonal T-cells belong to CD8+ and CD57+ population. No clonal Vβ-Jβ TCRB rearrangements were found in the control group, only Dβ-Jβ TCRB and TCRG. Given the high detectability (96.7%) of Vβ-Jβ TCRB monoclonal rearrangements in patients with αβ-T-LGL leukemia, this marker had the greatest specificity and positive predictive value (100%; 99.2%). The presence of clonal CD8+CD57+ cells in blood is common for healthy individuals and patients with reactive conditions and may not associate with any malignancy. Different specificity of TCRG/ Dβ-Jβ TRB/ Vβ-Jβ TCRB PCR reactions should be taken into account for T-cell clonality data interpretation.     

Use of Monoclonal Antibodies for the Diagnosis of T-cell Malignancies: Applications and Limitations

Biopsy samples from 136 peripheral T-cell lymphomas have been examined and compared with benign inflammatory T-cell infiltrates in an attempt to establish whether immunohistological methods may help to improve the distinction between these conditions. The results confirm and extend previous reports and indicate that the aberrant T-cell phenotypes constitute the single most reliable criterion for the distinction between benign and malignant T-cell infiltrates. These phenotypes are expressed frequently in T-cell malignancies in. lymphoid organs and are also seen in a substantial number of biopsy samples from advanced cutaneous T-cell lymphomas (CTCL). In contrast, early CTCL do not express aberrant T-cell phenotypes and are indistinguishable from benign cutaneous conditions in terms of their immunophenotypic properties. It is concluded that immunophenotypic techniques form a valuable supplement to routine histological methods for the diagnosis of T-cell lymphomas in lymphoid organs. The methods may also help to improve the diagnosis of advanced CTCL, but are of no or only limited help for the recognition of the early stages.     

鼻腔自然杀伤/T细胞淋巴瘤放射治疗的疗效和影响因素

 目的　回顾分析鼻腔自然杀伤（NK）／T细胞淋巴瘤的放射治疗效果,并分析其预后因素。方法　回顾分析9年间接受放射治疗的62例鼻腔NK／T细胞淋巴瘤的临床资料和疗效,单因素分析采用Kaplan-Meier法,多因素分析用COX比例风险模型。结果　全组中位生存时间69.7个月（95 % CI为63.0～78.0个月）,3、5年总生存率分别为66.1 %和46.8 %,远处转移导致治疗失败占61.8 %。T淋巴细胞CD3升高组和降低组的中位生存时间分别为72.6个月和39.6个月,两组比较差异有统计学意义（χ2＝4.9309,P＝0.0264）。多因素分析表明,修正后国际预后指数（IPI）为0～1（χ2＝7.5266,P＝0.0061）、CD3升高（χ2＝9.0912,P＝0.0266）和治疗结束达到CR（χ2＝9.0912,P＝0.0106）是影响鼻腔NK／T细胞淋巴瘤放疗总生存的有利预后因素。结论　放射治疗鼻腔NK／T细胞淋巴瘤疗效肯定,但远处转移治疗失败率高,全身治疗仍具有重要地位;修正后IPI为0～1、CD3升高、治疗结束达到CR是影响鼻腔NK／T细胞淋巴瘤放疗总生存的有利预后因素。     

Clonality of Primary Pulmonary Lymphoproliferative Disorders; Using in Situ Hybridization and Polymerase Chain Reaction for Immunoglobulin

Primary pulmonary lymphoproliferative disorders (PLDs) are histologically divided into a neoplastic state of high and low grade malignant lymphoma (ML), and a reactive state of fol-licular bronchitis/ bronchiolitis (FB) and lymphoid interstitial pneumonia (LIP). We reviewed 19 cases with PLDs, including 4 cases each of high and low grade B cell ML, 6 FB cases, and 5 cases of LIP. To clarify the clonality of the proliferating cells, we performed an immunohis-tochemical examination (IHC), in situ hybridization (ISH) for the immunoglobulin light chain and a polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain gene using DNA obtained from paraffin sections. In addition, a Southern blot analysis was also performed in 6 cases using fresh materials. In IHC, all ML were positive for L26 (CD20). while the monoclonality of the kappa light chain was observed in only one high grade case. However, using ISH we could detect the clonality in three of four high grade ML cases and in one of four low grade ML cases. In FE3 and LIP, no clonality of immunoglobulin by ISH was observed. In a PCR analysis for the immunoglobulin heavy chain gene, we could detect one or two prominent bands in all 8 cases of high and low grade ML. On the other hand, in all cases of FB and LIP, we could only detect either an oligoclonal or polyclonal population. In summary, the presence of monoclonality of ISH and/or PCR for the immunoglobulin heavy chain gene were limited in the neoplastic state, but not in the reactive state.     

血管免疫母细胞性T细胞淋巴瘤病因及发病机制研究进展

 【摘要】　血管免疫母细胞性T细胞淋巴瘤（AITL）是起源于滤泡生发中心辅助性T细胞（TFH）的外周T细胞性肿瘤,具有独特的临床病理特点和生物学行为,其病因及发病机制尚未明确。就近年AITL的病因及发病机制研究进展进行综述。     

外周T/NK细胞淋巴瘤治疗的进展

 外周T细胞淋巴瘤（PTCL）是一组异质性非常明显的淋巴瘤,起源于成熟T淋巴细胞,对传统化疗反应不佳,大部分患者预后差。对复发难治患者的治疗选择尚未达成共识。目前正在进行一些新的临床试验,探讨一些新的治疗方法和药物,如第一次缓解后采用自体干细胞移植进行巩固治疗的前瞻性临床研究,以及denileukin diftitox或者CD52单抗（alemtuzumab）等靶向治疗药物与化疗的联合等。文章对外周T／NK细胞淋巴瘤的流行病学、预后因子和目前的治疗进行了探讨。     

Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR

The definite and rapid diagnosis of invasive aspergillosis is necessary because of the high mortality caused. The objective of this study was to evaluate a real-time PCR assay to detect Aspergillus spp. in clinical samples, based on the Light Cycler technology. Specificity was assessed by using DNA extracted from pathogenic and non-pathogenic bacteria/fungi from Spanish Collection including: two Aspergillus flavus , four Aspergillus fumigatus , two Aspergillus nidulans , two Aspergillus niger and two Aspergillus terreus isolates. The analytical sensitivity was evaluated with different inocula (10¹–10⁵ conidia ml⁻¹), and serially diluted DNA of A. fumigatus. To assess clinical applicability, samples from patients at risk were analysed. Species identification was determined by analysing the melting curves. Reactions using genomic DNA from other species of different genera than Aspergillus were negative (specificity: 100%). Analytical sensitivity was 60 fg using DNA and 5–20 conidia using conidial suspensions. The linear range was from 60 to 6 × 10⁷ fg. The Tm ranged from 67.34 to 70.7 °C for the different Aspergillus spp. studied. Nine hundred and forty-eight consecutive blood samples from 127 patients were processed. In total, 10 (1%) of 948 samples from blood samples were PCR-positive. The real-time PCR assay provides a high sensitivity and specificity for detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species.     

伴噬血细胞综合征的T细胞淋巴瘤临床特点及生存分析

  目的　研究伴噬血细胞综合征（HPS）的T细胞淋巴瘤的临床特点及生存情况。方法　收集2006年1月至2011年12月上海交通大学医学院附属瑞金医院血液科就诊的30例伴HPS的T细胞淋巴瘤患者,以随机数字表法抽取的50例不伴HPS的T细胞淋巴瘤患者作对照,分析其临床特点、实验室检查及生存情况。结果　30例伴HPS的T细胞淋巴瘤组患者表现为高热100.00 %（30／30）、脾大96.67 %（29／30）,两系以上血细胞减少93.33 %（28／30）,骨髓噬血现象86.67 %（26／30）,乳酸脱氢酶（LDH）升高100.00 %（30／30）,高三酰甘油血症46.67 %（14／30）,低纤维蛋白血症60.00 %（18／30）,铁蛋白升高93.33 %（28／30）及肝损伤86.67 %（26／30）,以上指标与50例不伴HPS的T细胞淋巴瘤患者相比较,差异均有统计学意义（χ2值分别为23.11、22.50、36.05、64.20、21.82、5.31、16.54、26.82、46.43,均 P＜0.05）。但浅表淋巴结肿大比例的发生率却不高,仅有33.33 %,这使对原发病的诊断造成比较大的困难。治疗上主要以CHOP方案或依托泊苷+地塞米松的联合化疗,虽然能使症状和实验检查指标得到改善,但中位数生存时间仅20 d。伴与不伴HPS的T细胞淋巴瘤患者生存曲线差异有统计学意义（χ2＝35.05,P＜0.0001）。结论　伴HPS的T细胞淋巴瘤,临床表现复杂,常伴有多脏器受损的表现,联合化疗可使疾病暂时缓解,但总的生存时间短,预后差,发病机制和治疗手段有待进一步研究。     

不同治疗方案对鼻、鼻型NK/T细胞淋巴瘤疗效分析

目的：探讨鼻、鼻型NK/T细胞淋巴瘤的临床特征、治疗方法和预后。方法：收集自2004年1月-2010年1月在我院住院治疗的29例鼻、鼻型NK/T细胞淋巴瘤患者临床资料,分析其临床特点、治疗方案及预后。结果：治疗分CHOP方案组及L-ASP组,总有效率分别为56.3%和75.9%（P=0.045）。两组5年OS率和DFS率分别为43.75%、61.54%（P=0.038）及18.8%、46.15%（P=0.009）,具有统计学意义。难治组7例经VDLP方案补救化疗后总有效率为71.3%。B组症状、临床分期及KPS评分可能对患者的生存产生影响。结论：对于临床早期的鼻、鼻腔NK/T细胞淋巴瘤患者,初治时可以选择CHOP方案治疗,而中、晚期病例及CHOP方案治疗无效的病例,选择以左旋门冬酰胺酶为主的联合化疗结合放疗综合治疗可取得较好疗效。     

不同治疗方案对鼻、鼻型NK/T细胞淋巴瘤疗效分析

目的 探讨鼻、鼻型NK/T细胞淋巴瘤的临床特征、治疗方法和预后.方法 收集自2004年1月-2010年1月在我院住院治疗的29例鼻、鼻型NK/T细胞淋巴瘤患者临床资料,分析其临床特点、治疗方案及预后.结果:治疗分CHOP方案组及L-ASP组,总有效率分别为56.3%和75.9%（P=0.045）.两组5年OS率和DFS率分别为43.75%、61.54%（P=0.038）及18.8%、46.15%（P=0.009）,具有统计学意义.难治组7例经VDLP方案补救化疗后总有效率为71.3%.B组症状、临床分期及KPS评分可能对患者的生存产生影响.结论:对于临床早期的鼻、鼻腔NK/T细胞淋巴瘤患者,初治时可以选择CHOP方案治疗,而中、晚期病例及CHOP方案治疗无效的病例,选择以左旋门冬酰胺酶为主的联合化疗结合放疗综合治疗可取得较好疗效.     

Digital PCR analysis of plasma cell-free DNA for non-invasive detection of drug resistance mechanisms in EGFR mutant NSCLC: Correlation with paired tumor samples

As the development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has become an issue of concern, identification of the mechanisms responsible has become an urgent priority. However, for research purposes, it is not easy to obtain tumor samples from patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC) that has relapsed after treatment with EGFR-TKIs. Here, using digital PCR assay as an alternative and noninvasive method, we examined plasma and tumor samples from patients with relapsed NSCLC to establish the inter-relationships existing among T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification. Paired samples of tumor and blood were obtained from a total of 18 patients with NSCLC after they had developed resistance to EGFR-TKI treatment, and the mechanisms of resistance were analyzed by digital PCR. Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, the overall concordance between plasma and tissue samples being 83.3%. MET gene copy number gain in tumor DNA was observed by digital PCR in three patients, of whom one exhibited positivity for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA. Digital PCR analysis of plasma is feasible and accurate for detection of T790M mutation in NSCLC that becomes resistant to treatment with EGFR-TKIs.     

Hybrid capture-II and LCR-E7 PCR assays for HPV typing in cervical cytologic samples.

As part of an ongoing cohort study in the Hokuriku region of Japan, cervical cell samples from histologically confirmed normal (n = 114) or abnormal (n = 286) women were examined for the presence of HPV DNA using a second-generation hybrid capture assay (HCA-II) and LCR-E7 PCR. HCA-II detected low-risk (HPV-6, -11, -42, 43 and -44) and high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59 and -68) HPV types, while LCR-E7 PCR detected an additional 7 HPV types and some uncharacterized types. In screening of high-grade squamous intraepithelial lesions (HSILs) and invasive cervical cancer, the sensitivities of HCA-II and LCR-E7 PCR testing the high-risk HPV types were 83% and 81%, respectively, while the specificity of both assays was 93%. The sensitivity of LCR-E7 PCR increased to 87%, which was significantly higher than that in HCA-II, when testing both high-risk and other HPV types. Sixty-eight inconsistent results (17% of total tested) from HCA-II and LCR-E7 PCR were due to (i) low copy number of HPV genome (false-negative for HCA-II, 5.3% and for LCR-E7 PCR, 1.3%), (ii) infection with HPV types undetectable by HCA-II (4.8%), (iii) multiple HPV infections (5%) or (iv) unknown reasons (0.8%). LCR-E7 PCR revealed that infections with HPV-16, -18, -31, -33, -35, -51, -52, -56, -58 or -67 was a high risk for cancer since these types predominated in HSIL and invasive cervical cancer. Samples showing high relative light units (>20) with a high-risk probe in HCA-II also gave positive results in LCR-E7 PCR and were generally associated with abnormal cervical lesions. Thus, we propose that both HCA-II and LCR-E7 PCR are valuable screening tests for premalignant and malignant cervical lesions.     

Accessory role of autologous T lymphocytes and adherent cells for the in vitro proliferation of T-cell colony-forming cells from patients with T-cell acute lymphoblastic leukemia

Peripheral blood T colony-forming cells (T-CFC) from patients with T-cell malignancies can proliferate in methylcellulose in the absence of added growth factors or mitogenic stimulation. Mononuclear cells (MNC) from 7 patients with T-cell acute lymphoblastic leukemia were separated into cells forming rosettes with sheep erythrocytes (E+) or not (E-). E- cells were further depleted by complement-mediated cytotoxicity with OKT3 monoclonal antibody (E-OKT3- cells). The study of their spontaneous T-cell colony-forming ability suggested that proliferation of T-CFC in the absence of added growth factors requires cellular cooperation because: (1) No colony growth was observed at low cell concentrations (up to 2 X 10(4) cells/ml) whereas at higher cell densities the number of colonies increased exponentially; (2) The plating efficiency from unfractionated MNC was higher than that from E-OKT3- or E+ cells. Irradiated autologous E+ cells enhanced the plating efficiency from blast-enriched cell fractions (E-OKT3-) when co-cultured either directly in methylcellulose or separately in a two layer assay (agar-methylcellulose), suggesting that their activity could be due to diffusible factors; (3) Adherent-cell depletion of MNC decreased colony formation. Autologous irradiated adherent cells were able to restore the plating efficiency from MNCA- cells when co-cultured directly in methylcellulose but not in separate layers; however, media conditioned by patients' A+ cells could enhance the colony growth from patients' MNCA- cells, indicating that their activity could also be mediated by constitutively released soluble factors.