Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension

Adaptation of continuous cell lines to growth in suspension in a chemically defined medium has significant advantages for design and optimization in manufacturing of biologicals. In this work, changes in the protein expression level during a step-wise adaptation of an adherent Madin Darby canine kidney (MDCK) cell line to suspension growth were analyzed. Therefore, three cell line adaptations were performed independently. Two adaptations were monitored closely to characterize short term changes in protein expression levels after serum deprivation. In addition, initial stages of suspension growth were analyzed for both adaptations. The third adaptation involved MDCK suspension cells (MDCK_SUS2) grown over an extended time period to achieve robust growth characteristics. Here, cells of the final stage of adaptation were compared with their parental cell line (MDCK_ADH). A combination of two dimensional differential gel electrophoresis for relative protein quantification and tandem mass spectrometry for protein identification enabled insights into cellular physiology. The two closely monitored cell line adaptations followed different routes regarding specific changes in protein expression but resulted in similar proteome profiles at the initial stages of suspension growth analyzed. Compared to the MDCK_ADH cells more than 90% of all changes in the protein expression level were identified after serum deprivation and were related to cytoskeletal structure, genetic information processing and cellular metabolism. Myosin proteins, involved in cellular detachment by actin-myosin contractile mechanisms were also differentially expressed. Interestingly, for both of the two adaptations, proteins linked for tumorigenicity, like lactoylglutathione lyase and sulfotransferase 1A1 were differentially expressed. In contrast, none of these proteins were differentially expressed for the MDCK_SUS2 cell line. Overall, proteomic monitoring allowed identification of key proteins involved in adaptation from adherent to suspension growth. In addition, identified proteins related to tumorigenicity may represent markers to support cell clone selection at early stages of industrial cell line development.     

急性B细胞性白血性CD45阴性细胞的检测

目的 探讨细胞表面分化抗原４５阴性（ＣＤ４５）白血病细胞在急性Ｂ细胞性白血病微小残存病变监测中的意义。方法 利用３种不同荧光标记的单克隆抗体染凶及流式细胞仪检测２０例急性Ｂ淋巴细胞性白血病患儿和８名正常人骨骨ＣＤ１０和ＣＤ２２均阳性,ＣＤ阴性或弱阳性表现型细胞。结果 在２０例急性Ｂ细胞性白血病患儿中,均可检测到ＣＤ１０－Ｃ ８名正常人的ＣＤ１０－ＣＤ２２－ＣＤ４５表现型细胞表现频度均在０．００１％     

Pomalidomide-Based Regimens for Treatment of Relapsed and Relapsed/Refractory Multiple Myeloma: Systematic Review and Meta-analysis of Phase 2 and 3 Clinical Trials

IntroductionPomalidomide (Pom) has demonstrated synergistic antiproliferative activity in combination regimens as a result of its distinct anticancer, antiangiogenic, and immunomodulatory effects. This review aimed to compare outcome measures of different Pom regimens for relapsed/refractory multiple myeloma.MethodsA comprehensive literature search identified a total of 1374 studies. Thirty-five studies assessing 4623 subjects met the inclusion criteria: phase 2/3 trial, ≥ 2 prior lines of therapy, and clearly documented efficacy outcomes like overall response rate (ORR), overall survival, and progression-free survival. Statistical analyses for meta-analysis was performed by CMA version 3 and Cochrane Q statistics (P < .05 considered significant, I² index for heterogeneity). A random effects model was used if there was significant heterogeneity (P ≥ .05 over I² ≥ 50%).ResultsPooled analysis showed ORR 47.1% across all Pom-based (2- and 3-drug) regimens. Stratified analysis for efficacy outcomes (pooled ORR [%] and mean progression-free survival [months]) are reported. With doublet regimen, Pom with low-dose dexamethasone (LoDex) was the most common regimen (35.7%, 6.1 months), and overall survival was 14.37 months. With triplet regimens, pooled ORR was 61.9% (I² = 87.3%). These included bortezomib + Pom + LoDex (83.5%, 15.7 months), carfilzomib-Pom + LoDex (77.1%, 15.3 months), Pom + LoDex-bendamustine (74.2%), Pom-dexamethasone-daratumumab (64.5%), Pom + LoDex-cyclophosphamide (59.4%, 9.5 months), and Pom + LoDex-doxorubicin (32%). Leading adverse events were myelosuppression, with mean incidences of grade 3 or higher neutropenia, anemia, and thrombocytopenia of 47.6%, 26.5%, and 20.8%, respectively. Mean incidence of grade 3 or higher nonhematologic adverse events were infections 29.1%, pneumonia 13.8%, and fatigue 10%.ConclusionThree-drug Pom regimens yielded double the response rates compared to Pom + LoDex (pooled ORR, 61.9% vs. 35.7%), with bortezomib + Pom + LoDex and carfilzomib-Pom + LoDex demonstrating better outcomes than other regimens.     

舌鳞状细胞癌预后多因素分析

目的探讨影响舌鳞状细胞癌预后的相关临床等因素。方法回顾性分析175例舌鳞状细胞癌的长期随访资料,采用SPSS 13.0统计软件进行分析。频数资料组间比较采用χ2检验。临床病理指标单因素分析采用Kaplan-Meier法,组间曲线比较用Log-Rank检验,多因素分析采用Cox回归分析。结果全组患者3、5年生存率分别为76.5%、65.4%。单因素分析显示,肿瘤大小、肿瘤是否越过中线、舌外是否侵犯、组织学分级、临床分期、淋巴结转移、局部复发、治疗方式是影响预后的重要因素(P<0.05);多因素分析显示,临床分期、组织学分级、局部复发是影响舌鳞状细胞癌预后的重要因素(P<0.05)。结论影响舌鳞状细胞癌预后的主要因素是临床分期、组织学分级、局部复发。     

121例卵巢透明细胞癌临床病理及预后因素分析

目的探讨卵巢透明细胞癌（OCCA）的临床特点及预后影响因素。方法回顾性分析1999年2月至2009年9月间我院收治的121例OCCA患者的临床资料。结果本组发病年龄24～73岁,中位发病年龄48岁。FIGO分期：Ⅰ期57.0%（69/121）,Ⅱ期9.9%（12/121）,Ⅲ期30.6%（37/121）,Ⅳ期2.6%（3/121）。总的5年生存率（49.8%）：Ⅰ期68.7%,Ⅱ期76.4%,Ⅲ期13.9%,Ⅳ期0。18.2%（22/121）合并子宫内膜异位症;复发率41.7%（40/96）;残存肿瘤对紫杉醇联合卡铂化疗的反应率44.4%（8/18）。单因素分析结果显示：FIGO分期、淋巴结清扫、残存肿瘤大小、腹水/腹腔冲洗液细胞学结果、足够的化疗疗程、无进展时间（PFI）与OCCA的预后有关。治疗前和化疗3程后CA125水平也与OCCA的预后有关。本研究中,发病年龄、合并子宫内膜异位症、合并其他类型上皮癌、盆腔肿物大小和不同的化疗方案与OCCA的预后无关。Cox回归多因素分析结果显示,PFI是OCCA预后的独立影响因素（RR=0.289,P=0.002）。结论 OCCA以早期患者多见,易合并子宫内膜异位症,易复发,化疗反应率低,预后差。OCCA患者行理想肿瘤细胞减灭术,术后足够疗程的化疗可望改善预后。PFI是OCCA预后的独立影响因素。     

老年三阴性乳腺癌临床病理特征及预后分析

目的分析老年（≥60岁）三阴性乳腺癌（TNBC）患者的临床病理特点、生存情况及预后因素。方法收集2000年1月至2007年12月间在中国医学科学院肿瘤医院接受治疗的60岁以上雌激素受体（ER）、孕激素受体（PR）和人表皮生长因子受体2（HER-2）均阴性的TNBC患者的临床病理资料,观察其长期生存状况,分析其临床特点及预后因素。结果全组共84例年龄超过60岁的老年TNBC患者纳入分析,占同期收治老年乳腺癌患者的9.6%,中位年龄为66岁（60～88岁）。60.7%的患者合并有高血压、糖尿病、脑血管病等其他疾病,3例（3.6%）患者具有乳腺癌或卵巢癌的家族史。主要病理类型为浸润性导管癌,共75例（89.3%）。病理分期肿瘤大小为T1或T2的患者占92.9%,Ⅰ、Ⅱ、Ⅲ和Ⅳ期的患者分别有14例（16.7%）、51例（60.7%）、18例（21.4%）和1例（1.2%）,10例（11.9%）患者存在脉管瘤栓。全组患者5年无病生存率（DFS）和总生存率（OS）分别为68.0%和83.2%。单因素预后分析显示,临床分期和辅助化疗周期数对无病生存和总生存均有显著影响。多因素预后分析结果显示,年龄及临床分期是老年TNBC独立的预后因素。在随访期间共有10例患者出现局部复发或区域淋巴结转移,22例患者出现远处转移,7例患者同时出现局部复发和远处转移,其中13例（15.5%）患者同时出现2个部位以上的转移。至末次随访时已有13例患者（15.5%）死亡。结论老年TNBC具有独特的临床病理特点,年龄及临床分期是其独立的预后因素,对该特殊群体患者,亟需探索更为合理的个体化治疗策略。     

乳腺癌患者淋巴结CD4＋CD25＋T细胞和相关细胞因子的检测及相关性分析

目的探讨CD4＋CD25＋T细胞在乳腺癌发生、发展中的作用。方法将取自35例乳腺癌患者的105个肿大淋巴结,制备成单细胞悬液,应用流式细胞仪检测CD4＋CD25＋T细胞比例及CD4＋CD25-、CD8＋T细胞、NK细胞的相对水平。采用定量RT-PCR法,检测IL-2、IL-10、TGF-β1和IFN-γ的细胞因子水平。结果乳腺癌患者淋巴结中的CD4＋CD25＋T细胞水平（在CD4＋T细胞中的百分含量）与淋巴结转移相关,转移淋巴结中其水平明显高于未转移淋巴结。乳腺癌患者淋巴结中CD4＋CD25＋T细胞与CD4＋CD25-、CD8＋T细胞和NK细胞的水平呈负相关关系。乳腺癌患者淋巴结中CD4＋CD25＋T细胞水平与TGF-β1呈正相关,与IL-2、IL-10、IFN-γ无相关性。乳腺癌患者淋巴结中TGF-β1、IL-10、IFN-γ水平与淋巴结转移相关,转移淋巴结中TGF-β1、IL-10含量高而IFN-γ含量较低。IL-2与淋巴结转移无相关性。结论乳腺癌患者转移淋巴结中的CD4＋CD25＋T细胞水平高于未转移淋巴结。     

胃癌组织中p16基因甲基化状态及临床意义

[目的]探讨胃癌患者癌组织、癌旁组织以及对照正常胃黏膜组织中p16基因的甲基化状态,及其与临床病理参数及预后的关系。[方法]应用甲基化特异性实时荧光聚合酶链反应技术检测92例胃癌患者癌组织及配对癌旁组织中p16基因启动子区5′-CpG岛甲基化状态,分析p16基因异常甲基化与患者临床病理特征以及预后之间的关系。[结果]胃腺癌患者癌组织中检测到p16基因甲基化79例(85.9%),癌旁组织11例(12.0%),胃炎患者组织10例(20.8%),健康人未检测到甲基化。p16基因甲基化与肿瘤大小、分化程度、是否淋巴管侵犯、T分期、有无淋巴结转移、有无远处转移、临床分期有关(P<0.05)。p16基因甲基化影响患者预后,但不是胃癌患者无病生存的独立预后因子。[结论]胃腺癌患者p16基因甲基化与肿瘤的生物学行为有关,提示肿瘤进展,恶性程度高,预后欠佳。