EXPRESSION OF IL-2R ON PERIPHERAL BLOOD LYMPHOCYTES OF PATIENTS WITH COLORECTAL CANCER AND ITS CLINICAL SIGNIFICANCE

In recent years it has been noted that the levels of serum soluble interleukin-2 receptor (sIL-2R) in patients with malignancy were abnormally elevated and the levels of sIL-2R might be considered as an index to monitor patient's condition and assess treatment effects. But in medical literature it was reported that the levels of membrane interleukin-2 receptor (CD25) in peripheral blood lymphocytes (PBL) in patients with malignancy were various, some were elevated, some were dropped.[1] Th…     

肺癌患者外周血循环中癌细胞的流式细胞仪分析

目的：用流式细仪分析肺癌患者外周血循环中的癌细胞,方法：外周血经Ficoll梯度离心分离单核细胞组分,后者用CD45、细胞角蛋白（CK）及肺癌特异性抗体（2F／S5A）染色后,应用流式细胞仪检测CD45^-CK^ /S5A^ 细胞。结果检测了165例肺癌患者外周血,发现50例（30．3％）患者外周血癌细胞呈阳性。其中非小细胞肺癌阳性率为30．67％（45／150）,小细胞肺癌为33．33％,阳性率为与患者的病理分期有显著相关性（P＜0．05）。结论：利用流式细胞仪分析检测癌患者外周血循环中的癌细胞不驻有助于临床病理分期,而且对预测肺癌转移潜能具有应用价值。     

大肠癌患者外周血CK19和MUC-1 mRNA水平及其临床意义

目的 探讨大肠癌（CRC）患者外周血中角蛋白（CK）19、黏蛋白（MUC）-1 mRNA水平并分析两者与临床病理特征的关系。方法 采用巢式逆转录聚合酶链式反应（RT-PCR）检测20例健康体检者（健康组）、20例大肠良性腺瘤患者（良性疾病组）及90例CRC患者（CRC组）的外周血CK19和MUC-1 mRNA水平,并分析两者与CRC临床病理参数（Dukes分期、分化程度、远处转移及组织学类型）的关系。结果CRC组CK19 mRNA阳性率为58.9％（53／90）,均高于健康组的0和良性疾病组的5.0％（1／20）,差异有统计学意义（P＜0.05）;CRC组MUC-1 mRNA阳性率为52.2％（47／90）,与健康组的60.0％（12／20）和良性疾病组的50.0％（10／20）比较,差异无统计学意义（P＞0.05）;健康组和良性疾病组CK19 mRNA、MUC-1 mRNA阳性率的差异无统计学意义（P＞0.05）。CRC中CK19 mRNA和MUC-1 mRNA阳性率均与Dukes分期、分化程度及远处转移有关（P＜0.05）,但与组织学类型无关（P＞0.05）。 结论 CRC患者外周血中CK19 mRNA阳性率明显升高,且外周血中CK19mRNA、MUC-1 mRNA与Dukes分期、分化程度及远处转移有关,提示两者可能在CRC发生发展中均有重要意义。     

非小细胞肺癌外周血中树突状细胞含量降低及其意义

目的　探讨非小细胞肺癌 (NSCLC)外周血中树突状细胞 (DC)含量及其与肺癌临床病理特征的关系。方法　采用流式细胞技术检测了 4 1例NSCLC、8例肺部良性病变及 12例正常人外周血中的DC。结果　肺部良性病变者的外周血中DC含量与正常人相比无明显差异 (P >0 .0 5 ) ,但NSCLC患者DC含量显著降低 (P <0 .0 1)。上述三组的外周血DC与单个核细胞比值 (DC/PBMC)分别为 0 .6 0± 0 .0 5 %、0 .83± 0 .0 8%、1.0 2± 0 .11% ,DC计数分别为 2 1.0 0± 3.0 4× 10 6/L、2 9.0 0± 4 .0 1×10 6/L、16 .0 0± 1.5 2× 10 6/L。NSCLC外周血中DC含量与性别、年龄及病理类型无关 ,但与分化程度、淋巴结转移、临床分期及远道转移呈负相关 (P <0 .0 5 )。结论　NSCLC外周血中DC分化成熟严重缺陷 ,血中DC含量与肺癌恶性程度有关。     

大肠癌患者外周血癌细胞CK20检测及其意义

目的:探讨大肠癌患者外周血癌细胞角蛋白20(cytokeratin,CK20)检测与转移复发的关系。方法:以非同位 素RT-PCR方法,检测30例大肠癌患者术前和术后外周血中大肠癌细胞的标志物CK20,同时检测20例健康者外周血作对 照。结果:30例患者外周血中查出CK20阳性表达23例,阳性率76.67%,20例健康者外周血均无CK20表达。CK20阳性检 出与Dukes分期、淋巴结转移和肝转移存在显著性差异(P<0.05),术后3天内外周血CK20阳性检出较术前多出2例。随访 发现,外周血CK20扩增阳性者,术后发生肝转移的机会增加(33.3%)。结论:检测大肠癌患者外周血微转移癌细胞CK20可 提高大肠癌临床分期的准确性,帮助综合判断患者的预后,且可能有助于早期诊断大肠癌肝脏微小转移。     

A pilot study of mRNA expressions of 5-fluorouracil pathway genes in peripheral blood mononuclear cells and tumor tissues in patients with lung adenocarcinoma

To assess whether early lung cancer prediction might be informed by an mRNA assay for 5-fluorouracil pathway genes in peripheral blood mononuclear cells (PBMNCs), we examined specimens taken from 51 adenocarcinoma patients and 38 controls (including six patients with benign tumors). PBMNCs and tumor-tissue specimens were taken for measurement of the mRNAs of various 5-fluorouracil pathway genes [thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyl transferase (OPRT)]. By quantitative RT-PCR, all four mRNAs were detected in both PBMNCs and tumor tissues. In PBMNCs, TS mRNA/GAPDH mRNA levels were significantly higher in adenocarcinoma patients than in the controls, and significantly higher for pathological stages 2-4 and lymph-node involvement pN1-pN3 than for pathological stage 1 and pN0, respectively. No correlation between PBMNCs and tumor-tissue specimens was found for the level of any mRNA. Thus, the measurement of TS mRNA in PBMNCs might aid the diagnosis of lung adenocarcinoma.     

结直肠癌患者外周血中Survivin mRNA的表达及临床意义

目的 探讨 survivin mRNA 在结直肠癌患者外周血的表达及临床意义。方法 采用 SPSS16.0 软件进行统计分析。采 取 χ2 检验 ,收集保定市第一中心医院普通外科收治的结直肠癌患者90 例、结直肠腺瘤疾病患者60 例,同期选择本院体检 健康人60 例为对照组,运用巢式RT-PCR 技术检测并比较术前结直肠癌患者、结直肠腺瘤疾病患者、正常健康人3组血清 中survivin mRNA 表达水平。分析结直肠癌患者血清中survivin mRNA 水平与肿瘤术后分期、病理类型、分化程度和是否远 处转移等临床病理特征相关性。结果 健康人中 survivin mRNA 不表达,结直肠良性腺瘤疾病患者中有 2例survivin mRNA 表达,表达率为3.33%,结直肠癌患者外周血中survivin mRNA 有49 例,表达率为54.44%,survivin mRNA 表达率与肿瘤分期 及肿瘤转移密切相关（P＜0.05）,与肿瘤的病理类型及分化程度无明显相关（P＞0.05）。结论 结直肠癌患者外周 survivin mRNA 表达明显上调,其与结肠癌分期及是否远处转移密切相关,可以作为检测结直肠癌患者外周血转移和评价肿瘤是否进 展的良好的分子标记物。     

肺癌外周血细胞角蛋白联合2F7/S5A单抗检测及其临床应用

目的　应用流式细胞仪对肺癌外周血中的细胞角蛋白及 2F7/S5A单抗检测阳性细胞进行定量分析 ,探讨临床应用的可行性。方法　取外周血 5ml ,经Ficoll密度、梯度离心获取单个核细胞 ,CD45、细胞角蛋白 (CK)及肺癌单克隆抗体 ( 2F7/S5A)标记 ,应用流式细胞仪检测CD45 - CK+2F7/S5A+细胞 ,计数单位 (升 )血中的CD45 - CK+2F7/S5A+细胞含量。结果　共 15 1例进入研究 ,其中健康对照 2 0例 ,肺癌 116例 ,肺部良性疾病 15例。正常外周血单核细胞的表型为CD45 +CK- 2F7/S5A- ,而小细胞肺癌和非小细胞肺癌标记的表型均为CD45 - CK+2F7/S5A+。 2 0例健康对照均为阴性 ;15例良性肺部疾病中 1例为阳性 ;116例肺癌患者 39例为阳性 ,检测敏感性为 33.6 2 % ,特异性 93.33% ,阳性预测值 97.5 0 % ,阴性预测值 15 .38% ,检测阳性细胞含量的平均数为 0 .12× 10 6/L。结论　应用流式细胞仪检测肺癌患者外周血中CD45 - CK+2F7/S5A+细胞具有较高的特异性和阳性预测值 ,能定量反应肺癌患者外周血中的微量癌细胞 ,对肺癌的临床诊断及治疗具有一定的应用价值。     

Perioperative tumor cell dissemination in patients with primary or metastatic colorectal cancer

Introduction

Although there is general correlation between the TNM stage of colorectal cancer (CRC) and its prognosis, there is often significant variability of tumor behaviour and individual patient outcome, which is unaccounted for by pathologic factors alone. Our aim was to estimate perioperative tumor cell dissemination in patients with primary or CRC liver metastases as a possible factor influencing the outcome.

Methods

Forty patients were prospectively enrolled in the study from the year 2007 to 2008. Eighteen patients had histologically proven CRC (50% rectal, 44% colonic, 6% colonic and rectal). Sixteen patients (47%) had CRC liver metastases only. The remaining six patients who underwent colon or liver resection for benign conditions, acted as the control group. All patients with malignant pathologies had R0 resections. Blood samples were taken before the surgical incision (T0), immediately after tumor resection (T1) and at the end of the surgical intervention (T2). Data acquisition was performed using a dual-laser FACSCalibur flow cytometer. Circulating malignant cells were identified as being CD45−/cytokeratin+.

Results

The analysis of patients overall (CRC resection subgroup and hepatectomy subgroup) revealed that there was no statistically significant difference of the tumoral cell count in the blood per million of hematopoietic cells at T0, T1 and T2.

Conclusions

This study demonstrates no differences in the detected circulating numbers of tumor cells at different stages of surgical intervention.     

Anti-viral cytotoxic T cells inhibit the growth of cancer cells with antibody targeted HLA class I/peptide complexes in SCID mice

A number of experimental antibody mediated cancer therapies aim to redirect cytotoxic T cells (CTLs) of non-tumour specificity to cancer cells. It has been previously demonstrated that cancer cells targeted with recombinant HLA-class I/viral peptide complexes via antibody delivery systems can be killed by virus specific CTLs. This novel therapeutic system has been developed with a simple pre-clinical model using the recombinant anti-CD20 B9E9 scFvSA fusion protein to target HLA-A2/peptide complexes to CD20 +ve Daudi lymphoma cells. In vitro data confirmed that, although binding of the B9E9 scFvSA fusion protein alone to Daudi cells had no effect on their growth, effective CTL mediated killing of Daudi cells could be achieved by targeting with B9E9 sfvScSA and recombinant HLA-A2/MI complexes at dilutions as low as 100 pg/ml. In contrast the free HLA-A2/MI complexes only significantly inhibited CTL activity at concentrations in excess of 100 ng/ml. The in vivo tumour protection assays in SCID mice demonstrated that only 1 of the 4 mice that received anti-HLA-A2/M1 CTLs and Daudi cells targeted with the B9E9 scFvSA fusion protein and HLA-A2/M1 complexes developed a tumour. In contrast in the control mice that received CTL and native Daudi cells all 4 developed tumours, as did all 4 that received targeted Daudi cells but no CTLs. Similar results were obtained in a parallel experiment using Daudi cells targeted with B9E9 scFvSA and HLA-A2/BMLF1 complexes and a CTL line to HLA-A2/BMLF1. The demonstration of in vivo activity for targeted HLA class I/peptide complexes combined with anti-viral T cells, supports the further clinical development of the system where it may be combined with autologous CTLs produced by vaccination or ex vivo expansion.     

外周血T淋巴细胞亚群与NK细胞检测在肺癌诊断及治疗中的意义

 目的　通过研究肺癌患者外周血T淋巴细胞亚群与NK细胞的表达及动态变化,分析其与肺癌发生、发展的关系及其临床意义。方法　运用流式细胞术检测66例肺癌患者（肺癌组）、60例肺结核患者（肺结核组）和60名健康人（健康对照组）外周血中CD+3、CD+3 CD+8、CD+3 CD+4、Th／Ts、CD+16 CD+56的表达;肺癌组还检测了化疗前后第3、7和20天外周血CD+3、CD+3 CD+8、CD+3 CD+4、Th／Ts、CD+16 CD+56 的表达。结果　肺癌组CD+3、CD+3 CD+4、Th／Ts、CD+16 CD+56表达明显降低[（54.23±10.37）%、（34.23±8.03）%、1.35±0.20、（25.18±4.34）%],与肺结核组[（63.09±9.19）%、（39.46±12.74）%、1.51±0.41、（26.45±3.96）%]和健康对照组[（69.68±8.31）%、（42.31±13.29）%、1.89±0.48、（29.44±2.51）%]比较差异均有统计学意义（均P＜0.05）;CD+3 CD+8 表达三组间差异均无统计学意义（均P＞0.05）。在化疗组中,化疗缓解组化疗后第3天CD+3、CD+3 CD+4、Th／Ts和CD+16 CD+56 表达较化疗前均显著降低（均P＜0.01）,而CD+3 CD+8表达显著升高（P＜0.01）;第7天各项指标基本恢复到化疗前水平;第20天CD+3、CD+3 CD+4、Th／Ts和CD+16 CD+56 表达较化疗前均显著升高（均P＜0.05）,而CD+3 CD+8表达显著降低（P＜0.05）。化疗未缓解组化疗前后各项指标的表达差异均无统计学意义（均P＞0.05）。在肺癌组中,ⅢA期、ⅢB期与ⅠA期比较、淋巴结转移N3组与N0组比较,CD+3、CD+3 CD+4、CD+3 CD+8、Th／Ts和CD+16 CD+56 表达差异均有统计学意义（均P＜0.05）;不同病理分型间各项指标表达差异均无统计学意义（均P＞0.05）。结论　动态监测肺癌患者外周血T淋巴细胞亚群与NK细胞可指导临床诊断和治疗,并有助于评估患者免疫功能状态。     

Monitoring FTD in the peripheral blood mononuclear cells of elderly patients with metastatic colorectal cancer administered FTD plus bevacizumab as first-line treatment

Trifluridine/tipiracil (FTD/TPI) is an orally administrated anticancer drug with efficacy validated for patients with metastatic colorectal cancer (mCRC) or gastric cancer. FTD, a key component of FTD/TPI, exerts antitumor effects via its incorporation into DNA. Using specific antibodies against bromodeoxyuridine, FTD incorporation into DNA is detected in tumors and peripheral blood mononuclear cells (PBMC) of patients with mCRC who are administered FTD/TPI. The proportion of FTD-positive PBMC fluctuates according to the schedule of treatment, although the association between the proportion of FTD-positive PBMC and the clinical outcomes of patients is unknown. To answer this question, here we monitored the FTD-positive PBMC of 39 elderly patients with mCRC enrolled in KSCC1602, a single-arm phase 2 trial of FTD/TPI plus bevacizumab as a first-line treatment, for 1 month, during the first cycle of treatment. The median values and interquartile ranges of the percentage of FTD-positive PBMC on days 8, 15, and 29 were 39.3% (30.7%-52.2%), 66.9% (40.0%-75.3%), and 13.5% (5.7%-26.0%), respectively. Receiver operating characteristic analysis revealed that the percentage of FTD-positive PBMC on day 8 (the end of the first week of treatment) had moderate ability to accurately diagnose the occurrence of severe neutropenia and leukopenia within 1 month (area under the curve = 0.778 [95% confidence interval, 0.554-0.993]). This result suggests that excess FTD incorporation into PBMC at the initial phase of FTD/TPI plus bevacizumab treatment is a risk factor for early onset of severe hematological adverse events.     

乳腺癌外周血微转移与远处转移的相关性

背景与目的:乳腺癌是一种全身性疾病,早期就可发生血行转移.本研究采用流式细胞仪定量检测乳腺疾病患者外周血肿瘤细胞含量,探讨外周血肿瘤细胞含量与远处脏器转移的相关性.方法:以人细胞角蛋白抗体和CD45抗体双色免疫标记,应用多参数流式细胞仪方法检测65例乳腺癌患者术前和10例乳腺良性病变及8例健康献血者外周血标本中肿瘤细胞含量.结果:65例乳腺癌患者中,57例检测出肿瘤细胞,阳性率为87.7%;乳腺良性病变、健康献血者外周血中未检测到肿瘤细胞.外周血肿瘤细胞阳性率与肿瘤T分期及N分期具有相关性(r=0.271,P=0.017;r=0.393,P=0.002).除2例失访外,63例患者经过5年随诊,25例出现远处脏器转移,5年转移率为39.7%.所有远处转移病例均为外周血检测阳性患者,8例术前外周血检测阴性的患者均未发现远处转移,两组比较差异有统计学意义(P=0.014);而远处转移与肿瘤T分期、N分期均无明显相关性.结论:术前外周血肿瘤细胞数量与远处转移具有明显相关性,多参数流式细胞术检测乳腺癌患者外周血肿瘤细胞,对指导乳腺癌的个体化治疗具有潜在的应用价值.     

非小细胞肺癌患者外周血粘附分子表达的临床意义

目的 :探讨非小细胞肺癌患者外周血粘附分子CD4 4和CD6 2P的表达特征及其临床意义。方法 :应用流式细胞仪对 5 5例肺癌患者外周血CD4 4和CD6 2P表达进行荧光免疫检测 ,并与正常对照组 (n =30 )进行对比研究。结果 :5 5例肺癌患者外周血中CD4 4和CD6 2P表达明显高于正常对照组 (P <0 0 1)。Ⅲ期、Ⅳ期和Ⅰ期、Ⅱ期之间CD4 4和CD6 2P表达比较 ,有显著性差异 (P <0 0 1)。CD4 4和CD6 2P表达与肺癌组织学分级有明显相关性 (P <0 0 1)。结论 :应用流式细胞仪检测CD4 4和CD6 2P的表达水平可作为肺癌转移和预后的指标     

肺癌患者外周血淋巴细胞与癌细胞耐药基因表达的相关性研究

目的：探讨肺癌患者外周血淋巴细胞和癌细胞耐药基因表达的相关性。方法：采用RT—PCR法检测40例肺癌患者外周血淋巴细胞耐药基因MDR-1的表达,免疫组化法检测肺癌组织原代培养癌细胞耐药基因蛋白P—gp和MRP的表达。结果：肺癌患者外周血淋巴细胞耐药基因MDR-1的表达率37．5％（15／40）,与肺癌细胞耐药基因蛋白P—gp和MRP的表达具有相关性（P〈0．05）,与肺癌患者的年龄、分期、病理类型及性别无关（P〉0．05）。结论：外周血淋巴细胞耐药基因MDR-1的表达率较高,与癌细胞耐药基因蛋白P—gp和MRP的表达具有相关性,对肺癌临床化疗药物的选择具有重要的参考价值。     

大肠癌患者4种淋巴细胞表型的测定及其意义

背景与目的:机体淋巴细胞的功能状态与肿瘤的发生、发展过程密切相关。而淋巴细胞表面众多而复杂的膜蛋白是淋巴细胞膜信号传递、激活、凋亡等细胞分化调控机制重要的分子生物学基础。本研究探讨了大肠癌患者外周血淋巴细胞(peripheralbloodlymphocytes,PBL)CD8、CD28、CD49b、CD49d4种表型变化及其肿瘤生物学行为的关系。方法:应用CD8、CD28、CD49b、CD49d单克隆抗体,采用流式细胞术,对35例大肠癌患者(肿瘤组)及30例同期非肿瘤患者(对照组)的PBL上述4种表型进行监测。结果:肿瘤组淋巴细胞CD8的表达为(30.89±6.54)%,对照组的表达为(20.82±5.15)%,肿瘤组显著高于对照组(P<0.01);肿瘤组CD28、CD8+/CD28+及CD49b、CD49d的表达分别为(42.04±8.83)%、(9.29±2.81)%及(22.77±10.46)%、(65.59±13.15)%,对照组的表达分别为(56.05±7.32)%、(14.91±2.58)%及(33.18±17.18)%、(83.61±9.14)%,肿瘤组的表达显著低于对照组P均<0.05)。DukesD期大肠癌组CD8+/CD28+、CD49b、CD49d的表达分别为(7.62±1.63)%、(18.53±9.59)%、(59.85±16.18)%,DukesB期组的表达分别为(10.01±2.90)%、(9.60±11.01)%、(73.12±10.86)%,DukesD期大肠癌组的表达明显低于DukesB期组(P<0.05);淋巴结转移组CD28、CD8+/CD28+及CD49b、C     

Induction of viral and tumour specific CTL responses using antibody targeted HLA class I peptide complexes

The production of cytotoxic T cells with specificity for cancer cells is a rapidly evolving branch of cancer therapeutics. A variety of approaches aim to amplify anti-tumour cytotoxic T cell responses using purified peptides, tumour cell lysates or recombinant HLA/peptide complexes in differing antigen presenting systems. Using a two-step biotin-streptavidin antibody targeting system, recombinant HLA-class I/peptide complexes were attached to the surface of B cells via the anti-CD20 B9E9-scFvSA antibody-streptavidin fusion protein. Flow cytometry with a conformation dependant monoclonal antibody to HLA class I indicated that targeted HLA-class I/peptide complexes remain on the surface of B cells in culture for periods in excess of 72 h. PBMCs were stimulated in vitro for 8-14 days using the autologous B cells as antigen presenting cells. Following a single cycle of stimulation specific cytotoxic T cell responses to targeted HLA-A2 complexes containing the M1, BMLF1 and Melan A peptides could be demonstrated by tetramer staining and Cr release assays. With the HLA-A2/BMLF1 complex up to 2.99% of CD8+ve cells were tetramer positive producing 20% lysis (E : T 10 : 1) of CIR-A2 target cells in an in vitro cytotoxicity assay compared to baseline levels of 0.09% tetramer +ve and 2% lysis in the unstimulated population. PBMCs from a healthy donor treated with two cycles of stimulations with targeted HLA-A2/Melan A complexes, demonstrated expansion of the melanA tetramer +ve population from 0.03% to 1.4% producing 15% lysis of Melan A pulsed target cells. With further consideration to the key variables of HLA/peptide complex density, the ratio of stimulator to effector cells and optimum cytokine support, this system should offer an easy and effective method for the in vitro amplification of specific cytotoxic T cell responses and warrants development for the in vivo induction of cytotoxic T cell responses in cancer therapy.